Difference between revisions of "Part:BBa K2243023"

Latest revision as of 18:53, 1 November 2017

Bxb1 attB_435F_Bxb1 attP

To test the influence of attBP sites of Bxb1 to terminator ECK120034435 (abbreviation: 435) 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 ECK120034435 (abbreviation: 435) 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.

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

A set of desirable unidirectional terminators were selected based on Nasri H, et. al Nature Methods 2013 and they were then constructed on the pSB4C5 vector. Terminator strength was tested using flow cytometry and corresponding Ts value was calculated.

Then attB/P sites were then introduced.

Two systems- the system containing sandwiched terminators and the system expressing corresponding recombinases were co-transformed into DH5alpha cells. Ts values of terminators sandwiched with attB/P sites were measured with flow cytometry.

1. 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

2. We then characterized the inversion efficiency of Bxb1.

We transformed the testing system containing terminator ECK120034435 (abbreviation: 435) in the forward direction and expression system of Bxb1 recombinase into one single cell to see if the inversions happened, and if the leakage expression of Bxb1 would impact the system.

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.

Fig.5 Terminators flanked by one pair of attB/P sites Induced with 0.1M IPTG and 10mM Arabinose

The inversion efficiency was calculated according to the formula below.

Terminator Reference Table

Media:Peking_trt.xlsx

Sequence and Features