"

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 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 F2A can cleave LinA and LinB,however ,P2A can’t work like that.

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 did 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
We purified HheC and DhaA+P2A+HheC by AKTA FPLC and assayed the activity of them using 1,3-DCP. Special activity of HheC was 6.72(U/mg) and the chimeric protein was 5.28(U/mg).

Aug 20th~Aug 24th
RBS co-expression system constructed.
LinA-RBS-LinB DhaA-RBS-HheC

gel electrophoresis.

Aug 24th~Aug 27thgel
SDS-PAGE analysis.