Team:ZJU-China/Log

Contents 1 GENESOCKET DATA AND STRATEGY! 2 2013.11 3 2014.3 4 2014.3-6 5 2014.7 6 2014.8 7 2014.9 8 2014.10

GENESOCKET DATA AND STRATEGY!

GeneSocket uses a plasmid, a circuit and a strain to construct circuits on chromosome by lambda-red and ecombinase-derived bistable switch. BBa_K1433018, which is called SOCKET, is a circuit need to be recombined on chromosome by a helper plasmid BBa_K1433013, called SUPPORT DEVICE.!

TO achieve our project, we designed 8 exquisite circuits, 3 on plasmid and 5 on chromosome. See our Circuit Strategy.pdf!

TO construct this circuit, we use GBclonart Seamless Assembly Kit.

TO prepare segments which the kit required and to test circuit accuracy, we designed hundreds of primers. See our Primer.pdf!

TO use these segments which have prepared by using the primers through assembly kit , we did lots of PCR. See our PCR Strategy.pdf!

TO get some segments which cannot find in The 2014 DNA Distribution Kit, we designed 4 fragments and acquire them by de novo synthesis. See our Total Synthesis Strategy.pdf!

TO assembly these segments and construct our circuits by assembly kit, we did lots of work. See our Assembly Kit.pdf!

TO verify the accuracy of constructed circuits, we choose colony PCR. See our Colony PCR.pdf!

TO recombine these circuit on chromosome, we use lambda red homologous recombination in many different strains. See our lambda red.pdf and Strain.pdf.

TO deliver our circuit as parts, we use PCR, restriction enzymes or DNA ligases. See our Parts Strategy.pdf!

WHAT’S more, we helped team Peking construct a circuit and do some testing experiments. See our PKU primer.pdf!

WE have quite minute and clearly lab record every day during whole project period. See our Lab record demo.pdf and

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2013.11

Members of ZJU-China 2013 organized a teach-ins and we learnt iGEM competition and synthetic biology.

2014.3

We formed ZJU-China 2014 team with the help of previous team members.And we voted to choose a team leader.After that we started brain storm.

2014.3-6

We thought out multifarious idea and we took long time to do idea selection.In mid May,two of us went to Peking University and had a meets up. We primarily chose magnetosome selection as our project.

2014.7

After a lot of thinking, we determined to change the track of our project and named it GeneSocket!

Read papers about data and protocol,Then got down to Devise practical experiments.

2014.8

• We are divided into two groups: group PCR & group lambda-red.
• We prepared competent Escherichia coli cells successfully.
• We determined detailed protocol of lambda red.
• We amplified parts successfully,using the primers designed ourselves.
• We explored growth curves ofE.coli in different environments.
• We extracted and transformed plasmids of PKD46.
• We did blue/white plaques selection to screen homologous recombination. Validate transform result by PCR.
• We joined the 2014 iGEM Conference organized by NCTU in Taiwan.
• We communicated with XMU, SJTU-BioX-Shanghai, Fudan.
• We chose BL21 as our strain to do homologous recombination.

2014.9

• We prepared fragments to construct circuit.
• We change method of Lambda red recombination test.
• We did function check test by Fluorescence microscope and resistance.
• We refined protocol of recombineering-proficient cells preparation.
• We determined the best concentration of Ampicillin, Kanamycin, Tetracycline and Chloramphenicol through gradient analysis experiments.
• We changed our strain from BL21 to DH5alpha to do homologous recombination.

2014.10

• Reverse turn test succeed one by one.
• We described and submit parts used in our project.
• We refined our protocols of recombineering-proficient cells preparation to the final version.
• We made homologous recombination happen!
• We constructed a series of GeneSockets on plasmids.
• We did a series of experiments to check the function of GeneSocket ( on plasmids) step by step.
• We constructed our GeneSocket on chromosome!
• We changed our strain from DH5alpha to BW25113 and DH10beta.
• We determined DH10beta as our strain to construct our GeneSocket.

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