Flavin-based fluorescent protein (FbFP B2)

Background

We created this part as an improved version of the part BBa_K3013002 which was originally created by the iGEM team of Aachen 2019. The sequence of the protein originates from Pseudomonas putida (SB2) and was first improved by S. Ko et al [4].

Fluorescent proteins are a fundamental building block in synthetic biology research. For our “improve a part” project we decided to work with flavin-based fluorescent proteins (FbFPs), which are derived from bacterial and plant photosensory flavoproteins and commonly-used reporter genes. [2]
Flavoproteins have some relevant advantages compared to the green fluorescent protein – GFP. One oft the biggest advantage of FbFPs is their smaller size, compared to GFP, and their maintained functionality in oxygen-free environments. Thus, these reporter genes play a major role in bioimaging as they are independent of oxygen, unlike most other fluorescent proteins. In addition, FbFP have been genetically improved in various ways to make them more thermostable, more ph-stable and more luminous. [3]
The IGEM part BBa_K3013002 codes for the protein FbFP BS2 and is a flavin mononucleotide-based fluorescent protein containing a LOV domain. The previous team Aachen 2019 improved this part by changing the reporter gene from GFP to FbFP BS2 to use it for anaerobic metabolisms. A side-effect of this exchange, was a decrease in the brightness of fluorescence.
We aim to improve this part by increasing the luminescence of FbFPs, in order to aid further igem teams with projects regarding for example research in anaerobic organisms and therefore require the use of FbFPs.

Design of experiment

In the experiment we cloned three different CDS for FbFPs separately into the vector pJoe [5] and compared the fluorescence of the resulting colonies. For one of the sequences, we used the IGEM part BBa_K3013002 in order to be able to demonstrate later that our new part shows improved fluorescence.
The other two inserts are FbFP-variants from Pseudomonas putida. The original protein SB2 from p. putida has a low fluorescence activity. However, in one study, different SB2 variants were produced by random mutagenesis and selected regarding their fluorescence by flow-cytometry. The resulting variants (KOFP-1 to KOFP-13) have different mutations, which causes them to fluoresce at different intensities. Especially remarkable was the protein KOFP-7 which showed the strongest fluorescence in the publication [1]. An alignment of the sequences with the original SB2 sequence showed that KOFP-7 had a stop codon inserted at the 143rd amino acid, resulting in truncated protein. We assume that this mutation causes KOFP-7 improved fluorescence[4].
To validate the results from the publication, we cloned the FbFB gene from the parts, the original SB2 gene and KOFP-7 from p. putida separately into the expression vector pJoe.

Figure 1: Plasmid map of the vector pJoe with the insert SB2

Figure 2: Plasmid map of the vector pJoe with the insert original

Figure 3: Plasmid map of the vector pJoe with the insert KOFP-7.

Mechanism

First, we used a gradient PCR to identify the perfect temperature for the primer annealing for the three inserts. The primers had the best annealing at a temperature of 63,2°C. We then amplified the three inserts to use them later on for the Gibson Assembly.

Figure 4: Gel image for control of the PCR of KOFP-7, Original, parts and the vector pJoe.

The amplified inserts were cloned into the vector pJoe via Gibson Assembly. In the next step we transformed the E.coli strain DH5alpha with the newly assembled pIoe vector via heatshock transformation. As pIoe encodes a resistance against Ampicillin we used agar plates containing ampicillin, in order to select for colonied carrying the vector.
Afterwards we performed a colony PCR in order to determine clones carrying the vector with the full insert. The results are shown below.

Figure 5: Colony PCR from Original, Parts and KOFP-7 in three different gels.

Colony PCRs have shown that the cloning of parts was successful, and all colonies tested did indeed carry the insert. The cloning of KOFP-7 also showed success in seven colonies out of nine. However, cloning of original did not work and had to be repeated. In a second attempt under the same conditions the insert was successfully inserted in pJoe. The colony PCR results were validated by sequencing.
To measure the fluorescence, a retransformation from the strain DH5alpha to BL21 (D3) was performed, since pJoe carries a T7 promoter and requires a strain carrying a T7 polymerase for expression.

Characterization

For characterization, the liquid cultures were induced at an OD(600)=0.7 with 0.1mM IPTG and then incubated for 16 hours at 20°C. After 10 hours the protein expression was measured. For this purpose, the emission was recorded via plate reader at an excitation of 450nm and an emission of 495nm.
To measure the fluorescence, the cells had to be washed with PBS buffer beforehand, because the LB medium falsified the measurement. Therefore, the samples were centrifuged, decanted and resuspended in the PBS buffer. The fluorescence of the cells in PBS was then measured in the plate reader.

Figure 6: Results of fluorescence measurement after 10 to 16 hours after induction with IPTG in the Tecan reader.

The results show that the cells carrying the gene KOFP-7 show a significantly higher fluorescence compared to the original FbBP of p. putida and the protein encoded by the part (BBa_K3013002).

Conclusion

Our team set out to improve the part BBa_K3013002 by increasing the fluoresce without having any further impact on how the protein is expressed. In order to achieve this, we compared the three parts: original, part (BBa_K3013002) and KOFP-7 by fluorescence measurement. The result was that the fluorescence of SB-2 showed a similar intensity compared to the part BBa_K3013002. KOFP-7 on the other hand showed an increased fluorescence compared to the other parts, whereas all parts were cloned and expressed under the same conditions. Thus, the part BBa_K3410000 is an improved version of the part BBa_K3013002 . In the future, a dye needs to be identified to establish a concept for relative fluorescence units (RFU), like iGEM established fluorescein for measuring GFP in RFU and iGEM Bielefeld-CeBiTec 2019 established Texas red for measuring mCherry in RFU.

References

[1] L. H. Naylor, “Reporter Gene Technology: The Future Looks Bright,” p. 9.
[2] A. Mukherjee, J. Walker, K. B. Weyant, and C. M. Schroeder, “Characterization of Flavin-Based Fluorescent Proteins: An Emerging Class of Fluorescent Reporters,” PLoS ONE, vol. 8, no. 5, p. e64753, May 2013, doi: 10.1371/journal.pone.0064753.
[3] A. Mukherjee and C. M. Schroeder, “Flavin-based fluorescent proteins: emerging paradigms in biological imaging,” Curr. Opin. Biotechnol., vol. 31, pp. 16–23, Feb. 2015, doi: 10.1016/j.copbio.2014.07.010.
[4] S. Ko et al., “Discovery of Novel Pseudomonas putida Flavin-Binding Fluorescent Protein Variants with Significantly Improved Quantum Yield,” J. Agric. Food Chem., vol. 68, no. 21, pp. 5873–5879, May 2020, doi: 10.1021/acs.jafc.0c00121.
[5] K. Schmid, R. Ebner, J. Altenbuchner, R. Schmitt, and J. W. Lengeler, “Plasmid-mediated sucrose metabolism in Escherichia coli K12: mapping of the scr genes of pUR400,” Mol. Microbiol., vol. 2, no. 1, pp. 1–8, Jan. 1988, doi: 10.1111/j.1365-2958.1988.tb00001.x.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

color	green
emission	496
excitation	450
protein	FbFP B2