Difference between revisions of "Part:BBa K2243026"

Latest revision as of 15:41, 1 November 2017

Bxb1 attB_836F_Bxb1 attP

To test the influence of attBP sites of Bxb1 to terminator ECK120010836 (abbreviation: 836) in the forward direction.

Usage

We constructed this part to characterize the recombination efficiency of the recombinase Bxb1 gp35 (BBa_K2243012). It consists of the terminator ECK120010836 (abbreviation: 836) in the forward direction flanked by attB and attP sites of recombinase Bxb1 gp35. Upon recombination, the orientation of the terminator changes. As a result, expression of downstream sequence is initiated, and upstream sequence is transcribed.

Biology

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 terminator, attB and attP sites by oligo synthesis.

Characterization

We first characterized the terminator strength using the following formula:

Ts=〖GFP〗of the random sequence/〖[GFP]〗with the terminator

Fig.1 Terminator Strength Induced with 0.1mM IPTG

Fig.2 Terminator Strength Induced with 1mM IPTG

And we sifted out 6 desirable unidirectional terminators without potential cryptic promotor in both orientations.

Fig.3 Terminator Strength Induced with 0.1M IPTG

Fig.4 Forward and Reverse Terminator Strength Ratio Induced with 0.1mM and 1mM IPTG

Terminator Reference Table

Media:Peking_trt.xlsx

Sequence and Features