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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 ,
1.The construction of single enzyme carries.

To facilitate the overexpression of target genes, the four genes LinA, LinB, DhaA, and HheC were optimized, synthesized and cloned into the vector of PUC51 by Gene script Corporation. The resulting vector S1 contains gene fragments of LinA and LinB, while vector S2 contains gene fragments of DhaA and HheC.(Fig.1) After digestion using Xbal and Xhol, the four genes were cloned into a modified pBAD expression vector pOHC_00, respectively, resulting in the following four vectors, POHC_01 (LinA), POHC_02 (LinB), POHC_03 (DhaA), POHC_04 (HheC). E. coli strain DH5α was used as a host cell. (Fig.2) The isolated positive colonies were further confirmed by restriction digestion analysis.(Fig.3) Agarose gel showed cloning confirmation of the four target genes in pOHC plamid. Successful plasmid construction was confirmed by sequence analysis (Invitrogen).

LinA LinB
DhaA HheC

Fig. 1 Amplification of gene fragments by PCR. The primers used for am-plification are listed in Table 1. pOHC_00 (empty modified pBAD vector )

Fig. 2 Positive colonies identification. Positive colonies were confirmed by performing colony PCR using the primers listed in Table 1.

Fig. 3 Digestion the obtained plasmids with XbaI and XhoI.

Jul 9th~Jul 14th,
Construct co-expression system.

To facilitate the overexpression of target genes, the two genes LinA+F2A+LinB, DhaA+P2A+HheC were optimized, synthesized and cloned into the vector of PUC51 by Gene script Corporation. The resulting vector S1 contains gene fragments of LinA+F2A+LinB, while vector S2 contains gene fragments of DhaA+P2A+HheC.After digestion using Xbal and Xhol, the four genes were cloned into a modified pBAD expression vector pOHC_00, respectively, resulting in the following two vectors, POHC_05 (LinA+F2A+LinB), POHC_06 (DhaA+P2A+HheC). E. coli strain DH5α was used as a host cell.The isolated positive colonies were further confirmed by restriction diges-tion analysis. Agarose gel showed cloning confirmation of the four target genes in pOHC plamid. Successful plasmid construction was confirmed by sequence analysis (Invitrogen).

LinA-F2A-LinB DhaA-P2A-HheC

gel electrophoresis.

Fig. 1 Amplification of gene fragments by PCR. The primers used for am-plification are listed in Table 1. pOHC_00 (empty modified pBAD vector )

Fig. 2 Positive colonies identification. Positive colonies were confirmed by performing colony PCR using the primers listed in Table 1.

Fig. 3 Digestion the obtained plasmids with XbaI and XhoI.

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.

Add XhoI and PstI restriction digest sites in the opposite terminate of LinB and HheC gene.After digestion using Xbal and PstI, the two genes were cloned into pOHC_01, pOHC_03 respectively, resulting in the following two vectors, POHC_07 (LinA+RBS+LinB), POHC_08 (DhaA+RBS+HheC). E. coli strain DH5α was used as a host cell.The isolated positive colonies were further confirmed by restriction digestion analysis. Agarose gel showed cloning confirmation of the four target genes in pOHC plamid. Successful plasmid construction was confirmed by sequence analysis (Invitrogen).

LinA-RBS-LinB DhaA-RBS-HheC

Fig. 1 Amplification of gene fragments by PCR. The primers used for am-plification are listed in Table 1.

Fig. 2 Positive colonies identification. Positive colonies were confirmed by performing colony PCR using the primers listed in Table 1.

Fig. 3 Digestion the obtained plasmids with XbaI and XhoI.

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