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.

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

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,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).

Figure. 2 tryptophan sensor construction with lacZ as output signal

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.

Week4(7.15-7.22)

In this week, we tried to clone mutD and sfGFP cluster downstream of araBAD promoter (pBAD). We amplified pSC101-Cm-pBAD and sfGFP fragment from plasmid AraC_pBAD_CI_OR222-sfGFP<ref>Lou, C. B., Stanton, B., Chen, Y. J., Munsky, B. & Voigt, C. A. Ribozyme-based insulator parts buffer synthetic circuits from genetic context. Nature Biotechnology 30, 1137-+, doi:10.1038/nbt.2401 (2012).</ref> which was kindly provided by Professor Ouyang Qi in Peking University. mutD was introduced by three different ribozyme binding site (RBS) sequence upstream and also overlap homology with primers. All the primers information was listed in the appendix, primer information. The three fragments were assembled by infusion cloning as described above. The partial sequence of RBS-mutD op-sfGFP was sequenced to confirm. The three constructed plasmids were named after pBAD_B0030-mutD-sfGFP, pBAD_B0032-mutD-sfGFP and pBAD_SDA_RBS-mutD-sfGFP, respectively.

Figure. 3 in vivo mutation machine construction with mutD and sfGFP series expression

In this week, we also tested our synthetic tryptophan sensor performance by galactosidase activity. Some protocol seemed not to work for some culture condition and measurement conditions. The final successful protocol was shown as below. Firstly, we picked up E. Coli JM109 carrying pTrc99A_trp sensor_lacZ single clone and cultured it in Luria-Bertani (LB) medium (Tryptone 10g/L, Yeast Extract 5g/L, NaCl 10g/L) with concentration of ampicillin at 50mg/L for overnight. On the next morning, the 600nm optical density (OD600) of the culture was measured to be 3.090. This seed was transferred into 2mL LB medium with the same concentration of ampicillin by a ratio of 5% in a 5mL 48 well culture plate (Huayang Chengxun Cor. Beijing China). After 1.6h, OD600 of every well reached about 0.6. Each well was also supplemented with 720uL fresh LB medium. Isopropylβ-D-1-Thiogalactopyranoside (IPTG) and tryptophan stock solution was also added into each well to the final concentration of 0.4mM and a gradient of 0.4, 0.5, 0.6, 0.8, 1, 2, 3mM, respectively. After 21h, the galactosidase activity was measured as described below.

Firstly, OD600 of the final culture was measured. Then mix 400uL culture with 2.8mL Z buffer (Na2HPO4.7H2O 16.1g/L, NaH2PO4.H2O 5.5g/L, KCl 0.75g/L, MgSO4.7H2O 0.25g/L, 2-thiol ethanol 2.7ml/L, adjusted pH to 7.0). Nextly, mix 2mL mixture with 250uL 1mg/mL MUG DMSO solution to react for 15min. Finally, 300uL 1M NaOH solution was added to quench the reaction and the fluorescence was measured with excitation and emission wavelength at 360 and 445 nm, respectively. The fluorescence intensity was normalized by culture OD600 and reaction time 15min as the activity of galactosidase (MUG units=F360/445/OD600/t). The results of the test were shown in Figure. 4.

Figure. 4 measurement of novel translating ribosome based tryptophan sensor by galactosidase activity

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