Team:NAU-CHINA/Basic Part

Template:2018_NAU-CHINA

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Basic Parts

PARTS

Basic Parts

Our Best Basic Part:BBa_K2557001

This year, our team has selected a series of recombinases through literature searches and characterized a number of them. Here we present the best characterized recombinase - Bxb1 (BBa_K2557001) as our best basic part for the award.

Background

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.

Fig. 1 Bxb1 structure homology-model provided by SWISS-MODEL.

Function In our Genetic Circuit

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

Method: Co-transfected with six different numbers of plasmids containing recombinase genes(miniCMV-Bxb1 and miniCMV-TP901) and fixed numbers of plasmids containing corresponding recombinase recognition sites. After 36 hours of plasmid co - transfection, the proportion of fluorescent cells and the average fluorescence intensity of cells were detected by flow cytometry. The experiment was repeated three times.

Result:

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.

In conclusion, Bxb1 works well in HEK 293T as expected, so we present Bxb1(BBa_K2557001) as our best basic part.

Basic Part List

Name Type Description Length
BBa_K2557001 Coding Bxb1 recombinase 1565
BBa_K2557000 Coding Anti-GFP-mnotch-TEV protease-NLS 2409
BBa_K2557003 DNA Bxb1 attB-Bxb1 attP 200
BBa_K2557004 Coding PhiC31 recombinase 1881
BBa_K2557006 DNA PhiC31 attB-PhiC31 attP 204
BBa_K2557007 Coding TP901 recombinase 1604
BBa_K2557009 DNA TP901 attB-TP901 attP 210
BBa_K2557032 promoter miniCMV promoter 38
BBa_K2557033 promoter EF1α promoter 212
BBa_K2557034 promoter Ubc promoter 400
BBa_K2557035 Reporter TagRFP 711
BBa_K2557036 Reporter mCherry 708
BBa_K2557037 Reporter EGFP 738
BBa_K2557038 DNA TetO 271
BBa_K2557041 DNA Bxb1 attB 96
BBa_K2557042 DNA Bxb1 attP 112
BBa_K2557043 DNA PhiC31 attB 45
BBa_K2557044 DNA PhiC31 attP 135
BBa_K2557045 DNA TP901 attB 113
BBa_K2557046 DNA TP901 attP 112
BBa_K2557047 Coding Bxb1-RDF 2345
BBa_K2557048 Coding PhiC31-RDF 2630
BBa_K2557049 Coding TP901-RDF 1796
BBa_K2557050 Coding TetR with TEV cleavage site 651

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