Difference between revisions of "Part:BBa K2243018"

Revision as of 12:58, 1 November 2017

Bxb1 attB_322F_Bxb1 attP

To test the influence of Bxb1 attB/P to terminator L3S3P22 (abbreviation: 322) 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 L3S3P22 (abbreviation: 322) 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

1. We first characterized the terminator strength using the following formula:

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

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

Terminator Strength Induced with 0.1M IPTG

Terminator Strength Induced with 1M IPTG

2. We then characterized the inversion efficiency.

We transformed the testing system and expression system of corresponding recombinase into one single cell to see if the inversions happened, and if the leakage expression of recombinases would impact the system. In the end, couples of terminators and recombinases with near complete inversions and minimal leakage were selected.

Microplate spectrophotometer was used to conduct preliminary measurements. Bacterial culture was added into 96-well plate(200ul for each well). OD600 and fluorescence intensity were measured. Background OD600 and fluorescence of plate, culture medium ,and autofluorescence should be eliminated through setting control groups. Fluorescence intensity/OD600 was cauculated using the net fluorescence intensity and net OD600. The transcription barrier strength of attBP/Terminator can be characterized quantitatively referring to the Ts formula.

===Sequence and Features===