Difference between revisions of "Part:BBa K2557001"

Latest revision as of 17:38, 17 October 2018

Bxb1 recombinase

Bxb1 is a widely studied recombinase that we use to mediate intracellular signals.In order to allow the recombinase to enter the nucleus and function, we added SV40 NLS to the front end. To detect protein content, we added the FLAG tag.

Usage and Biology

Bxb1, also known as Bxb1 gp35, is an recombinase comeing from Mycobacterium Phage Bxb1 and can bind to specific attB/P sites to catalyze DNA recombination. It helps the phage to integrate its genome into bacterial genome naturally. By constructing the attB/P sites in different directions, Bxb1 gp35 can catalyze the recombination of DNA between their sites, leading to inversion when attB/P are in opposite directions and excision when attB/P are in the same directions. The resulting attL and attR sequences cannot be recognized and bound by integrases alone, so the state after integration is stable. Bxb1 gp35 is widely used to construct combinational logic gate and synthetic biology.

In our genetic circuit, Bxb1 regulated by TetR. When the inhibition released by TEV protease, Bxb1 can bind to specific attB/P sites to catalyze DNA recombination, leading to the expression of RFP.

Characterization

Fig.2 Function verification of reversal efficiency and threshold characteristics of different recombinase in HEK 293 T Cells (A)Fluorescence microscope observation of HEK 293T undergone different experimental treatments (B)The statistical chart of average fluorescence intensity of cells shows that the cells with Bxb1 recombinase have a higher fluorescence intensity than those with TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. (C)The statistics of the proportion of fluorescent cells show that the proportion of fluorescent cells has a sudden jump discontinuity between low concentration and high concentration of Bxb1 and TP901 recombinases. Similar results were obtained in all three repetitions.

The results of image B show that the reverse efficiency of Bxb1 recombinase is higher than TP901 recombinase under the same promoter strength and recombinase concentration. However, if the concentration of recombinase is low, there is no significant difference in fluorescence intensity. The results of the image C show that Bxb1 and TP901 recombinases have a threshold property. So, the proportion of fluorescent cells have a jump discontinuity between low concentration and high concentration of recombinase.

Improvement

The Peking iGEM 2017 team identified the properties of Bxb1 gp35(BBa_K2243012)in E. coli. We obtained the sequence of this part and requested the plasmid from them. Since our chassis organism is mammalian cell, we optimized the codon and then we identified BBa_K2557001 in detail in HEK 293T cells. Although Bxb1 works well in HEK 293T cells, we did not repeat the activity reported by Peking iGEM 2017 team in E. coli.

Pronuclear verification of recombinase Two plasmids with different resistances and origins of replication were used for function verification of the recombinase. One of them is a reporter gene plasmid, which uses the constitutive promoter J23119. The recombination site is located on both sides of the promoter: one side is sfGFP, and the other is mRFP. The other plasmid is a recombinase expression plasmid using P_BAD, an inducible promoter, which is induced by arabinose. When the two plasmids were co-transfected into E. coli, the reporter plasmid expressed sfGFP, a kind of green fluorescent protein; when the inducer arabinose was added, the recombinant enzyme was expressed, the promoter was inverted, and the mRFP , a kind of red fluorescent protein, was expressed.

Due to the use of two different resistant plasmids, kana and chloramphenicol, we used a plate containing two resistances of kana and chloramphenicol for screening, grew more colonies, and randomly selected 9 singles. After the colonies, we made colony PCR (Fig. 1) and the results showed that both plasmids were transferred.

Fig. 3 A total of 9 single colonies were verified. Lane 1-9, recombinase expression plasmid validation; line 10, DL2000 DNA Marker; line 11-19, reporter gene expression plasmid validation;line 20, DL2000 DNA Marker.

The verified E. coli was separately placed in a 1.5 ml centrifuge tube containing antibiotic-containing LB medium, and the culture was grown at 37° C and 200 RPM for 6 hours, and then the culture was aliquoted into two portions, one of which was added with an inducer (10 mM Arabinose). Two cultures were grown for 12 hours at 37°C and 200 RPM, and the mixture was incubated for 1 hour at room temperature prior to testing. Both lasers are used to excite both sfGFP and mRFP.

Fig. 4 Two repetitions were selected and the results showed no obvious green fluorescence

Fig.5 Two replicates were selected after addition of the inducer and the results showed no obvious red fluorescence The result shows no obvious fluorescence. We changed some conditions, such as lowering the temperature, adjusting the rotation speed, adjusting the time, etc. But we still did not get the expected results. We consulted the teacher and the teacher replied that there might be weak fluorescence but our instrument couldn't detect it.

References

1.Blechl, A., Lin, J., Shao, M., Thilmony, R. & Thomson, J. The Bxb1 Recombinase Mediates Site-Specific Deletion in Transgenic Wheat. Plant Mol. Biol. Report. 30, 1357–1366 (2012).

2.Rutherford, K. & Van Duyne, G. D. The ins and outs of serine integrase site-specific recombination. Curr. Opin. Struct. Biol. 24, 125–131 (2014).

Sequence and Features