Difference between revisions of "Part:BBa K2243006"

Revision as of 14:22, 1 November 2017

Bxb1 attB_J23119_Bxb1 attP

The constitutive promoter J23119 is between Bxb1 gp35 specific sites and will be inverted by the Bxb1 gp35 recombinase, thus leading to the direction of promoter and change of gene expression. This unit can be used as standard reporter of recombinase bxb1 gp35 characterization.

Usage and Biology

We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of a constitutive promoter (BBa_J23119) flanked by attB and attP sites of recombinase Bxb1 gp35. Different orientation of attB and attP allows the sequence to be flipped, excised, or inserted between recognition sites, which makes it useful for gene editing. Upon recombination, the orientation of the constitutive promoter change. As a result, expression of downstream sequence is shut down, and upstream sequence is transcribed.

Biology

J23119 is the consensus sequence of a combinatorial constitutive promoter library family. The attP site of Mycobacteriophage Bxb1 is used to integrate phage DNA at the host attB site of Mycobacterium smegmatis, generating the recombinant junctions attL and attR. DNA cleavage and re-ligation occur at the central crossover region at attB and attP, which allows the sequence to be flipped, excised, or inserted between recognition sites. We obtained the promoter, attB and attP sites by oligo synthesis.

Design note

We construct this structure by Gibson Assembly.

Characterize

We used this part to characterize the recombination efficiency of the integrase Bxb1 gp35 (BBa_K2243012).

Figure 1. The standard genetic structure used to characterize the recombinases.

Fig 2. Bxb1 gp35 recombination efficiency with a variety of calculated RBS and RBS sequences from iGEM (B0030~B0035). T.I.R = Translation Initiation Rate

Source

Mycobacterium Phage Bxb1

References

Sequence and Features