Difference between revisions of "Part:BBa E1010"
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__NOTOC__
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<b>Summary</b>
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<partinfo>BBa_E1010 short</partinfo>
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In order to better and more comprehensive understand our favorite reporter part whose name is BBa_E1010, this year NAU-CHINA uses SWISS MODEL (https://swissmodel.expasy.org/) to model and simulate the tertiary structure of protein. We hope that the prediction of the structure will help other teams to better understand the nature and characteristics of this part and be able to use the reporter gene more skillfully.
  
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monomeric RFP:
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The following model was built (see Materials and Methods "Model Building"):
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Red Fluorescent Protein.
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Excitation peak: 584 nm
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Emission peak: 607 nm
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Fig.1. Model #01                          Fig.2. The Active center
  
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===Usage and Biology===
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Fig.3. Local quality                      Fig.4. Comparison with Non-redundant Set of PBD Structures
  
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Robert E. Campbell started with Discosoma RFP (DsRed) and evolved a faster folding, monomeric variant.  See paper listed in source.  Codon optimized for expression in bacteria (?? DE)
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<b>Target</b>
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MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA
  
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[[Image:AmilCP_amilGFP_RFP.jpg|300px]]
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<b>Template 2qli.1.A</b>
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[[Image:On cultures BYR small.jpg|300px]]
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VSKGEEVIKEFMRFKQHMEGSVNGHEFEIEGEGEGRPYEGTQTARLKVTKGGPLPFAWDILSPQIX—SKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKVRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEMKMRLRLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA
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[[Image:Pellets BYR.jpg|300px]]
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'''iGEM11_Uppsala-Sweden:''' Expression of chromoproteins. The images above show ''E coli'' <partinfo>constitutively</partinfo> expressing amilCP <partinfo>BBa_K592009</partinfo> (blue), amilGFP <partinfo>BBa_K592010</partinfo> (yellow) and RFP <partinfo>BBa_E1010</partinfo> (red).  
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<b>Materials and Methods</b>
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<b>Template Search</b>
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Template search with BLAST and HHBlits has been performed against the SWISS-MODEL template library (SMTL, last update: 2020-09-23, last included PDB release: 2020-09-18).
  
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Peking iGEM 2016 has fused this part with triple spytag. The fused protein is participate in Peking’s polymer network. By adding this protein, the whole polymer network become visible in most conditions. If you want to learn more about Peking’s polymer network and the role of mRFP in this network, please click here https://parts.igem.org/Part:BBa_K1989004".
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The target sequence was searched with BLAST against the primary amino acid sequence contained in the SMTL. A total of 670 templates were found.
  
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An initial HHblits profile has been built using the procedure outlined in (Steinegger et al.), followed by 1 iteration of HHblits against Uniclust30 (Mirdita, von den Driesch et al.). The obtained profile has then be searched against all profiles of the SMTL. A total of 720 templates were found.
  
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Allergen characterization of BBa_E1010: NOT a potential allergen
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<b>Template Selection</b>
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For each identified template, the template's quality has been predicted from features of the target-template alignment. The templates with the highest quality have then been selected for model building.
  
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The Baltimore Biocrew 2017 team discovered that proteins generated through biobrick parts can be evaluated for allergenicity. This information is important to the people using these parts in the lab, as well as when considering using the protein for mass production, or using in the environment. The allergenicity test permits a comparison between the sequences of the biobrick parts and the identified allergen proteins enlisted in a data base.The higher the similarity between the biobricks and the proteins, the more likely the biobrick is allergenic cross-reactive. In the full-length alignments by FASTA, 30% or more amount of similarity signifies that the biobrick has a Precaution Status meaning there is a potential risk with using the part. A 50% or more amount of identity signifies that the biobrick has a Possible Allergen Status. In the sliding window of 80 amino acid segments, greater than 35% signifies similarity to allergens. The percentage of similarity implies the potential of harm biobricks’ potential negative impact to exposed populations. For more information on how to assess your own biobrick part please see the “Allergenicity  Testing Protocol” in the following page http://2017.igem.org/Team:Baltimore_Bio-Crew/Experiments
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<b>Model Building</b>
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Models are built based on the target-template alignment using ProMod3. Coordinates which are conserved between the target and the template are copied from the template to the model. Insertions and deletions are remodelled using a fragment library. Side chains are then rebuilt. Finally, the geometry of the resulting model is regularized by using a force field. In case loop modelling with ProMod3 fails, an alternative model is built with PROMOD-II (Guex et al.).
  
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<b>Model Quality Estimation</b>
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The global and per-residue model quality has been assessed using the QMEAN scoring function (Studer et al.).
  
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For the biobrick part, BBa_E1010, there was a 27.6% of identity match and 56.9% of similarity match compared to the allergen database. This means that the biobrick part is not of potential allergen status. In the 80 amino acid alignments by FASTA, no matches found that are greater than 35% for this biobrick.
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<b>Ligand Modelling</b>
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Ligands present in the template structure are transferred by homology to the model when the following criteria are met: (a) The ligands are annotated as biologically relevant in the template library, (b) the ligand is in contact with the model, (c) the ligand is not clashing with the protein, (d) the residues in contact with the ligand are conserved between the target and the template. If any of these four criteria is not satisfied, a certain ligand will not be included in the model. The model summary includes information on why and which ligand has not been included.
  
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<b>Oligomeric State Conservation</b>
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The quaternary structure annotation of the template is used to model the target sequence in its oligomeric form. The method (Bertoni et al.) is based on a supervised machine learning algorithm, Support Vector Machines (SVM), which combines interface conservation, structural clustering, and other template features to provide a quaternary structure quality estimate (QSQE). The QSQE score is a number between 0 and 1, reflecting the expected accuracy of the interchain contacts for a model built based a given alignment and template. Higher numbers indicate higher reliability. This complements the GMQE score which estimates the accuracy of the tertiary structure of the resulting model.
  
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<b>References</b>
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BLASTWaterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., Schwede, T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46(W1), W296-W303 (2018). 
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Guex, N., Peitsch, M.C., Schwede, T. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis 30, S162-S173 (2009). 
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Bienert, S., Waterhouse, A., de Beer, T.A.P., Tauriello, G., Studer, G., Bordoli, L., Schwede, T. The SWISS-MODEL Repository - new features and functionality. Nucleic Acids Res. 45, D313-D319 (2017). 
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Studer, G., Rempfer, C., Waterhouse, A.M., Gumienny, G., Haas, J., Schwede, T. QMEANDisCo - distance constraints applied on model quality estimation. Bioinformatics 36, 1765-1771 (2020). 
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Bertoni, M., Kiefer, F., Biasini, M., Bordoli, L., Schwede, T. Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology. Scientific Reports 7 (2017). 
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Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T.L. BLAST+: architecture and applications. BMC Bioinformatics 10, 421-430 (2009). 
  
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>Internal Priming Screening Characterization of BBa_E1010: Has no possible internal priming sites between this BioBrick part and the VF2 or the VR primer.
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<b>HHblits</b>
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Steinegger, M., Meier, M., Mirdita, M., Vöhringer, H., Haunsberger, S. J., Söding, J. HH-suite3 for fast remote homology detection and deep protein annotation. BMC Bioinformatics 20, 473 (2019).
  
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The 2018 Hawaii iGEM team evaluated the 40 most frequently used BioBricks and ran them through an internal priming screening process that we developed using the BLAST program tool. Out of the 40 BioBricks we evaluated, 10 of them showed possible internal priming of either the VF2 or VR primers and sometime even both. The data set has a range of sequence lengths from as small as 12 bases to as large as 1,210 bases. We experienced the issue of possible internal priming during the sequence verification process of our own BBa_K2574001 BioBrick and in the cloning process to express the part as a fusion protein. BBa_K2574001 is a composite part containing a VLP forming Gag protein sequence attached to a frequently used RFP part (BBa_E1010). We conducted a PCR amplification of the Gag-RFP insert using the VF2 and VR primers on the ligation product (pSB1C3 ligated to the Gag + RFP). This amplicon would serve as template for another PCR where we would add the NcoI and BamHI restriction enzyme sites through new primers for ligation into pET14b and subsequent induced expression. Despite gel confirming a rather large, approximately 2.1 kb insert band, our sequencing results with the VR primer and BamHI RFP reverse primer gave mixed results. Both should have displayed the end of the RFP, but the VR primer revealed the end of the Gag. Analysis of the VR primer on the Gag-RFP sequence revealed several sites where the VR primer could have annealed with ~9 - 12 bp of complementarity. Internal priming of forward and reverse primers can be detrimental to an iGEM project because you can never be sure if the desired construct was correctly inserted into the BioBrick plasmid without a successful sequence verification.
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<b>Uniclust30</b>
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Mirdita, M., von den Driesch, L., Galiez, C., Martin, M.J., Söding, J., Steinegger, M. Uniclust databases of clustered and deeply annotated protein sequences and alignments. Nucleic Acids Research 45, D170–D176 (2016).
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===Contribution===
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Group: USAFA iGEM 2019
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<br>
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Summary: We have characterized the fluoresence of this part over time and at different temperatures in order to determine optimal incubation conditions and times for best mRFP expression. Our results indicate that 37 degrees C is optimal for both growth and mRFP expression. At 12 hours of incubation at 37C, the RFP was detectable, but after 24 and 48 hours, the expression was much more robust.
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<br>
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[[File:T--USAFA iGEM--BBaE1010 characterization.jpg|900px|thumb|none|alt=mRFP fluorescence.|Figure 1. Quantification of mRFP expression at different incubation temperatures]]
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[[File:T--USAFA iGEM--BBaE1010 time.jpg|900px|thumb|none|alt=mRFP fluorescence.|Figure 2. Picture of visible mRFP expression at different incubation times]]
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<br>
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Conclusion: BBaE1010 is a strong reporter that has RFP expression that can be detected with a fluorescent plate reader or visualized by the unaided eye. 37C was the optimal growth temperature for E. coli expressing the BBaE1010 mRFP and the expression/detection of the mRFP increased over time with 48 hours showing the most robust red color.
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<br>
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<h2></h2>
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===Pre-experiment===
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E. coli Nissle 1917 strains were grown overnight in Lysogeny Broth (LB) containing ampicilin (100 µg/mL) at 37°C and 200 rpm. Cultures were diluted in fresh LB until achieve 0,1 OD with the corresponding antibiotic and transferred to a 96-well plate (50 µL/well). Samples were always made in triplicates and a blank of LB. During 8h the absorbance at OD600 and fluorescence (excitation 488 nm and emission 530 nm) were measured with intervals of 1 hour.
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<html>
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<img src="https://2019.igem.org/wiki/images/3/3a/T--NAU-CHINA--Characterization-1.jpg"width="600"/>
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</html>
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<br>
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===Contribution===
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Group: USAFA iGEM 2019
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<br>
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Summary: We have characterized the fluoresence of this part over time and at different temperatures in order to determine optimal incubation conditions and times for best mRFP expression. Our results indicate that 37 degrees C is optimal for both growth and mRFP expression. At 12 hours of incubation at 37C, the RFP was detectable, but after 24 and 48 hours, the expression was much more robust.
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<br>
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[[File:T--USAFA iGEM--BBaE1010 characterization.jpg|900px|thumb|none|alt=mRFP fluorescence.|Figure 1. Quantification of mRFP expression at different incubation temperatures]]
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[[File:T--USAFA iGEM--BBaE1010 time.jpg|900px|thumb|none|alt=mRFP fluorescence.|Figure 2. Picture of visible mRFP expression at different incubation times]]
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<br>
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Conclusion: BBaE1010 is a strong reporter that has RFP expression that can be detected with a fluorescent plate reader or visualized by the unaided eye. 37C was the optimal growth temperature for E. coli expressing the BBaE1010 mRFP and the expression/detection of the mRFP increased over time with 48 hours showing the most robust red color.
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<br>
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<h2></h2>
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===Contribution===
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Group:NAU_CHINA 2019
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<br>
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Summary: We measured the fluorescence signal of mRFP in buffer under different pH values and temperatures.
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<br>
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Report genes are essential for every team to construct genetic pathways. We believe that every team has a J04450 complex. After all, it is our favorite report gene!<br>
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This year, the NAU iGEM characterized E1010. Based on the work of the previous team work, we explored the tolerance of E1010 expression products at different pH values and temperatures.
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===Pre-experiment===
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We dispensed a small amount of the sample into 12 PCR tubes, and a gradient test at 65-95℃ was conducted in a PCR machine for 10 minutes.<br>
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We found that the protein samples showed significant denaturation when the temperature was higher than 75℃. So, we set the experimental temperature later at and above 75℃.<br>
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The purified sample was dispensed into four PCR tubes and the pH  was）  adjusted to 4.0, 8.0, 10.28, respectively.<br>
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Under each pH value, we dispensed each sample into 12 PCR tubes（50uL sample + 50uL buffer）, and a gradient test at 75-95℃ was conducted in a PCR machine for 10 minutes.<br>
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We measured the fluorescence intensity of the product under the microplate reader (excitation wavelength was 584 nm, and absorption wavelength was 611 nm).<br>
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<html>
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<img src="https://2019.igem.org/wiki/images/3/3a/T--NAU-CHINA--Characterization-1.jpg"width="600"/>
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</html>
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<br>
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===Analysis===
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The results show that the product of J04450 begins to show significant denaturation at 84.4℃, and the fluorescence value of the protein no longer changes significantly at the temperature of around 92.5℃, which means E1010 product is completely denatured (shown in our characterization map with a small amount of protein precipitation and the color is pale yellow). <br>
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The temperature (from 75.4℃ to 90.3℃) and fluorescence values show a significant linear relationship .<br>
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At the same temperature, the pH=4.0 and pH=8.0 curves were basically consistent. The stability of the E1010 expression products at these two pH values was significantly better than that under the condition of pH=10.28.
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===Discussion===
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Report genes  are required for the construction of most gene pathways. In the future iGEM journey, other teams and we may use some bacteria, like thermophiles, that grow in extreme conditions. We hope we can still use our familiar report  gene in these bacteria to detect protein secretion and other related circumstances, so we conducted these tests.<br>
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We simulated the extreme environments in which we released the expression products to the extracellular—acid, weak acid, alkaline, and high temperatures—by adjusting the temperature gradients and pH values.<br>
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We hope that our experimental results can provide other teams with a reference for the stability of report  gene expression products in extreme environments.
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===Reference===
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[1] Sudhagar S A , Prasad K P , Makesh M , et al. Characterization and production of polyclonal antisera against pangasius (Pangasianodon hypophthalmus) serum immunoglobulin IgM derived from DEAE cellulose based ion exchange chromatography[J]. Aquaculture Research, 2015, 46(6):1417-1425.
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<h2></h2>
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===Contribution===
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Group: Hong_Kong_LFC_PC 2019
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<br>
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Summary: We designed a uric-acid-sensitive uricase generator for treating the gout disease. Since we will use the fluorescence protein to track the gene expression of the device, we would like to check the effect of uric acid on the signal of this E1010 RFP. Since uric acid can alter the pH value, it may interfere the folding of RFP and lower its fluorescence signal. We treat the extracted RFP with different concentration of uric acid, and found that uric acid has a very little effect on the fluorescence signal. This indicate that our system is possible to track the gene expression.
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<br>
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https://2019.igem.org/wiki/images/9/91/T--Hong_Kong_LFC_PC--rFP2.jpg
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<br>
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===Contribution===
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Group: Valencia_UPV iGEM 2018
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<br>
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Author: Adrián Requena Gutiérrez, Carolina Ropero
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<br>
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Summary: We have adapted the part to be able to assemble transcriptional units with the Golden Gate method and we have done the characterization of this protein.
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<br>
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Documentation:
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<br>
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Part BBa_K2656014 is the monomeric Red Fluorescent Protein 1 coding sequence [https://parts.igem.org/Part:BBa_E1010 BBa_E1010] adapted into the [http://2018.igem.org/Team:Valencia_UPV/Design Golden Braid assembly method]. Thus, this sequence is both compatible with the BioBrick and GoldenBraid 3.0. grammar.
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This coding sequence can be combined with other Golden Braid compatible parts from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Valencia UPV IGEM 2018 Printeria Collection] to assemble transcriptional units in a one-step BsaI reaction with the [http://2018.igem.org/Team:Valencia_UPV/Protocols Golden Gate assembly protocol].
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The characterization of this protein (and by extension of all the other part that codify for the mRFP1) was performed with our transcriptional unit [https://parts.igem.org/Part:BBa_K2656109 BBa_K2656109].
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This transcriptional unit was assembled in a GoldenBraid alpha1 plasmid including the following parts:
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<html>
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<ul>
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<li></html>[https://parts.igem.org/Part:BBa_K2656004 BBa_K2656004]: the [https://parts.igem.org/Part:BBa_J23106 J23106] promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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<li></html>[https://parts.igem.org/Part:BBa_K2656009 BBa_K2656009]: the [https://parts.igem.org/Part:BBa_B0030 B0030] ribosome biding site in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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<li></html>[https://parts.igem.org/Part:BBa_K2656014 BBa_K2656014]: coding sequence <html></li>
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<li></html>[https://parts.igem.org/Part:BBa_K2656026 BBa_K2656026]: the [https://parts.igem.org/Part:BBa_B0015 B0015] transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]<html></li>
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</ul>
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</html>
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In order to carry out a correct characterization of the protein and to be able to use it to make measurements of the different transcriptional units that we assembled with it, we obtained the emission and excitation spectra in the conditions of our equipment. By using this protocol  [http://2018.igem.org/Team:Valencia_UPV/Experiments#spectra] with the parameters of Table 1, Figure 1 was obtained.
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{|class='wikitable'
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|'''Parameter'''
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|'''Value'''
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|Number of samples
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|3
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|Excitation Wavelength measurement range 1 (nm)
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|[450-620]
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|Excitation Wavelength measurement range 2 (nm)
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|[620-700]
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|-
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|Emission wavelenght 1 (nm)
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|650
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|-
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|Emission wavelenght 2 (nm)
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|590
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|-
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|Emission Wavelength measurement range (nm)
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|[565-700]
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|-
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|Excitation wavelenght (nm)
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|540
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|Gain (G)
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|70
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|colspan=4|Table 1. Parameters used to obtain the spectra
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|}
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[[File:T--Valencia_UPV--mRFP_spectrum.png|900px|thumb|none|alt=mRFP spectra.|Figure 1. mRFP emission and excitation spectra]]
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===Contribution===
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Group: Hong Kong-CUHK iGEM 2017
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<br>
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Author: Yuet Ching Lin
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<br>
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Summary: We measured the fluorescent signal of mRFP in buffers with different pH.
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<br>
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Documentation:
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<br>
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Charaterization of mRFP pH stabillity:
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We grew C41 bacteria with parts BBa_J61002 in 2XYT for 24 hours. After purifying the mRFP by Ion Exchange Chromatography and Hydrophobic Interaction Chromatography, we measured the fluoresece (ex ,em ) of purified mRFP, which is diluted to 10µg/100µl (total 200µl) in triplicates, into different buffers (ranges from pH2 to pH12; Volume of mRFP:buffer = 1:1.8). To facilitate reproducibility of the experiment, we correlated the relative fluorescent intensity to an absolute fluorophore concentration by referring it to a standard curve of the fluorophores(Rhodamine) using the interlab study protocol.  The result shows that the stability drops dramatically in pH condition below 6 and relatively stable in pH 6-10.
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[[File:Mrfp.PNG|center|thumb|350px|''<b>Fig.1</b> Vary pH attributed to different fluorescent intensity of RFP.]]
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<table cellpadding="2" border="1px" cellspacing="0" align="center" width="70%">
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<caption><p align="justify"><b>Table 1 Plate reader setting of fluorescent measurement</b></p></caption>
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    <td><b>Measurement Type</b></td>
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    <td>Fluorescence</td>
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  <tr>
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    <td><b>Microplate name</b></td>
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    <td>COSTAR 96</td>
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  </tr>
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  <tr>
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    <td><b>Scan mode</b></td>
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    <td>orbital averaging</td>
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  </tr>
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  <tr>
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    <td><b>Scan diameter [nm]</b></td>
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    <td>3</td>
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  </tr>
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  <tr>
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    <td><b>Excitation</b></td>
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    <td>550-20</td>
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  </tr>
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  <tr>
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    <td><b>Emission</b></td>
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    <td>605-40</td>
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  </tr>
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  <tr>
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    <td><b>Dichronic filter</b> </td>
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    <td>auto 572.5</td>
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  </tr>
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    <tr>
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    <td><b>Gain </b></td>
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    <td>500</td>
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  </tr>
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      <tr>
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    <td><b>Focal height [nm]</b></td>
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    <td>9</td>
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  </tr>
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</table>
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===Contribution2===
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Part name:<partinfo>BBa_K2382013</partinfo>
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<br>
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Group: iGEM17_CSMU_NCHU_Taiwan  2017
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<br>
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Author: SHAO-CHI LO
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<br>
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Summary: We add a His Tag at the end. Therefore, a fusion protein with this part may have red color and the ability to be purified easily.
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<br>
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Documentation:
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===<span class='h3bb'>Sequence and Features</span>===
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<partinfo>BBa_E1010 SequenceAndFeatures</partinfo>
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===Parts table===
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<html><!--- Please copy this table containing parameters for BBa_E1010 at the end of the parametrs section ahead of the references. ---><style type="text/css">table#AutoAnnotator {border:1px solid black; width:100%; border-collapse:collapse;} th#AutoAnnotatorHeader { border:1px solid black; width:100%; background-color: rgb(221, 221, 221);} td.AutoAnnotator1col { width:100%; border:1px solid black; } span.AutoAnnotatorSequence { font-family:'Courier New', Arial; } td.AutoAnnotatorSeqNum { text-align:right; width:2%; } td.AutoAnnotatorSeqSeq { width:98% } td.AutoAnnotatorSeqFeat1 { width:3% } td.AutoAnnotatorSeqFeat2a { width:27% } td.AutoAnnotatorSeqFeat2b { width:97% } td.AutoAnnotatorSeqFeat3 { width:70% } table.AutoAnnotatorNoBorder { border:0px; width:100%; border-collapse:collapse; } table.AutoAnnotatorWithBorder { border:1px solid black; width:100%; border-collapse:collapse; } td.AutoAnnotatorOuterAmino { border:0px solid black; width:20% } td.AutoAnnotatorInnerAmino { border:1px solid black; width:50% } td.AutoAnnotatorAminoCountingOuter { border:1px solid black; width:40%;  } td.AutoAnnotatorBiochemParOuter { border:1px solid black; width:60%; } td.AutoAnnotatorAminoCountingInner1 { width: 7.5% } td.AutoAnnotatorAminoCountingInner2 { width:62.5% } td.AutoAnnotatorAminoCountingInner3 { width:30% } td.AutoAnnotatorBiochemParInner1 { width: 5% } td.AutoAnnotatorBiochemParInner2 { width:55% } td.AutoAnnotatorBiochemParInner3 { width:40% } td.AutoAnnotatorCodonUsage1 { width: 3% } td.AutoAnnotatorCodonUsage2 { width:14.2% } td.AutoAnnotatorCodonUsage3 { width:13.8% } td.AutoAnnotatorAlignment1 { width: 3% } td.AutoAnnotatorAlignment2 { width: 10% } td.AutoAnnotatorAlignment3 { width: 87% } td.AutoAnnotatorLocalizationOuter {border:1px solid black; width:40%} td.AutoAnnotatorGOOuter {border:1px solid black; width:60%} td.AutoAnnotatorLocalization1 { width: 7.5% } td.AutoAnnotatorLocalization2 { width: 22.5% } td.AutoAnnotatorLocalization3 { width: 70% } td.AutoAnnotatorGO1 { width: 5% } td.AutoAnnotatorGO2 { width: 35% } td.AutoAnnotatorGO3 { width: 60% } td.AutoAnnotatorPredFeat1 { width:3% } td.AutoAnnotatorPredFeat2a { width:27% } td.AutoAnnotatorPredFeat3 { width:70% } div.AutoAnnotator_trans { position:absolute; background:rgb(11,140,143); background-color:rgba(11,140,143, 0.8); height:5px; top:100px; } div.AutoAnnotator_sec_helix { position:absolute; background:rgb(102,0,102); background-color:rgba(102,0,102, 0.8); height:5px; top:110px; } div.AutoAnnotator_sec_strand { position:absolute; background:rgb(245,170,26); background-color:rgba(245,170,26, 1); height:5px; top:110px; } div.AutoAnnotator_acc_buried { position:absolute; background:rgb(89,168,15); background-color:rgba(89,168,15, 0.8); height:5px; top:120px; } div.AutoAnnotator_acc_exposed { position:absolute; background:rgb(0, 0, 255); background-color:rgba(0, 0, 255, 0.8); height:5px; top:120px; } div.AutoAnnotator_dis { position:absolute; text-align:center; font-family:Arial,Helvetica,sans-serif; background:rgb(255, 200, 0); background-color:rgba(255, 200, 0, 1); height:16px; width:16px; top:80px; border-radius:50%; } </style><div id='AutoAnnotator_container_1397080705152'><table id="AutoAnnotator"><tr><!-- Time stamp in ms since 1/1/1970 1397080705152 --><th id="AutoAnnotatorHeader" colspan="2">Protein data table for BioBrick <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_E1010">BBa_E1010</a> automatically created by the <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">BioBrick-AutoAnnotator</a> version 1.0</th></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Nucleotide sequence</strong> in <strong>RFC 10</strong>: (underlined part encodes the protein)<br><span class="AutoAnnotatorSequence">&nbsp;<u>ATGGCTTCC&nbsp;...&nbsp;ACCGGTGCT</u>TAATAACGCTGATAGTGCTAGTGTAGATCGC</span><br>&nbsp;<strong>ORF</strong> from nucleotide position 1 to 675 (excluding stop-codon)</td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid sequence:</strong> (RFC 25 scars in shown in bold, other sequence features underlined; both given below)<br><span class="AutoAnnotatorSequence"><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqNum">1&nbsp;<br>101&nbsp;<br>201&nbsp;</td><td class="AutoAnnotatorSeqSeq">MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE<br>DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLD<br>ITSHNEDYTIVEQYERAEGRHSTGA*</td></tr></table></span></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Sequence features:</strong> (with their position in the amino acid sequence, see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software/FeatureList">list of supported features</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorSeqFeat1"></td><td class="AutoAnnotatorSeqFeat2b">None of the supported features appeared in the sequence</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Amino acid composition:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Ala (A)</td><td class="AutoAnnotatorInnerAmino">12 (5.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Arg (R)</td><td class="AutoAnnotatorInnerAmino">9 (4.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asn (N)</td><td class="AutoAnnotatorInnerAmino">4 (1.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Asp (D)</td><td class="AutoAnnotatorInnerAmino">14 (6.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Cys (C)</td><td class="AutoAnnotatorInnerAmino">0 (0.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gln (Q)</td><td class="AutoAnnotatorInnerAmino">8 (3.6%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Glu (E)</td><td class="AutoAnnotatorInnerAmino">22 (9.8%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Gly (G)</td><td class="AutoAnnotatorInnerAmino">23 (10.2%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">His (H)</td><td class="AutoAnnotatorInnerAmino">5 (2.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ile (I)</td><td class="AutoAnnotatorInnerAmino">9 (4.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Leu (L)</td><td class="AutoAnnotatorInnerAmino">12 (5.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Lys (K)</td><td class="AutoAnnotatorInnerAmino">22 (9.8%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Met (M)</td><td class="AutoAnnotatorInnerAmino">9 (4.0%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Phe (F)</td><td class="AutoAnnotatorInnerAmino">10 (4.4%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Pro (P)</td><td class="AutoAnnotatorInnerAmino">12 (5.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Ser (S)</td><td class="AutoAnnotatorInnerAmino">12 (5.3%)</td></tr></table></td><td class="AutoAnnotatorOuterAmino"><table class="AutoAnnotatorWithBorder"><tr><td class="AutoAnnotatorInnerAmino">Thr (T)</td><td class="AutoAnnotatorInnerAmino">14 (6.2%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Trp (W)</td><td class="AutoAnnotatorInnerAmino">3 (1.3%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Tyr (Y)</td><td class="AutoAnnotatorInnerAmino">11 (4.9%)</td></tr><tr><td class="AutoAnnotatorInnerAmino">Val (V)</td><td class="AutoAnnotatorInnerAmino">14 (6.2%)</td></tr></table></td></tr></table></td></tr><tr><td class="AutoAnnotatorAminoCountingOuter"><strong>Amino acid counting</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Total number:</td><td class="AutoAnnotatorAminoCountingInner3">225</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Positively charged (Arg+Lys):</td><td class="AutoAnnotatorAminoCountingInner3">31 (13.8%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Negatively charged (Asp+Glu):</td><td class="AutoAnnotatorAminoCountingInner3">36 (16.0%)</td></tr><tr><td class="AutoAnnotatorAminoCountingInner1"></td><td class="AutoAnnotatorAminoCountingInner2">Aromatic (Phe+His+Try+Tyr):</td><td class="AutoAnnotatorAminoCountingInner3">29 (12.9%)</td></tr></table></td><td class="AutoAnnotatorBiochemParOuter"><strong>Biochemical parameters</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Atomic composition:</td><td class="AutoAnnotatorBiochemParInner3">C<sub>1135</sub>H<sub>1749</sub>N<sub>299</sub>O<sub>347</sub>S<sub>9</sub></td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Molecular mass [Da]:</td><td class="AutoAnnotatorBiochemParInner3">25423.7</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Theoretical pI:</td><td class="AutoAnnotatorBiochemParInner3">5.65</td></tr><tr><td class="AutoAnnotatorBiochemParInner1"></td><td class="AutoAnnotatorBiochemParInner2">Extinction coefficient at 280 nm [M<sup>-1</sup> cm<sup>-1</sup>]:</td><td class="AutoAnnotatorBiochemParInner3">32890 / 32890 (all Cys red/ox)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Plot for hydrophobicity, charge, predicted secondary structure, solvent accessability, transmembrane helices and disulfid bridges</strong>&nbsp;<input type='button' id='hydrophobicity_charge_button' onclick='show_or_hide_plot_1397080705152()' value='Show'><span id="hydrophobicity_charge_explanation"></span><div id="hydrophobicity_charge_container" style='display:none'><div id="hydrophobicity_charge_placeholder0" style="width:100%;height:150px"></div><div id="hydrophobicity_charge_placeholder1" style="width:100%;height:150px"></div></div></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Codon usage</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Organism:</td><td class="AutoAnnotatorCodonUsage3"><i>E. coli</i></td><td class="AutoAnnotatorCodonUsage3"><i>B. subtilis</i></td><td class="AutoAnnotatorCodonUsage3"><i>S. cerevisiae</i></td><td class="AutoAnnotatorCodonUsage3"><i>A. thaliana</i></td><td class="AutoAnnotatorCodonUsage3"><i>P. patens</i></td><td class="AutoAnnotatorCodonUsage3">Mammals</td></tr><tr><td class="AutoAnnotatorCodonUsage1"></td><td class="AutoAnnotatorCodonUsage2">Codon quality (<a href="http://en.wikipedia.org/wiki/Codon_Adaptation_Index">CAI</a>):</td><td class="AutoAnnotatorCodonUsage3">excellent (0.84)</td><td class="AutoAnnotatorCodonUsage3">good (0.72)</td><td class="AutoAnnotatorCodonUsage3">good (0.68)</td><td class="AutoAnnotatorCodonUsage3">good (0.74)</td><td class="AutoAnnotatorCodonUsage3">good (0.78)</td><td class="AutoAnnotatorCodonUsage3">good (0.71)</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Alignments</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorAlignment1"></td><td class="AutoAnnotatorAlignment2">SwissProt:</td><td class="AutoAnnotatorAlignment3"><a href='http://www.uniprot.org/uniprot/Q9U6Y8'>Q9U6Y8</a> (86% identity on 221 AAs), <a href='http://www.uniprot.org/uniprot/P83690'>P83690</a> (63% identity on 215 AAs)</td></tr><tr><td class="AutoAnnotatorAlignment1"></td><td class="AutoAnnotatorAlignment2">TrEML:</td><td class="AutoAnnotatorAlignment3"><a href='http://www.uniprot.org/uniprot/Q5S3G8'>Q5S3G8</a> (95% identity on 225 AAs), <a href='http://www.uniprot.org/uniprot/D0VWW2'>D0VWW2</a> (94% identity on 220 AAs)</td></tr><tr><td class="AutoAnnotatorAlignment1"></td><td class="AutoAnnotatorAlignment2">PDB:</td><td class="AutoAnnotatorAlignment3"><a href='http://www.rcsb.org/pdb/explore/explore.do?structureId=2h5q'>2h5q</a> (94% identity on 216 AAs), <a href='http://www.rcsb.org/pdb/explore/explore.do?structureId=2qlg'>2qlg</a> (94% identity on 215 AAs)</td></tr></table></td></tr><tr><th id='AutoAnnotatorHeader' colspan="2"><strong>Predictions</strong> (obtained from <a href='http://predictprotein.org'>PredictProtein.org</a>)</th></tr><tr><td class="AutoAnnotatorLocalizationOuter"><strong>Subcellular Localization</strong> (reliability in brackets)<table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorLocalization1"></td><td class="AutoAnnotatorLocalization2">Archaea:</td><td class="AutoAnnotatorLocalization3">secreted (100%)</td></tr><tr><td class="AutoAnnotatorLocalization1"></td><td class="AutoAnnotatorLocalization2">Bacteria:</td><td class="AutoAnnotatorLocalization3">cytosol (52%)</td></tr><tr><td class="AutoAnnotatorLocalization1"></td><td class="AutoAnnotatorLocalization2">Eukarya:</td><td class="AutoAnnotatorLocalization3">cytosol (20%)</td></tr></table></td><td class="AutoAnnotatorGOOuter"><strong>Gene Ontology</strong> (reliability in brackets)<br><table class="AutoAnnotatorNoBorder"><tr><td class='AutoAnnotatorGO1'></td><td class='AutoAnnotatorGO2'>Molecular Function Ontology:</td><td class='AutoAnnotatorGO3'> - </td></tr><tr><td class='AutoAnnotatorGO1'></td><td class='AutoAnnotatorGO2'>Biological Process Ontology:</td><td class='AutoAnnotatorGO3'><a href='http://amigo.geneontology.org/cgi-bin/amigo/term_details?term=GO:0018298'>GO:0018298</a> (40%), <a href='http://amigo.geneontology.org/cgi-bin/amigo/term_details?term=GO:0008218'>GO:0008218</a> (27%)</td></tr><tr><td class='AutoAnnotatorGO1'> </td><td class='AutoAnnotatorGO2'> </td><td class='AutoAnnotatorGO3'>&nbsp;</td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"><strong>Predicted features:</strong><table class="AutoAnnotatorNoBorder"><tr><td class="AutoAnnotatorPredFeat1"></td><td class="AutoAnnotatorPredFeat2a">Disulfid bridges:</td><td class="AutoAnnotatorPredFeat3">&nbsp;- </td></tr><tr><td class="AutoAnnotatorPredFeat1"></td><td class="AutoAnnotatorPredFeat2a">Transmembrane helices:</td><td class="AutoAnnotatorPredFeat3">&nbsp;- </td></tr></table></td></tr><tr><td class="AutoAnnotator1col" colspan="2"> The BioBrick-AutoAnnotator was created by <a href="http://2013.igem.org/Team:TU-Munich">TU-Munich 2013</a> iGEM team. For more information please see the <a href="http://2013.igem.org/Team:TU-Munich/Results/Software">documentation</a>.<br>If you have any questions, comments or suggestions, please leave us a <a href="http://2013.igem.org/Team:TU-Munich/Results/AutoAnnotator">comment</a>.</td></tr></table></div><br><!-- IMPORTANT: DON'T REMOVE THIS LINE, OTHERWISE NOT SUPPORTED FOR IE BEFORE 9 --><!--[if lte IE 8]><script language="javascript" type="text/javascript" src="http://2013.igem.org/Team:TU-Munich/excanvas.js"></script><![endif]--><script type='text/javascript' src='http://code.jquery.com/jquery-1.10.0.min.js'></script><script type='text/javascript' src='http://2013.igem.org/Team:TU-Munich/Flot.js?action=raw&ctype=text/js'></script><script>var jqAutoAnnotator = jQuery.noConflict(true);function show_or_hide_plot_1397080705152(){hydrophobicity_datapoints = [[2.5,-0.28],[3.5,-1.36],[4.5,-0.88],[5.5,0.18],[6.5,-0.44],[7.5,-0.44],[8.5,0.82],[9.5,0.36],[10.5,-1.44],[11.5,-0.10],[12.5,-0.18],[13.5,0.10],[14.5,-1.18],[15.5,0.10],[16.5,-1.16],[17.5,-0.46],[18.5,-1.46],[19.5,0.28],[20.5,-0.80],[21.5,-0.18],[22.5,-0.74],[23.5,-1.28],[24.5,-1.56],[25.5,-1.56],[26.5,-0.58],[27.5,-0.64],[28.5,-0.02],[29.5,-1.28],[30.5,-0.66],[31.5,-2.26],[32.5,-1.64],[33.5,-2.46],[34.5,-2.08],[35.5,-2.26],[36.5,-2.26],[37.5,-2.26],[38.5,-1.50],[39.5,-1.88],[40.5,-1.76],[41.5,-0.70],[42.5,-1.40],[43.5,-0.50],[44.5,-0.58],[45.5,0.40],[46.5,-0.10],[47.5,-0.10],[48.5,-0.94],[49.5,-0.24],[50.5,-1.40],[51.5,-0.50],[52.5,-0.04],[53.5,0.60],[54.5,1.04],[55.5,1.18],[56.5,-0.28],[57.5,0.94],[58.5,1.14],[59.5,0.62],[60.5,0.48],[61.5,0.48],[62.5,0.14],[63.5,-1.32],[64.5,-1.42],[65.5,-1.18],[66.5,-0.64],[67.5,-1.98],[68.5,-0.92],[69.5,-0.92],[70.5,-0.00],[71.5,-0.62],[72.5,-0.48],[73.5,-1.16],[74.5,-0.54],[75.5,-2.08],[76.5,-0.40],[77.5,-0.08],[78.5,-0.46],[79.5,-1.08],[80.5,0.38],[81.5,-1.30],[82.5,-0.22],[83.5,0.32],[84.5,1.14],[85.5,0.06],[86.5,0.14],[87.5,-0.70],[88.5,0.02],[89.5,-1.32],[90.5,-1.18],[91.5,-1.18],[92.5,-2.00],[93.5,-1.72],[94.5,-0.56],[95.5,-1.08],[96.5,0.18],[97.5,0.38],[98.5,-1.16],[99.5,-1.62],[100.5,-1.00],[101.5,-0.72],[102.5,0.82],[103.5,1.38],[104.5,2.30],[105.5,2.24],[106.5,0.70],[107.5,-0.84],[108.5,-0.86],[109.5,-1.86],[110.5,-0.96],[111.5,-0.96],[112.5,-0.96],[113.5,-0.88],[114.5,-1.42],[115.5,-1.62],[116.5,-0.02],[117.5,0.42],[118.5,-0.28],[119.5,1.26],[120.5,-0.08],[121.5,-0.22],[122.5,-0.86],[123.5,-0.16],[124.5,-1.14],[125.5,-1.06],[126.5,-1.26],[127.5,-0.68],[128.5,-0.76],[129.5,-1.32],[130.5,-0.70],[131.5,-1.58],[132.5,-0.42],[133.5,0.12],[134.5,0.12],[135.5,-0.58],[136.5,-1.04],[137.5,-2.02],[138.5,-2.02],[139.5,-1.40],[140.5,-0.80],[141.5,-0.72],[142.5,-0.22],[143.5,-0.76],[144.5,-0.82],[145.5,-1.34],[146.5,-1.54],[147.5,-1.52],[148.5,-1.62],[149.5,-1.80],[150.5,-1.80],[151.5,-1.60],[152.5,-2.06],[153.5,-1.44],[154.5,-0.36],[155.5,-0.44],[156.5,0.18],[157.5,-0.44],[158.5,0.10],[159.5,-1.44],[160.5,-0.28],[161.5,-1.10],[162.5,0.36],[163.5,-1.32],[164.5,0.22],[165.5,-0.94],[166.5,-0.74],[167.5,-1.58],[168.5,-0.88],[169.5,-2.28],[170.5,-1.76],[171.5,-1.76],[172.5,-1.32],[173.5,-1.94],[174.5,-0.46],[175.5,-0.98],[176.5,-0.42],[177.5,-0.92],[178.5,-0.48],[179.5,-0.94],[180.5,0.20],[181.5,-0.44],[182.5,-1.08],[183.5,-1.14],[184.5,-0.68],[185.5,-1.74],[186.5,-0.20],[187.5,0.26],[188.5,0.50],[189.5,0.02],[190.5,0.46],[191.5,-1.08],[192.5,-0.90],[193.5,-1.52],[194.5,-0.98],[195.5,-1.50],[196.5,0.04],[197.5,-0.52],[198.5,1.08],[199.5,0.04],[200.5,0.66],[201.5,-0.74],[202.5,-0.74],[203.5,-2.34],[204.5,-2.90],[205.5,-3.00],[206.5,-2.50],[207.5,-0.90],[208.5,0.64],[209.5,0.64],[210.5,0.20],[211.5,0.08],[212.5,-1.52],[213.5,-3.26],[214.5,-2.20],[215.5,-2.20],[216.5,-2.02],[217.5,-2.22],[218.5,-1.96],[219.5,-2.48],[220.5,-1.92],[221.5,-1.92],[222.5,-0.66]];charge_datapoints = [[2.5,-0.20],[3.5,-0.40],[4.5,-0.40],[5.5,-0.40],[6.5,-0.20],[7.5,-0.20],[8.5,0.00],[9.5,0.00],[10.5,0.20],[11.5,0.00],[12.5,0.40],[13.5,0.40],[14.5,0.60],[15.5,0.40],[16.5,0.20],[17.5,0.00],[18.5,0.00],[19.5,-0.20],[20.5,-0.20],[21.5,0.00],[22.5,0.10],[23.5,-0.10],[24.5,-0.10],[25.5,-0.30],[26.5,-0.30],[27.5,-0.60],[28.5,-0.40],[29.5,-0.60],[30.5,-0.40],[31.5,-0.60],[32.5,-0.40],[33.5,-0.20],[34.5,0.00],[35.5,0.00],[36.5,0.00],[37.5,0.00],[38.5,-0.20],[39.5,-0.20],[40.5,-0.20],[41.5,0.00],[42.5,0.20],[43.5,0.20],[44.5,0.40],[45.5,0.40],[46.5,0.40],[47.5,0.40],[48.5,0.40],[49.5,0.20],[50.5,0.20],[51.5,0.20],[52.5,0.00],[53.5,0.00],[54.5,0.00],[55.5,0.00],[56.5,-0.20],[57.5,-0.20],[58.5,-0.20],[59.5,-0.20],[60.5,-0.20],[61.5,0.00],[62.5,0.00],[63.5,0.00],[64.5,0.00],[65.5,0.00],[66.5,0.00],[67.5,0.20],[68.5,0.20],[69.5,0.20],[70.5,0.20],[71.5,0.40],[72.5,0.30],[73.5,0.30],[74.5,0.30],[75.5,0.10],[76.5,-0.10],[77.5,-0.20],[78.5,-0.40],[79.5,-0.40],[80.5,-0.20],[81.5,0.00],[82.5,0.00],[83.5,0.20],[84.5,0.20],[85.5,0.20],[86.5,-0.20],[87.5,-0.20],[88.5,-0.20],[89.5,0.00],[90.5,0.00],[91.5,0.00],[92.5,0.20],[93.5,0.20],[94.5,0.00],[95.5,0.00],[96.5,0.20],[97.5,-0.20],[98.5,-0.40],[99.5,-0.40],[100.5,-0.40],[101.5,-0.40],[102.5,-0.20],[103.5,0.00],[104.5,0.00],[105.5,0.00],[106.5,0.00],[107.5,-0.20],[108.5,-0.20],[109.5,-0.20],[110.5,-0.20],[111.5,-0.20],[112.5,-0.20],[113.5,-0.20],[114.5,-0.40],[115.5,-0.40],[116.5,-0.40],[117.5,-0.20],[118.5,0.00],[119.5,0.20],[120.5,0.40],[121.5,0.40],[122.5,0.60],[123.5,0.40],[124.5,0.40],[125.5,0.20],[126.5,0.20],[127.5,0.00],[128.5,0.00],[129.5,-0.20],[130.5,-0.20],[131.5,-0.20],[132.5,-0.20],[133.5,-0.20],[134.5,0.00],[135.5,0.20],[136.5,0.40],[137.5,0.40],[138.5,0.40],[139.5,0.40],[140.5,0.20],[141.5,-0.20],[142.5,-0.20],[143.5,-0.20],[144.5,-0.20],[145.5,-0.40],[146.5,0.00],[147.5,0.00],[148.5,0.00],[149.5,0.00],[150.5,0.00],[151.5,-0.40],[152.5,-0.40],[153.5,-0.40],[154.5,-0.40],[155.5,0.00],[156.5,0.20],[157.5,0.00],[158.5,0.00],[159.5,0.20],[160.5,0.00],[161.5,0.20],[162.5,0.40],[163.5,0.60],[164.5,0.40],[165.5,0.60],[166.5,0.20],[167.5,0.20],[168.5,0.00],[169.5,0.10],[170.5,-0.10],[171.5,-0.10],[172.5,-0.10],[173.5,-0.30],[174.5,-0.40],[175.5,-0.20],[176.5,0.00],[177.5,0.00],[178.5,0.20],[179.5,0.20],[180.5,0.00],[181.5,0.20],[182.5,0.40],[183.5,0.40],[184.5,0.40],[185.5,0.40],[186.5,0.20],[187.5,0.00],[188.5,0.00],[189.5,0.00],[190.5,0.00],[191.5,0.20],[192.5,0.20],[193.5,0.00],[194.5,0.00],[195.5,0.20],[196.5,0.00],[197.5,-0.20],[198.5,0.00],[199.5,0.00],[200.5,-0.20],[201.5,-0.10],[202.5,0.10],[203.5,-0.10],[204.5,-0.30],[205.5,-0.30],[206.5,-0.40],[207.5,-0.40],[208.5,-0.20],[209.5,-0.20],[210.5,-0.20],[211.5,-0.20],[212.5,-0.40],[213.5,-0.20],[214.5,0.00],[215.5,-0.20],[216.5,-0.20],[217.5,0.20],[218.5,0.10],[219.5,0.10],[220.5,0.30],[221.5,0.30],[222.5,0.10]];dis_datapoints = [];trans_datapoints = [];sec_helix_datapoints = [[8,10],[57,66],[79,85]];sec_strand_datapoints = [[13,22],[25,31],[41,49],[93,100],[104,113],[117,126],[148,152],[156,168],[172,183],[195,200],[201,203],[210,221]];acc_exposed_datapoints = [[1,6],[9,10],[13,13],[15,15],[19,19],[23,23],[26,26],[32,32],[34,35],[37,37],[39,39],[41,41],[50,51],[53,53],[70,70],[74,74],[77,78],[81,81],[84,84],[88,90],[92,92],[110,110],[114,117],[128,128],[131,132],[134,134],[137,140],[142,142],[144,145],[148,148],[154,155],[158,158],[168,169],[171,171],[178,178],[182,182],[184,186],[188,188],[191,191],[200,200],[203,203],[205,207],[209,209],[212,212],[215,216],[218,218],[223,225]];acc_buried_datapoints = [[7,8],[12,12],[14,14],[16,16],[18,18],[20,20],[22,22],[24,24],[27,27],[29,29],[31,31],[33,33],[40,40],[44,44],[46,46],[48,48],[54,58],[60,65],[67,68],[71,72],[79,79],[82,83],[87,87],[91,91],[93,93],[95,97],[99,99],[103,107],[111,111],[113,113],[118,118],[120,120],[122,122],[124,124],[126,126],[129,129],[133,133],[135,136],[146,146],[150,151],[156,157],[159,159],[161,161],[163,163],[165,165],[167,167],[173,173],[175,175],[177,177],[179,179],[183,183],[187,187],[189,190],[194,195],[197,197],[199,199],[211,211],[217,217],[219,219]];flot_plot_options = []; flot_plot_options[0] = {grid: {borderWidth: {top: 0,right: 0,bottom: 0,left: 0}},legend: {show: false},xaxes: [{show: true,min: 0,max: 200,ticks: [[0.5, '1'], [24.5, '25'], [49.5, '50'], [74.5, '75'], [99.5, '100'], [124.5, '125'], [149.5, '150'], [174.5, '175'], [199.5, '200']],tickLength: -5}],yaxes: [{show: true,ticks: [[0, '0'], [4.5,'hydro-<br>phobic&nbsp;&nbsp;'], [-4.5,'hydro-<br>philic&nbsp;&nbsp;']],min: -4.5,max: +4.5,font: 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[plot_num*200 + 149.5, (plot_num*200 + 150).toString()], [plot_num*200 + 174.5, (plot_num*200 + 175).toString()], [plot_num*200 + 199.5, (plot_num*200 + 200).toString()] ],tickLength: -5}];};try {if( jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_button').val() =='Show' ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_container').css('display','block');jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_button').val('Hide');var description_html = '<div id=\'AutoAnnotator_plot_selectors\'>';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'hydrophobicity_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for hydrophobicity (<img src=\'https://static.igem.org/mediawiki/2013/e/e9/TUM13_hydrophobicity_icon.png\' alt=\'blue graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'charge_checkbox\' checked=\'checked\'>&nbsp;Moving average over 5 amino acids for charge (<img src=\'https://static.igem.org/mediawiki/2013/3/3e/TUM13_charge_icon.png\' alt=\'red graph\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'dis_checkbox\' checked=\'checked\'>&nbsp;Predicted disulfid bridges (<img src=\'https://static.igem.org/mediawiki/2013/2/28/TUM13_dis_icon.png\' alt=\'yellow circle\' height=\'10\'></img>) with the number of the bridge in the center';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'trans_checkbox\' checked=\'checked\'>&nbsp;Predicted transmembrane helices (<img src=\'https://static.igem.org/mediawiki/2013/7/78/TUM13_trans_icon.png\' alt=\'turquois bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'sec_checkbox\' checked=\'checked\'>&nbsp;Predicted secondary structure: Helices (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_helix_icon.png\' alt=\'violet bars\' height=\'10\'></img>) and beta-strands (<img src=\'https://static.igem.org/mediawiki/2013/b/bf/TUM13_strand_icon.png\' alt=\'yellow bars\' height=\'10\'></img>)';description_html = description_html + '<br>&nbsp;<input type=\'checkbox\' id=\'acc_checkbox\' checked=\'checked\'>&nbsp;Predicted solvent accessability: Exposed (<img src=\'https://static.igem.org/mediawiki/2013/1/16/TUM13_exposed_icon.png\' alt=\'blue bars\' height=\'10\'></img>) and buried (<img src=\'https://static.igem.org/mediawiki/2013/0/0b/TUM13_buried_icon.png\' alt=\'green bars\' height=\'10\'></img>) residues';description_html = description_html + '<br></div>';jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_explanation').html(description_html);plot_according_to_selectors_1397080705152();jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #AutoAnnotator_plot_selectors').find('input').click(plot_according_to_selectors_1397080705152);}else{jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_container').css('display','none');jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_button').val('Show');jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_explanation').html('');}}catch(err){txt='There was an error with the button controlling the visibility of the plot.\n';txt=txt+'The originating error is:\n' + err + '\n\n';alert(txt);}};function plot_according_to_selectors_1397080705152(){try{var plot_datasets = [[],[]];if(jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_checkbox').prop('checked') == true){plot_datasets[0] = { color: 'rgba(100,149,237,1)',data: hydrophobicity_datapoints,label: 'Hydrophobicity',lines: { show: true, fill: true, fillColor: 'rgba(100,149,237,0.1)' },yaxis: 1};}if(jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #charge_checkbox').prop('checked') == true){plot_datasets[1] = {color: 'rgba(255,99,71,1)',data: charge_datapoints,label: 'Charge',lines: { show: true, fill: true, fillColor: 'rgba(255,99,71,0.1)' },yaxis: 2};}for (plot_num = 0 ; plot_num < number_of_plots ; plot_num ++){jqAutoAnnotator.plot('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder'+ plot_num.toString(), plot_datasets, flot_plot_options[plot_num] );}var screen_width = jqAutoAnnotator('canvas.flot-base').width(); var pos_of_first_tick = 46;var pos_of_last_tick = screen_width - 51;var tick_diff = (screen_width - 97)/199;if(jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #dis_checkbox').prop('checked') == true){for ( j = 0 ; j < dis_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_dis\' style=\'left:' + ((pos_of_first_tick - 8 + (dis_datapoints[j][0] - 1)*tick_diff - Math.floor((dis_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px;\'><b>' + (j+1) + '</b></div>');jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((dis_datapoints[j][1] - 1)/200) ).append('<div class=\'AutoAnnotator_dis\' style=\'left:' + ((pos_of_first_tick - 8 + (dis_datapoints[j][1] - 1)*tick_diff - Math.floor((dis_datapoints[j][1] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px;\'><b>' + (j+1) + '</b></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #trans_checkbox').prop('checked') == true){for ( j = 0 ; j < trans_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((trans_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_trans\' style=\'width:' + (((trans_datapoints[j][1] - trans_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px ;left:' + ((pos_of_first_tick + (trans_datapoints[j][0] - 1.5)*tick_diff - Math.floor((trans_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #sec_checkbox').prop('checked') == true){for ( j = 0 ; j < sec_helix_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((sec_helix_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_sec_helix\' style=\'width:' + (((sec_helix_datapoints[j][1] - sec_helix_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (sec_helix_datapoints[j][0] - 1.5)*tick_diff - Math.floor((sec_helix_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}for ( j = 0 ; j < sec_strand_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((sec_strand_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_sec_strand\' style=\'width:' + (((sec_strand_datapoints[j][1] - sec_strand_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (sec_strand_datapoints[j][0] - 1.5)*tick_diff - Math.floor((sec_strand_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}if(jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #acc_checkbox').prop('checked') == true){for ( j = 0 ; j < acc_buried_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((acc_buried_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_acc_buried\' style=\'width:' + (((acc_buried_datapoints[j][1] - acc_buried_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (acc_buried_datapoints[j][0] - 1.5)*tick_diff - Math.floor((acc_buried_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}for ( j = 0 ; j < acc_exposed_datapoints.length ; j++ ){jqAutoAnnotator('#AutoAnnotator_container_1397080705152 #hydrophobicity_charge_placeholder' + Math.floor((acc_exposed_datapoints[j][0] - 1)/200) ).append('<div class=\'AutoAnnotator_acc_exposed\' style=\'width:' + (((acc_exposed_datapoints[j][1] - acc_exposed_datapoints[j][0] + 1)*tick_diff).toFixed(0)).toString() + 'px; left:' + ((pos_of_first_tick + (acc_exposed_datapoints[j][0] - 1.5)*tick_diff - Math.floor((acc_exposed_datapoints[j][0] - 1)/200)*200*tick_diff).toFixed(0)).toString() + 'px\'></div>');}}}catch(err){txt='There was an error while drawing the selected elements for the plot.\n';txt=txt+'The originating error is:\n' + err + '\n\n';throw(txt);}}</script></html>
+
−
 
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−
 
+
−
 
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==Functional Parameters: Austin_UTexas==
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<html>
+
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<body>
+
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<partinfo>BBa_E1010 parameters</partinfo>
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<h3><center>Burden Imposed by this Part:</center></h3>
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<figure>
+
−
<div class = "center">
+
−
<center><img src = "https://static.igem.org/mediawiki/parts/f/fa/T--Austin_Utexas--no_burden_icon.png" style = "width:160px;height:120px"></center>
+
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</div>
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<figcaption><center><b>Burden Value: -1.1 ± 2.5% </b></center></figcaption>
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</figure>
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<p> Burden is the percent reduction in the growth rate of <i>E. coli</i> cells transformed with a plasmid containing this BioBrick (± values are 95% confidence limits). This BioBrick did not exhibit a burden that was significantly greater than zero (i.e., it appears to have little to no impact on growth). Therefore, users can depend on this part to remain stable for many bacterial cell divisions and in large culture volumes. Refer to any one of the
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<a href="https://parts.igem.org/Part:BBa_K3174002">BBa_K3174002</a> - <a href="https://parts.igem.org/Part:BBa_K3174007">BBa_K3174007</a> pages for more information on the methods, an explanation of the sources of burden,  and other conclusions from a large-scale measurement project conducted by the <a href="http://2019.igem.org/Team:Austin_UTexas">2019 Austin_UTexas team</a>.</p>
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<p>This functional parameter was added by the <a href="https://2020.igem.org/Team:Austin_UTexas/Contribution">2020 Austin_UTexas team.</a></p>
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Revision as of 07:37, 3 October 2020

Summary In order to better and more comprehensive understand our favorite reporter part whose name is BBa_E1010, this year NAU-CHINA uses SWISS MODEL (https://swissmodel.expasy.org/) to model and simulate the tertiary structure of protein. We hope that the prediction of the structure will help other teams to better understand the nature and characteristics of this part and be able to use the reporter gene more skillfully.

The following model was built (see Materials and Methods "Model Building"):

Fig.1. Model #01 Fig.2. The Active center

Fig.3. Local quality Fig.4. Comparison with Non-redundant Set of PBD Structures

Target MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

Template 2qli.1.A VSKGEEVIKEFMRFKQHMEGSVNGHEFEIEGEGEGRPYEGTQTARLKVTKGGPLPFAWDILSPQIX—SKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKVRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEMKMRLRLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA

Materials and Methods Template Search Template search with BLAST and HHBlits has been performed against the SWISS-MODEL template library (SMTL, last update: 2020-09-23, last included PDB release: 2020-09-18).

The target sequence was searched with BLAST against the primary amino acid sequence contained in the SMTL. A total of 670 templates were found.

An initial HHblits profile has been built using the procedure outlined in (Steinegger et al.), followed by 1 iteration of HHblits against Uniclust30 (Mirdita, von den Driesch et al.). The obtained profile has then be searched against all profiles of the SMTL. A total of 720 templates were found.

Template Selection For each identified template, the template's quality has been predicted from features of the target-template alignment. The templates with the highest quality have then been selected for model building.

Model Building Models are built based on the target-template alignment using ProMod3. Coordinates which are conserved between the target and the template are copied from the template to the model. Insertions and deletions are remodelled using a fragment library. Side chains are then rebuilt. Finally, the geometry of the resulting model is regularized by using a force field. In case loop modelling with ProMod3 fails, an alternative model is built with PROMOD-II (Guex et al.).

Model Quality Estimation The global and per-residue model quality has been assessed using the QMEAN scoring function (Studer et al.).

Ligand Modelling Ligands present in the template structure are transferred by homology to the model when the following criteria are met: (a) The ligands are annotated as biologically relevant in the template library, (b) the ligand is in contact with the model, (c) the ligand is not clashing with the protein, (d) the residues in contact with the ligand are conserved between the target and the template. If any of these four criteria is not satisfied, a certain ligand will not be included in the model. The model summary includes information on why and which ligand has not been included.

Oligomeric State Conservation The quaternary structure annotation of the template is used to model the target sequence in its oligomeric form. The method (Bertoni et al.) is based on a supervised machine learning algorithm, Support Vector Machines (SVM), which combines interface conservation, structural clustering, and other template features to provide a quaternary structure quality estimate (QSQE). The QSQE score is a number between 0 and 1, reflecting the expected accuracy of the interchain contacts for a model built based a given alignment and template. Higher numbers indicate higher reliability. This complements the GMQE score which estimates the accuracy of the tertiary structure of the resulting model.

References BLASTWaterhouse, A., Bertoni, M., Bienert, S., Studer, G., Tauriello, G., Gumienny, R., Heer, F.T., de Beer, T.A.P., Rempfer, C., Bordoli, L., Lepore, R., Schwede, T. SWISS-MODEL: homology modelling of protein structures and complexes. Nucleic Acids Res. 46(W1), W296-W303 (2018). Guex, N., Peitsch, M.C., Schwede, T. Automated comparative protein structure modeling with SWISS-MODEL and Swiss-PdbViewer: A historical perspective. Electrophoresis 30, S162-S173 (2009). Bienert, S., Waterhouse, A., de Beer, T.A.P., Tauriello, G., Studer, G., Bordoli, L., Schwede, T. The SWISS-MODEL Repository - new features and functionality. Nucleic Acids Res. 45, D313-D319 (2017). Studer, G., Rempfer, C., Waterhouse, A.M., Gumienny, G., Haas, J., Schwede, T. QMEANDisCo - distance constraints applied on model quality estimation. Bioinformatics 36, 1765-1771 (2020). Bertoni, M., Kiefer, F., Biasini, M., Bordoli, L., Schwede, T. Modeling protein quaternary structure of homo- and hetero-oligomers beyond binary interactions by homology. Scientific Reports 7 (2017). Camacho, C., Coulouris, G., Avagyan, V., Ma, N., Papadopoulos, J., Bealer, K., Madden, T.L. BLAST+: architecture and applications. BMC Bioinformatics 10, 421-430 (2009).

HHblits Steinegger, M., Meier, M., Mirdita, M., Vöhringer, H., Haunsberger, S. J., Söding, J. HH-suite3 for fast remote homology detection and deep protein annotation. BMC Bioinformatics 20, 473 (2019).

Uniclust30 Mirdita, M., von den Driesch, L., Galiez, C., Martin, M.J., Söding, J., Steinegger, M. Uniclust databases of clustered and deeply annotated protein sequences and alignments. Nucleic Acids Research 45, D170–D176 (2016).