Team:Tsinghua-E/Notebook

Notice:Except for specially mentioned, all the following vectors were constructed by In-Fusion HD Cloning Kit (produced by Clontech). To be brief, every fragment of desired DNA was linearized by primers designed following the protocol provided by Takara infusion cloning guide to ensure accurately 15 base pair (bp) overlap homology with each adjacent fragments. The gel-purified fragments were mixed with 2uL infusion cloning kit Premix solution and ddH2O was used to adjust the mixture to 10uL. The amount of the added fragments was based on the Takara infusion cloning guide.

Week1(6.23-6.30)

Literature was reviewed carefully and the corresponding gene candidates were confirmed. Some of them were submitted to be synthesized. Some of students were trained for basic molecule biology experiment operation.

Week2(7.1-7.7)

The primers needed in our project is designed and synthesized. Some chemicals were also purchased. Some of students were trained for basic molecule biology experiment operation and further some characterization experiment operation.

Week3(7.8-7.14)

We cloned dnaQ gene from E. Coli BL21(DE3) by PCR and utilized overlap extension PCR to introduce mutagenesis in dnaQ by the following protocol.

Firstly, we used E. Coli BL21 (DE3) strain as template to amplify dnaQ gene by PCR with mut-F and mut-R primers, respectively. After purification of PCR product (the 〖E.Z.N.A.〗^TM Gel Extraction Kit, produced by Omega Bio-tek), it was used as template to introduce point mutation by two parallel PCRs with the following two sets of primers: mut-L73W-F and mut-L73W-R (generating L73W); mut-A164V-F and mut-A164V-R (generating A164V). The three PCR products were gel-purified and combined by 5ng,

Figure.1 dnaQ PCR amplification from E. Coli BL21(DE3)

respectively and assembled with mut-F and mut-R primers. The resulting overall product was T-cloned into Takara pMD-19 T-vector (produced by Takara BioTechnology (DALIAN)CO.,LTD.) and sequenced to confirm the sequence. The L73W and A164V mutant BL21 (DE3) dnaQ was named after mutD.

Maltose hydrolase gene malQ was cloned from E. Coli JW3995 (citation a malQ PCR is needed).

In parallel, we also constructed tryptophan sensor circuit. According to the report about the translating ribosome by nascent peptide based tryptophan degradation gene cluster regulation mechanism, we previously synthesized the corresponding tnaC coupling Rho sequence. This gene cluster was assembled with lacZ gene by overlap PCR and the gel-purified fragment was cloned between NcoI and HindIII sites of pTrc99A vector by restriction enzyme digest and T4 ligase induced ligation. All the information about primers was shown in “primer information”. This plasmid was named after pTrc99A_trp sensor_lacZ.

Figure. 2 tryptophan sensor construction with lacZ as output signal

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