Team:Tsinghua/Parts

2012 iGEM Parts by Tsinghua

 

1.Plac-LuxR-Plux
BBa_K766001
There is something wrong with the previous part BBa_J09855, which doesn't work well. After we sequenced the part, we find that the sequence of Plac promoter and Plux promoter is incorrect. So that we rebuild this part using overlap PCR and DNA systhesis. Finally, our part can work normally.

2.RFP with RBS and T1 terminator
BBa_K766002
We build this part as a reporter to detect the activation of upstream promoter. This fluorescence protein is very easily detectable even by eyes. Besides, we have added a strong RBS and T1 terminator to the end. You can use this part to construct a more complicated part further more.

3. C6HSL sensor and reporter
BBa_K766003
We build this part to work as a receiver. C6HSL from the neighbor cell can diffuse across the membrane and activate the transcription from Plux promoter. There is something wrong with original Part:BBa_J09855. So we rebuild the reporter part which can be activated by AHL and add the reporter part RFP which is easily detectable. The expression of it is very clear.

 

4. His-Tag-LuxI without LVA-tag
BBa_K766000
This part is a protein coding sequence which encodes a LuxI protein without LVA-tag and added a His-tag at the amino terminus. As a result, not only can this LuxI be degraded at a shorter time but it can be purified by affinity chromatography.

 

 

5. T7 promoter-His tag-LuxI
BBa_K766005
We build this part to work as the signal sender in our project. T7 promoter is a high-efficient promoter which can bind with T7 polymerase and start the transcription. Besides, we add the lac operator between T7 promoter and CDS. If you use this part in a luxI-coding plasmid like pet15b, the expression of gene will be repressed. However, IPTG can be used to activate the expression. His tag is on the N-terminal, which consists of six histidine amino acid. We can use Ni-column to purify the protein. Besides, you can use Hig-tag antibody to detect the protein in western blot. This design makes the detection of luxI gene become very easy.

 

6. Plux-LacI without LVA-tag
BBa_K766006

         This device is a protein-expression unit which contains a Plux promoter and a transcriptional unit of LacI without LVA-tag. Plux can be activated by luxR-AHL complex and the promoter can activate the expression of downstream genes. We add LacI in this part. As we all know, LacI is a repressor which can bind with Lac operator. With the help of this feature, many logic gates or gene expression modulation can be achieved.

There is not LVA-tag in this part so that the repressor can keep working for a relatively long time.

 

 

7. Plas/cI hybrid Promoter-RFP
BBa_K766007
This device is composed of a Plac/cI hybrid promoter and a translational unit of mRFP. The Plas/cI hybrid promoter is made up of Plas promoter and cI operator. The promoter can be activated by the LasR-C12HSL complex and repressed by cI repressor. This part can be used in building some logic gates. The RFP segment is used as a signal to show the result of activation or repression. This kind fluorescent protein is very easy to be detected even by eyes.

 

8. AndNot gate based on AHL
BBa_K766004

We design this A and not B gate. This part is based on the two kinds of AHL：C6HSL (signal A) and C12HSL(signal B). C6HSL can activate the promoter Plux and C12HSL can activate the promoter Plas. In this part, we add cI operator downstream of Plas. As a result, the promoter Plas can be activated by signal B but repressed by cI protein. The expression of cI protein is under the control of Plux, which can be activated by signal A. RFP is controlled by Hybrid promoter (Plas and cI operator), which will work as a reporter.
In a word, the expression of RFP is only on the condition that A is absent and B is present.