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From 2013.igem.org

Notebook

Jun 20th~Jul 1st,
We met with our mentors and got an overview of the basics of synthetic biology, namely characterization, which will be the focus of our works during the summer. We did a survey of previous iGEM teams that got the Best Experimental Measurements award, then presented the findings to our mentors.Then we did some experiment technologies trainings, such as making gel, gel electrophoresis.

Jul 1st~Jul 8th ,
We construct our single enzyme carriers
LinA LinB
DhaA HheC

Gels analysis

PCR colonies ,and keep them in refrigerator.

Jul 9th~Jul 14th,

Construct our 2A-mediated co-expression system.
LinA-F2A-LinB DhaA-P2A-HheC

gel electrophoresis.

Jul 15th~Jul 21st

Transformed those genes into MC1061 , and growth of of strain MC1061 transformed pOHC_05, pOHC_06 in LB medium with inducer and 5mM substrate in batch culture .As the growing of the E.coli ,the substrate disappears .So new clones show the activity predicted and our co-expression system worked .

Jul 22nd~Jul 27th

From the gel ,we can see that P2A can cleave LinA-2A-LinB into single genes ,however ,F2A can’t wok in the cleaviation .

Jul 27th
Today ,we just put down our assays in the lab ,and went to a senior high school to have fun with them with the goal of promoting IGEM and Synthetic biology .

Jul 29th~Aug 7th

We do GC analysis , Growth of E.coli strain MC1061 transformed LinA+F2A+LinB and LinA gene in LB medium with inducer and 5mM γ-HCH respectively, and Growth of E.coli strain MC1061 transformed DhaA+P2A+HheC and DhaA gene in LB medium with inducer and 5mM TCP respectively.

Aug 8th~Aug 12th
Crude activity detecting in supernatant and sediment.

Aug 13th~Aug 19th
The purified enzyme, HheC and DhaA+P2A+HheC confusion protein was isolated by AKTA FPLC. Only if four single HheC compose as a tetramer can it catalyze dehalogenation. Assaying HheC activity in fusion protein could be an ideal way to detect the influence of 2A peptide. It indicates that the 2A peptide not only doesn’t break the initial space structure, but as an assistant linker compose the two enzymes’ function together.

Aug 20th~Aug 24th
construct our RBS co-expression system
LinA-RBS-LinB DhaA-RBS-HheC

gel electrophoresis.

Aug 24th~Aug 27thgel
SDS-PAGE