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AttB/P Flanking Region Inversion and Different RBS Modulation

Bistable switch plays a core role in our whole device, linking and orchestrating the chromosome circuit and plasmid circuit together. An efficient and convenient Socket bacteria throws out a high standard requirements for stability and efficiency of the bistable switch. Only when the bistable switch reaches a highly controllable and reliably level, can the Socket bacteria realize time-sabing and infinite rounds of gene knock-in. Out of the consideration, we selected Bxb1 system rather than the traditional pre-transcriptional leveled with.

Figure 1 - Plate colony fluorescence stimulated by ultraviolet light. As negative controls, INT-6N and INT-B0031 shows no fluorescence, and SET shows nearly all the green fluorescence. Co-transformation of INT-6N and SET gets almost all the red fluorescent colonies; however transformation with INT-B0031 exhibits partial inversion of fluorescence.

Figure 2 - Fluorescence of bacteria cells involved in the attB/P inversion assay. GFP and RFP are both positive control; SET, INT-6N and INT-B0031 serve as negative control; co-transformation of Int-B0031,SET, and , INT-6N, SET are experimental groups which displays distinct inversion rate.

The Bxb1 system brackets two important reombinases: Int & Xis. From functionality perspective, the switch owns two opposing inversion behaviors, one of which is AttB/P flanking promoter inversion, and the other is attL/R flanking promoter inversion.

We built an attB/P inversion assay to demonstrate the inversion of the crucial flanking element, the promoter J23110. During Int expression, Int inverts the kind of fluorescence output from green to red. Initially, we set up a B0031-invovled Int circuit, and observed partially inversion of the promoter. However, incomplete inversion of the promoter is not preferable for its insufficient efficiency of inversion. So different strength of RBS are considered to be incorporated to enhance the inversion efficiency.

Figure 3 - Fluorescence results of attB/P inversion assay under fluorescent microscope. GFP and RFP are positive controls; 6N, B0031 and SET are negative controls; cotransformation groups certify the inversion activity of INT and exhibit different efficiency with different RBS.

Figure 4 - Fluorescence results of attB/P inversion assay under fluorescent microscope. GFP and RFP are positive controls; 6N, B0031 and SET are negative controls; cotransformation groups certify the inversion activity of INT and exhibit different efficiency with different RBS.

Fig. 5. PCR confirmation of promoter direction. Lane 1 and 2 are based on the same template, SET (negative control); lane 3,4 are INT-B0031+SET; lane 5 and 6 are INT-6N+SET. Each templates are proliferated with two primer pairs (GFP-detect/J23110-detect and RFP-detect/J23110-detec) .

For this purpose, another RBS of higher strengeth, called 6N, is introduced the circuit. With the increase of RBS strength, inversion rate got obvious promotion, approximately up to 100%. Comparison with two different RBS fluorescent images, we can attain expected results and know the different inversion rate of two different RBS-involved inversion. To achieve highly-efficient inversion, there’s no doubt to employ 6N RBS circuit.

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