iGEM 2011 — Team UST-Beijing

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# Team:UST-Beijing/Notebook

### From 2011.igem.org

# Project I.

## 1.1：The construction of pSB1AC3（BBa\_K603000）/PR

|  |  |  |
| --- | --- | --- |
| ![](https://static.igem.org/mediawiki/2011/7/71/1.1PR%E7%89%87%E6%AE%B5.jpg) | ![](https://static.igem.org/mediawiki/2011/9/91/1.2PsB1AC3_map.jpg) | ![](https://static.igem.org/mediawiki/2011/a/ab/1.3%E5%B5%8C%E5%85%A5PR%E8%B4%A8%E7%B2%92.jpg) |

PR + pSB1AC3 = pSB1AC3/PR (new part)

Cut PR w/EcoRI & SpeI

Cut pSB1AC3 w/EcoRI & SpeI

Combine the insert and vector with T4 ligase

## 1.2: Gel electrophoresis

|  |  |
| --- | --- |
| ![](https://static.igem.org/mediawiki/2011/2/25/2pSB1AC3_EcoRI%26SpeI%E5%8F%8C%E9%85%B6%E5%88%87%E9%AA%8C%E8%AF%81.jpg) | 1: wide range maker  2、3、4、5：the constructed plasmid cutted with SpeI and EcoRI restriction enzymes |

## 1.3: DNA sequencing

![](https://static.igem.org/mediawiki/2011/3/39/3%E6%B5%8B%E5%BA%8F%E7%BB%93%E6%9E%9C.PNG)
The result of DNA sequencing demonstrates that there is no mutation in the new part by comparing to the original sequence.

## 2.1 The construction of pSG5/PR which is used for eukaryotic expression

![](https://static.igem.org/mediawiki/2011/c/c4/PR_for_pSG5.jpg)![](https://static.igem.org/mediawiki/2011/f/fb/PSG5.JPG)![](https://static.igem.org/mediawiki/2011/3/33/PSG5PR.jpg)

PR + pSG5 = pSG5/PR
Cut PR w/EcoRI & BamH I
Cut pSG5 w/EcoRI & BamH I
Combine the insert and vector with T4 ligase

## 2.2 Gel electrophoresis

| ![](https://static.igem.org/mediawiki/2011/8/8b/%E5%A4%A7%E7%B4%AB_%E5%8F%8C%E5%88%87.jpg) | 1: wide range marker  2: the constructed plasmid pSG5/PR cut with EcoR1 and BamH1 |

## 2.3 DNA sequencing

![](https://static.igem.org/mediawiki/2011/1/15/2.3.PNG)

The result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

## 3.1  The construction of pBABE/PR

**1) PCR for amplifying more insert**
Primer 1:         5'-ggg gaa ttc taa acc acc atg ctt gcc-3'
Primer 2:         5'-aaa gaa ttc tca cta tta ggc gtt gct-3'
**Reaction system:**
10XPCR buffer      5ul
dNTP               4ul
primer 1           1ul
primer 2           2ul
pfu                1ul
template           1ul
DMSO               5ul
ddH2O              31ul
Total              50ul
**Reaction condition:**
95℃       5min

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*
95℃       15s
52℃       15s
72℃       2min

25cycles

\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*\*
72℃       10min
4℃        ∞

**2) Ligation**

![](https://static.igem.org/mediawiki/2011/b/ba/PR.jpg)![](https://static.igem.org/mediawiki/2011/a/a0/PBABE.jpg)![](https://static.igem.org/mediawiki/2011/4/48/PBABEPR.jpg)

PR + pBABE = pBABE/PR
Cut PR w/EcoRI
Cut pBABE w/EcoRI
Combine the insert and vector with T4 ligase

## 3.2 Gel electrophoresis

![](https://static.igem.org/mediawiki/2011/2/21/EcoR1%E9%AA%8C%E8%AF%81.jpg)![](https://static.igem.org/mediawiki/2011/5/52/Sal1%E9%AA%8C%E8%AF%81.jpg)

1:Middle range maker 　　　 1:DL2,000 DNA Marker

2:cut with EcoR I　　　　　 2:cut with Sal I

Figure 1 indicates that the insert is combined with the vector successfully.
Figure 2 indicates that the insert is in the right direction.

## 3.3 DNA sequencing

![](https://static.igem.org/mediawiki/2011/2/21/3.3.jpg)

As before, the result of DNA sequencing demonstrates that there is no mutation in PR by comparing to the original sequence.

## 4.1 The construction of BBa\_K603001

[![](https://static.igem.org/mediawiki/2011/f/fa/BBa_K603001.jpg)](https://static.igem.org/mediawiki/2011/f/fa/BBa_K603001.jpg)

fig.1 Proteorhodopsin(PR)digested at EcoRI and PstI and ligased to pSB1C3(Linearized plasmid backbone Pre-cut at EcoRI and PstI ends).

## 4.2 Gel electrophoresis

[![](https://static.igem.org/mediawiki/2011/8/8f/Cut_gel.jpg)](https://static.igem.org/mediawiki/2011/8/8f/Cut_gel.jpg)

fig.2 In this fig,NO.1 is Wide range DNA marker,NO.2-4 are plasmid BBa\_K603001 digested at EcoRI and PstI,demonstrate that the plasmid is correct.

# Project II

## 1.The construction of lacI-DBD-LXR-LBD

fig.1 Amino acid sequence of lacI-DBD-LXR-LBD chimeric protein,the DNA binding domain of lacI is boxed.

The DNA sequencing(bottom left)and the gel electrophoresis of the digested pET28(bottom right) both demostrate that

the construction of lacI-DBD-LXR-LBD is exactly what we designed before.

## 2.The gene worknet

[![](https://static.igem.org/mediawiki/2011/3/3e/Shuangzhilixitong.jpg)](https://static.igem.org/mediawiki/2011/3/3e/Shuangzhilixitong.jpg)

## 3.The experiment

![Circuit.JPG](https://static.igem.org/mediawiki/2011/a/a5/Circuit.JPG)

fig.1 The gene circuit of the two-plasmid system.

![IPTG%2BKAN_2.jpg](https://static.igem.org/mediawiki/2011/c/c5/IPTG%2BKAN_2.jpg)

fig.2 The key element that affect the RFP are IPTG,Kan and our compound.

![Quxiantu.jpg](https://static.igem.org/mediawiki/2011/1/1d/Quxiantu.jpg)

fig.3 We have tried five compounds,compound B,C,D and E can active the gene and express rfp.The pET28a is

control that have no lacI-DBD-LXR-LBD gene.

## Notebook

You should make use of the calendar feature on the wiki and start a lab notebook. This may be looked at by the judges to see how your work progressed throughout the summer. It is a very useful organizational tool as well.

Retrieved from "[http://2011.igem.org/Team:UST-Beijing/Notebook](http://2011.igem.org/Team%3AUST-Beijing/Notebook)"

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