Difference between revisions of "Part:BBa K2243006"

Revision as of 14:23, 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.

Flow cytometry were used to evaluate the recombination efficiency, the expression vector and reporter of a recombinase were used to co-transform E. coli Top10 and samples were prepared for flow cytometry reading. Single colonies were picked and used to inoculate 1ml of LB media with antibiotics in a V-bottom 96-well plate. The cultures were grown at 37°C and 1000 RPM for 12h. Subsequently, an aliquot comprising 2 μL of the culture was transferred into 1ml of M9 glucose media with antibiotics and inducer (1mM IPTG or 10mM arabinose for RBS tuning, gradient concentration for transfer curve) in a V-bottom 96-well plate. The cultures were grown at 37°C and 1000 RPM for 15h. An aliquot comprising 2μL of the cul-ture was transferred into 198 μL of phosphate buffered saline (PBS) containing 2 mg/mL kanamycin in a 96-well plate. This mixture was incubated for one hour at room temperature before testing. Two lasers were used to excite GFP and RFP simultaneously. Single-cell fluorescence distribution at both emission wavelengths was recorded.

The counted cells were gated to eliminate the population which showed no fluorescence. The remaining cells were divided into two subsets by a diagonal: RFP sub-set and GFP subset. The recombination efficiency was estimated from the proportion of the RFP subset in the total fluorescent population.

Figure 2. Gating of the RFP and GFP subsets and change of fluorescence after induction. Left: no induc-er. Right: 10 mM arabinose for 15h.

Figure 3. 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