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C. imager
June
1.With the help of Mr. Wanshun Ma and Qiong Guo from Beijing Institute of Nanoenergy and Nanosystems, Chinese Academy of Science(CAS), we got Caulobacter crescentus CB15 from ATCC.
2.We got E. coli S17-1 from Professor Tao Zhu in University of Science and Technology of Tianjin and also as key scientist of Cansino, which would be utilized for conjugation with C. crescentus.
3.Experiment Practice.
July 30th 1. Preparation of competent bacteria S17-1 and using it, red fluorescent protein (mRFP) was successfully transformed.
July 2nd 1. Start construction of the first shuttle plasmid for conjugation OriT-RFP.
July 4th With bacterial colony PCR, we were attempting to get DgrA and DgrB, which are indispensable for flagellum synthesis and fortunately, DgrA band appeared.
July 5th Again using bacterial colony PCR, we got DgrB band.
July 22nd Construct OriT-RFP plasmid using 3A assembly methods.
July 26th Successfully constructed Orit-RFP plasmid and made a conjugation using it. Though it seemed worked, results from electrophoresis were not exactly correct. The reason was unknown.
August 6th Trying to guide the plasmid into* C. crescentus* by electrotransformation, including the preparation of competent cell and electrotransformation procedure. This method was tried 6 times in August, but it failed again and again. Finally, we had no alternative but to try on conjugation again.
August 8th Redesign the primer used for standardization of DgrA, DgrB and HfiA which controls the biosynthesis of holdfast.
August 9th Add promoter on DgrA, DgrB and HfiA.
August 21st Failing to get the correct sequence of R0051-DgrA, we all ascribe to promoter in Parts.
August 24th Ligate DgrA with K592020.
September 25th Construction of R0082-HfiA, OriT-K592020 and OriT-K592016 have been accomplished. September 26th Alter the carrier and standardization of DgrA, DgrB and HfiA.
September Conjugate OriT-K592020 into C. crescentus
E.coli Photography
June: We identified gene source of light sensing and imaging system and sent list to Addgene through Beijing Zhongyuan LTD.
July: Getting familiar with protocols and procedures for experiment. We used J04450, PJT119b and K592020 plasmid to produce mRFP, sfGFP and amilCP respectively. The combination between color was quite beautiful.
July 15th: pDwan(containing FixJ, YF1 necessary for blue light sensing) and PJT119b( containing cCas, cCar and sfGFP)
End of July: Construction of P-ho1-pcyA-Cph8 circuit and Cph8 and PCB and PompC –lamdaCI-T-Plamda-mRFP downstream respond circuit. The circuit will feel red light and produce mRFP. Construction of P-YF1-FixJ-T-PFixk2-LamdacI-Plamda-amilCP which can feel blue light and express amilCP.
August: Cotransformation of pDawn and K592020. We finally get the bacteria culture sensing blue light and producing amilCP. Cotransformation of P-ho1-pcyA-Cph8 and PJT119b so that bacteria are able to express sfGFP controlled by green light.
Middle of August: Test on blue light sensing bacteria. Pattern based on projector and bacteria went well as we wished.
RNA Logic Gates and Recombinase
July: Start to synthesis 11 riboswitch sequences and we successfully get all of them by the end of July.
July 20th: We set about combining riboswitch-t with plasmid pSB1A2 which contains GFP coding sequence.
July 27th: We attempt to test the fluorescence intensity but it seems not work.
August 7th: We start to construct gate-yes, gate-no.
August 10th: We start to construct gate-or, gate-and.
August 14th: We test the gate-yes and gate-no, but there is no obvious GFP expressed.
August 25th: We test the gate-or and gate-and, but there is no obvious GFP expressed
September: We complete our parts’ standardization.
Kill Switch
July 28th: Verify the lac operon regulatory situation. Leak expression was found downstream of the lac operon blue pigment protein in E. coli in the absence of IPTG induction conditions.
July 29th: Start construction of Lacl-plac plasmid, it is desirable to suppress the leakage of the expression of downstream genes plac Lacl by increasing the concentration of protein.
August 1st:We are constantly extracting plasmid containing Lac gene. We tried to extract five kinds of plasmids, spent a lot of time, and finally BBa-P0412 plasmid extraction success.
August 2nd-August 15th:We conducted plasmid enzyme digestion, trying to ligate RBS-Lacl-Terminator plasmid (BBa-P0412) with plasmid (K541505) containing LALF gene using the 3A France, conventional method,. However, after several times tests , it appeared several false positives.
September: Rearranged the experimental data and found that genes playing a role in suicide LALF need assistance with the signal peptide. We asked company to synthesize plac-RBS- signal peptide.
October 1st: Extract K737051 plasmid for promoter-RBS-lacl-terminator2-plac-RBS-GFP-teminator2, accounting 2298bp.
October 10th: The bacteria containing the plac-RBS- signal peptide were synthesized by company and put in IPTG concentration gradient panel coated plate, with the increase of bacteria found in IPTG concentration quickly disappear, indicating that the expression of the IPTG-induced E. coli signal peptide, which cuts down the prosperity of E. coli, being able to induce the death of bacteria by IPTG.
