Difference between revisions of "Part:BBa K1433005"

Latest revision as of 05:49, 14 October 2018

RBS-gamma protein-beta protein-exonuclease

This part is constructed to improve BBa_K1223012, pKD46 functional unit.

BBa_K1223012 is not conformed to any RFC because there are three restrict enzyme sites of EcoRI and PstI. To improve the compatibility of this part and make the modules enable to be used in future assemblies, we removed these restrict enzyme sites by synonymous mutations.

Composition

1. RBS: an RBS from PKD46.
2. Exo: a 5’→3’ double-strand DNA specific exonuclease.
3. Beta: a single strand binding protein.
4. Gam: a protein inhibits the E. coli RecBCD exonuclease.

Lambda red composition

Lambda red is a Lambda-phage-derived recombination system which contains three proteins: Exo, Beta and Gam. It can recombine dsDNA/ssDNA into different kinds of DNA molecules such as chromosome, bacteria artificial chromosome (BAC) or even multicopy plasmids, as long as each side of the donor dsDNA/ssDNA is flanked by 36-50bp homologous arms. The homologous arms also determine the accurate sites of recombination, which ensures that the donor fragment can be inserted into any sites by choosing and adding homologous arms using PCR. 



• Exo: Exo is a 5’→3’ double-strand DNA specific exonuclease and is only required for dsDNA recombination. It can degrade one of the whole strand of dsDNA to allow a single strand annealing recombination



• Beta: Beta is a single strand binding protein, who is the central player in red system for both dsDNA and ssDNA recombination. It binds and aims the donor fragment to homologous sites.



• Gam: The Gam protein inhibits the E. coli RecBCD exonuclease that normally degrades all linear dsDNA. It’s important for using dsDNA as donor fragment.



• Homologous arm : Homologous arm are short sequence flank over each side of the donor sequence. It also matches the recombination site on acceptor region. For high possibility of recombination, the homologous arm maybe the longer the better, but actually 36-50bp are enough for antibiotic resistance screening as most researchers has been reported. the sequence of homologous arm are not limited by specific recombination site like many recombinases demand. But there are also some factors we need to consider when choosing this arm. Fortunately, ZJU-China has design a tool to help you carry out this process easily! For more information, please see our “GS-BOX”.

Usage and Biology

Usage

IISc-Bangalore 2018

For their Phage Assisted Imune Recruitment (PAIR) the IISc 2018 iGEM Team used a composite part (BBa_K2609026) made from this part to recombine the Bacteriophage T4 genome to produce a lysis deficient phage. This was done using the parts BBa_K2609008 and BBa_K2609009 which have flanking sequences homologous to the flanking regions of Endolysin gene (e gene) on the T4 genome. A recombinant T4 Endolysin (BBa_K2609017) was used in conjuction with this part to screen for recombinants. Following successful recombination, their lysis deficient phage would trigger it's host to secrete mcp-1 (BBa_K2609000).

Characterisation

IISc-Bangalore 2018

Expression with BBa_K2609026

The part was transformed into E. coli BL21(DE3) since it contains the T7 polymerase under a lac promoter. It was induced with IPTG in mid log phase and the cell pellet was loaded onto an SDS PAGE. The following are the exact conditions:

• Initial Growth Temperature: 37°C
• Growth Medium: LB
• Chloramphenicol Concentration: 35μg/mL
• Induction OD600: 0.6
• IPTG Concentration: 500μM
• Growth Temperature Following Induction: 37°C
• Growth Time Following Induction: 3hrs

Bands were observed slightly below 32kDa ladder band and at the 25kDa ladder band. These two correspond to the bet protein (29.7 kDa) and the exo protein (25.9 kDa). The other protein - gam (16.3 kDa) - cannot be seen on the gel.