iGEM 2013 — Team UESTC_Life

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# Team:UESTC Life/Notebook

### From 2013.igem.org

| Notebook |
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![](https://static.igem.org/mediawiki/igem.org/8/8a/Uestclifebiot_logoarrow.png)

##

**Jun 20th~Jul 1st,**
After having a meeting with our instructors and got an overview of the basics of synthetic biology. We did a survey of previous iGEM teams that got the Best Experimental Measurements award and read many papers. Base on the condition of our college and our expectation, we decided our project and create a schedule. 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* ![Uestclifenote1.png](https://static.igem.org/mediawiki/igem.org/5/5a/Uestclifenote1.png)

![Uestclifenote5.png](https://static.igem.org/mediawiki/igem.org/5/5b/Uestclifenote5.png)
Fig. 1 Amplification of gene fragments by PCR. pOHC\_00 (empty modified pBAD vector )

![Uestclife1.2.png](https://static.igem.org/mediawiki/igem.org/2/2d/Uestclife1.2.png)
Fig. 2 Positive colonies identification. Positive colonies were confirmed by performing colony PCR.

![Uestclife1.3.png](https://static.igem.org/mediawiki/igem.org/0/07/Uestclife1.3.png)
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* ![Uestclifenote6.png](https://static.igem.org/mediawiki/igem.org/a/ae/Uestclifenote6.png) *DhaA+P2A+HheC* ![Uestclifenote7.png](https://static.igem.org/mediawiki/igem.org/b/bc/Uestclifenote7.png)

gel electrophoresis.
![Uestclifenote8.png](https://static.igem.org/mediawiki/igem.org/6/64/Uestclifenote8.png)
Fig. 1 Amplification of gene fragments by PCR. pOHC\_00 (empty modified pBAD vector )

![Uestclife2.2.png](https://static.igem.org/mediawiki/igem.org/e/e8/Uestclife2.2.png)
Fig. 2 Positive colonies identification. Positive colonies were confirmed by performing colony PCR.

![Uestclife2.3.jpg](https://static.igem.org/mediawiki/2013/8/88/Uestclife2.3.jpg)
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 .
![Uestclifenote9.png](https://static.igem.org/mediawiki/igem.org/d/df/Uestclifenote9.png) ![Uestclifenote10.png](https://static.igem.org/mediawiki/igem.org/0/0d/Uestclifenote10.png)

**Jul 22nd~Jul 27th**

![Uestclifenote11.jpg](https://static.igem.org/mediawiki/igem.org/0/05/Uestclifenote11.jpg)
![Uestclifenote12.jpg](https://static.igem.org/mediawiki/igem.org/6/65/Uestclifenote12.jpg)
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 .**
![Uestclifenote13.jpg](https://static.igem.org/mediawiki/igem.org/c/c5/Uestclifenote13.jpg)

![Uestclifenote14.jpg](https://static.igem.org/mediawiki/igem.org/4/4d/Uestclifenote14.jpg)

![Uestclifenote15.jpg](https://static.igem.org/mediawiki/igem.org/c/ce/Uestclifenote15.jpg)

**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.
![Uestclife00.jpg](https://static.igem.org/mediawiki/igem.org/4/48/Uestclife00.jpg)

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

![Uestclifenote16.jpg](https://static.igem.org/mediawiki/igem.org/6/65/Uestclifenote16.jpg) ![Uestclifenote17.png](https://static.igem.org/mediawiki/igem.org/f/f9/Uestclifenote17.png)

**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* ![Uestclifenote18.png](https://static.igem.org/mediawiki/igem.org/9/92/Uestclifenote18.png) *DhaA-RBS-HheC* ![Uestclifenote19pOHC.png](https://static.igem.org/mediawiki/igem.org/3/37/Uestclifenote19pOHC.png)

![Uestclifenote20.png](https://static.igem.org/mediawiki/igem.org/1/15/Uestclifenote20.png)
Fig. 1 Amplification of gene fragments by PCR.

 ![Uestclife3.2.png](https://static.igem.org/mediawiki/igem.org/f/f3/Uestclife3.2.png)
Fig. 2 Positive colonies identification. Positive colonies were confirmed by performing colony PCR.

 ![Uestclifenote3.3.png](https://static.igem.org/mediawiki/igem.org/0/01/Uestclifenote3.3.png)
Fig. 3 Digestion the obtained plasmids with XbaI and XhoI.

**Aug 24th~Aug 27th**gel
SDS-PAGE analysis.
![Uestclifenote21.png](https://static.igem.org/mediawiki/igem.org/c/c7/Uestclifenote21.png)
![Uestclifenote22.png](https://static.igem.org/mediawiki/igem.org/8/85/Uestclifenote22.png)

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