Part:BBa_E0020

engineered cyan fluorescent protein derived from A. victoria GFP

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<* Allergen characterization of BBa_E0020: Not a potential allergen

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_E0020, there was a 0% of identity match and 0% similarity match to the top allergen in the allergen database. This means that the biobrick part is not of potential allergen status. In 80 amino acid alignments by FASTA window, no matches found that are greater than 35% for this biobrick. This also means that there is not of potential allergen status.

>Internal Priming Screening Characterization of BBa_E0020: Has 2 possible internal priming site between this BioBrick part and the VF2 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.

For the BioBrick part BBa_E0020, the first location of the internal priming site is on the 111-118 base number of the BioBrick and on the 1-8 base number of the VF2 primer. The second location of the internal priming site is on the 449-455 base number of the BioBrick and on the 10-16 base number of the VF2 primer.

Improvement by Fudan-CHINA 2018

In iGEM 2018, Fudan-CHINA improves this part by taking it apart using BiFC (bimolecular fluorescence complementation).We have compared the improves part with BBa_E0020 and proved our work. You can use our parts as labels for interaction of two target proteins and the location of two proteins in subcellular level, tremendously expanding the usage of the original part and equipping iGEMers another powerful tool for biological visualization.

Feel free to check out our new basic parts Part:BBa_K2886004 & Part:BBa_K2886005!

Usage and Biology

Sequence and Features

Assembly Compatibility:

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Characterisation by IISc-Bangalore 2019

Fig (2) : i²mCherry, untransformed BL21DE3 and ECFP protein lysates observed under UV

Spectral Characterisation

Artificially derived from GFP, the engineered cyan fluorescent protein (ECFP) is commonly used as a fluorescent marker in molecular biology studies due to its photostability, high lifespan and relatively low maturation times. To characterise the fluorophore we first performed a full wavelength scan to obtain the excitation and emission spectra of the protein.

Fig (1) : Excitation and Emission Spectra for ECFP

From the experimental data, the excitation λ is found out to be 429 nm while the emission λ is observed to be 478 nm.

Protein Purification using Ammonium Sulphate Precipitation

In order to purify the fluorescent protein from the crude cell lysate, we performed an ammonium sulphate protein precipitation assay. In this assay we estimate the percentage saturation of ammonium sulphate at which our protein of interest (ECFP) precipitates. In order to do this, we perform several rounds of protein precipitation via centrifugation while increasing the amount of ammonium sulphate at each round of precipitation. The precipitate obtained after each centrifugation (at different percentage saturation of ammonium sulphate) is dissolved in PBS and stored at 4^oC.
We run a SDS PAGE, to estimate the concentration at which the protein of interest precipitates. The concentration of ammonium sulphate at which we obtain the darkest band on the polyacrylamide gel is the percentage saturation at which ECFP precipitates.

Fig (2) : SDS PAGE for ECFP induced and uninduced samples

On running the induced and uninduced ECFP crude protein lysates, on a polyacrylamide gel, we obtain bands between 25 kDa and 32 kDa. ECFP has a molecular weight of around 27 kDa. The SDS PAGE exhibits a band at the required size indicating the presence of the gene of interest. The induced sample has a much more pronounced band in comparison to the uninduced sample, signifying minimal leaky transcription.

On running the ammonium sluphate precipitate fractions on a SDS PAGE, we observe the following :

Fig (3) : SDS PAGE for varying percentage saturation fractions of Ammonium Sulphate

From the SDS gel, we can infer that the ECFP protein precipitates at around 55% - 60% saturation of Ammonium Sulphate. The bands of appropriate sizes can be observed in the lanes containing 55% and 60% saturated ammonium sulphate protein precipitates. Thus, we can get an idea of isolating our protein of interest at a specific concentration of salt by differential precipitation.

Functional Parameters: Austin_UTexas

BBa_E0020 parameters

Burden Imposed by this Part:

Burden Value: 0.3 ± 2.8%

Burden is the percent reduction in the growth rate of E. coli 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 BBa_K3174002 - BBa_K3174007 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 2019 Austin_UTexas team.

This functional parameter was added by the 2020 Austin_UTexas team.

Estonia_TUIT 2021 team contribution

Enhanced cyan fluorescent protein (ECFP)

ECFP was obtained by mutation of a tyrosine into tryptophan (Y66W) in the part of the GFP molecule that determines the color (Golub et al., 2019). Even though ECFP possesses low quantum yield, indicating poor efficiency of photon absorption and emission, it is still a widely used fluorescent protein. The reasoning for the use of ECFP is its photostability (85 s) and a shorter maturation time than that of GFP (49 min(2)). ECFP exhibits an excitation peak at 434 nm wavelength and an emission peak at 477 nm. The emission and excitation spectra of ECFP are shown in Figure 1.

Figure 1. The emission and excitation spectra of ECFP (ECFP :: Fluorescent Protein Database). The violet line corresponds to the excitation spectrum, the blue line corresponds to the emission spectrum. The excitation maximum of 434 nm, the emission maximum of 477 nm, the extinction coefficient of 32500M^-1cm^-1 and the quantum yield of 0.4 were recorded.

The settings for the Nikon microscope A1 HD25/A1R HD25 for imaging of ECFP can be seen in Table 1 (Imaging Fluorescent Proteins | Nikon’s MicroscopyU).

Table 1. Fluorescent Nikon microscope A1 HD25/A1R HD25 settings for ECFP.

Excitation Laser (nm)	Excitation Filter CWL / BW (nm)	Dichromatic Mirror Cut-On (nm)	Barrier Filter CWL / BW (nm)	Relative Brightness (% of EGFP)
Diode (440)	435/40	460LP	495/50	39

References

• Golub, M., Guillon, V., Gotthard, G., Zeller, D., Martinez, N., Seydel, T., Koza, M. M., Lafaye, C., Clavel, D., Stetten, D. von, Royant, A., & Peters, J. (2019). Dynamics of a family of cyan fluorescent proteins probed by incoherent neutron scattering. Journal of the Royal Society Interface, 16(152). https://doi.org/10.1098/RSIF.2018.0848
• ECFP :: Fluorescent Protein Database. (n.d.). Retrieved October 15, 2021, from https://www.fpbase.org/protein/ecfp/
• Imaging Fluorescent Proteins | Nikon’s MicroscopyU. (n.d.). Retrieved October 15, 2021, from https://www.microscopyu.com/techniques/fluorescence/fluorescent-protein-imaging-parameters