Difference between revisions of "Part:BBa E0040"

Revision as of 09:35, 16 October 2019

green fluorescent protein derived from jellyfish Aequeora victoria wild-type GFP (SwissProt: P42212

GFP (mut3b) [note that this part does not have a barcode]

Usage and Biology: Characterization

Group: Valencia_UPV iGEM 2018
Author: Adrián Requena Gutiérrez, Carolina Ropero
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.
Documentation:
The characterization of this protein (and by extension of all the other part that codify for the GFP) was performed with our transcriptional unit BBa_K2656105. This transcriptional unit was assembled in a Golden Braid alpha1 plasmid including the following parts:

• BBa_K2656004: the J23106 promoter in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
• BBa_K2656009: the B0030 ribosome biding site in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]
• BBa_K2656022: This part.
• BBa_K2656026: the B0015 transcriptional terminator in its Golden Braid compatible version from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection Part Collection]

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 have obtained the emission and excitation spectra in the conditions of our equipment. By using this [http://2018.igem.org/Team:Valencia_UPV/Experiments#spectra protocol] with the parameters of Table 1, Figure 1 was obtained.

Figure 1. GFP emission and excitation spectra

Usage and Biology

Untagged version of gfp from Repressilator reporter. See the design page for more source information.

The original citation for GFPmut3b is as follows:

Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996).

Here's the link: http://www.sciencedirect.com/science/article/pii/0378111995006850

Fluorescence wavelengths

Cormack et al.Cormack report the following excitation and emission data for GFPmut3 -

• Excitation max - 501nm
• Emission max - 511nm

Latency

Cormack et al.Cormack report detectable fluorescence within 8 mins. Please add maturation time data for E0040 here.

References

<biblio>

1. Cormack pmid=10659856

</biblio>

Allergen characterization of BBa_E0040

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

For the biobrick part, BBa_E0040, there was a 28.7% of identity match and 47.1% of similarity match compared to the top allergen in the 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.

>Internal Priming Screening Characterization of BBa_E0040: Has no possible internal priming sites between this BioBrick part and the VF2 or the VR primer.

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.

Part Characteristics in Cell-Free Chassis

Purificaton

GFPmut3b can be purified for calibration after the addition of a his-tag. The detailed [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.6 protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.6 results]for the purification can be found.

Calibration

The fluorescence of purified GFPmut3B was calibrated in the cell-free chassis. The derived [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.3 calilbration curve]allows the determination of the concentration of GFPmut3b in the cell-free chassis. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.3 Detailed protocols]for generating the calibration curve are available. Other calibration curves for are also available on the results page.

Calibration curve of concentration of GFP against fluorescence. The conversion factor is 79.429.

Degradation

The degradation of GFPmut3B in the cell-free chassis was also characterized. Purified GFPmut3B was allowed to degrade in the cell-free chassis and the fluorescence was measured over time. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.4 Detailed protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.4 results]are attached.

Degradation curve of different GFP concentrations.

Semi-log plot of GFP degradation at different GFP concentrations

From the semi-log plot, the degradation constant (in minutes) was derived to be 0.0003501, which is equivalent to GFPmut3b having a half-life of 33 hours in the cell-free chassis.

Expression of GFP-mut3b in cell lysate

Cell-free GFP-mut3b synthesis was analyzed in self-made E.Coli lysate from strain BL21(DE3). Fluorescence was measured at 37°C for five hours on a plate reader. For details on how the lysate and the energy solution were made and which components went into the final reaction volume of 10uL, check out our [http://2017.igem.org/Team:EPFL/Protocols protocols]. Shown are three repeats with a negative control as well as a shaded error graph (control was subtracted) summarizing the result. GFP-mut3b expression yields high signals in lysate and thus is a good choice of reporter while working on a cell-free chassis. Saturation occurs after about five hours.

Three repeats and control of GFP-mut3b expression in cell lysate.

Shaded error graph of GFP-mut3b expression in cell lysate (control was subtracted).

IIT Delhi 2017 - Characterization of Photobleaching

Photobleaching is the phenomenon of irreversible damage to the fluorophore, such that after certain number of electronic transitions on absorption of photons, it cannot fluoresce anymore. This hinders the ability to continuously image a sample over a long period of time, thus acting as a bottleneck to the characterization pipeline. Therefore, it is of paramount importance to understand and characterize the bleaching effect so that an optimum time gap between successive images could be chosen. This would ensure that the fluorophores do not bleach and at the same time we don’t have to compromise on the amount of collected data due to the time gap.

Here, we characterize the photobleaching effect in wildtype GFP (E0040) using fluorescent microscopy with the etaluma Lumascope 500 microscope. Cells expressing GFP under the PhlF repressible promoter (BBa_K2525016) in the absence of PhlF, so that it constitutively expressed GFP. Cells were loaded in microfluidic chambers and droplet encapsulation was performed to capture a small number of cells. This droplet was continuously exposed to light corresponding to the excitation wavelength of GFP (~485 nm) and the emission was captured continuously as well. The real time video for photobleaching in the cells encapsulated in the droplet is shown in GIF 1. ImageJ was used to analyze the images to obtain the rate of photobleaching as shown in Fig 1. Where we have fitted an exponential curve to the total intensity over time. It is known that photobleaching has a first order decay. We obtain a photobleaching rate of 0.002 per second (7.2 per hour).

GIF. 1 Real time video showing photobleaching in cells encapsulated in a droplet. To access the video in the GIF you'll have to visit the GIF at its link on the wiki; for that, click twice over the image in succession.

Fig. 1 Total Intensity in the encapsulated droplet over time. Since photobleaching is known to be a first order process, we have fitted an exponential curve to the data. The high R-squared value implies a good fit of the exponential model to the data. The rate of photobleaching turns out to be 0.002 per second (7.2 per hour).

Kyoto 2018 - Characterization

We tested nonspecifically interaction of GFP(E0040) by immunoprecipitation for characterization. The figure shows the result of immunoprecipitation by GFPnanobody.(link) Without GFP’s band, we can see same bands pattern in lane 2 and 6. This result shows GFP has no nonspecifically interaction with proteins of S.cerevisiae under physiological salt concentration. So, there is no problem when we identify the location of localized proteins that fused with this GFP. Otherwise, in lane 8, there is a unique band.(④) This band might shows GFP’s nonspecifically interaction under high salt concentrations. So, when we use this part, we have to experiment under physiological salt concentration.

Secondly,TDH3 promoter and CYC1 terminator were added to both ends of ORF and cloned into pRS316 which is a shuttle vector of S. cerevisiae and E. coli. The resulting plasmid was transformed into wild-type yeast strain BY 4741 to overexpress GFP in yeast. Photographs of pellets recovered from the culture medium of yeast cells is below.As can be easily seen, the yeast pellet overexpressing GFP was colored in a pale yellow color.From this, it was confirmed that GFP of BBa_E0040 can be expressed in large amounts in yeast cells without changing the codon and that the expression level thereof is so large as to be visually observed under visible light without breaking the yeast.

Improvements

• Chlamydomonas reinhardtii chloroplast optimised: BBa_K2148009
• Yeast- and FACS optimized GFP: BBa_K194001
• Yeast- and FACS optimized, fast degradable GFP: BBa_K194002
• RFC[1000] compatible GFP: BBa_K2294444

Improved by BNU-China 2019

Nanjing 2019 - Characterization

We adapted the part to be able to assemble our gene of interest (BBa_K3253003) with the cloning vector (BBa_K3253005) to test whether our gene of interest has already been successfully transferred to the Agrobacterium and our target plant. This florescent gene was expected to have its greatest excitement wavelength at 510nm, which is an indicator of the result of our transfer. Due the delay of the gene posting from the IGEM official for a month. The official promised that the gene would arrive at 10th August, but the actual Ghchi6 sequence arrived at China at 7th September. This is the main reason that we haven't finished our experiment on time. Please take this into consideration. We have shown our progress in our demonstration.

NYMU-Taipei 2019 - Characterization

We were searching for fluorescent protein-coding genes to be used in our project. This BBa_E0040 iGEM part was listed in the well 13L of Plate 4 of the 2018 Spring iGEM Distribution Kit. The plasmid backbone listed was pSB1A2. We had tried several times to do transformation with the provided plasmid DNA on E. coli DH5_alpha cells and grow the transformed cells on Ampicillin plates. However, no colonies had showed up on the Ampicillin plates. Because most iGEM teams submitted their parts on pSB1C3 backbone plasmid, we then tried to plate the transformed cells on Chloramphenicol plates to see if we could find any growth, To our surprise, we did see colonies grow on the top of our Chloramphenicol plates. We were able to purify plasmid DNA out of liquid cultures from these transformed colonies. We therefore would like to share our characterization of this BBa_E0040 part with other iGEM teams trying to use this part in their project.