Team:NPU-China/Notebook

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NoteBook

Chronological List of Progression

Week 1
15/07/2018 - 21/07/2018
Project Handover.
Instruction of lab notices and experimental skills.
Test remaining DNA fragments and re-prepare first-stage fragments with miscellaneous bands and degradation.
Amplification of well preserved second-stage fragments.
Confirm the skeleton and strain for homologous recombination in vivo: Y33-ura and By4741(S.c).
Design a new series of primers especially for recombination in vivo: LL33-X-F/R.

Week 2
22/07/2018 - 28/07/2018
Confirm the skeleton and strain for homologous recombination in vivo: Y33-ura and By4741(S.c).
Design a new series of primers especially for recombination in vivo: LL33-X-F/R.
Prepare the materials used for yeast transformation.
Explore the PCR conditions for fragments with less amplification effect.
Prepare new first-stage fragments with new series of primers: LLX.

Week3
29/07/2018 - 04/08/2018
Prepare new first-stage fragments with new series of primers: LLX.
Yeast transformation but failed.
Find out the cause of failure and make it up.
Prepare phosphorylated fragments(DX) for DATEL.
Try DATEL and obtained much larger DNA fragments in vito.
Determined to use new S.c strain: By4743 for transformation in vivo.

Week 4
05/08/2018 - 11/08/2018
Design a new series of plasmids for yeast transformation verification
Yeast transformation for Plasmid 1 and Plasmid 2 by using By4743. Some groups are verified successfully by
verification primers. But encounter with obstacles during amplification of plasmids with in E.coli.
Try to find out possible cause of failure.
Continue DATEL but encounter with some obstacles.

Week 5
12/08/2018 - 18/08/2018
Re-transformation in yeast for Plasmid 1 and Plasmid 2.
Yeast transformation for Plasmid 3.
Test transformation results.

Week 6
19/08/2018 - 25/08/2018
Determine to use new skeleton stabilizing large geomic fragments: pGF.
Re-construct the whole genome into 3 parts: All(All genomic fragments transformed in once), H1(Front part of genomic
fragments transformed in once), and H2(Latter part of genomic fragments transformed in once).

Week 7
26/08/2018 - 01/09/2018
Design new primers.
Amplification of new fragments used for transformation.
Explore the PCR conditions for new fragments.

Week 8
02/09/2018 - 08/09/2018
Explore the PCR conditions for new fragments.
Continue standardizing parts.

Week 9-10
09/09/2018 - 22/09/2018
Prepare enough fragments for transformation.
Debug failures during standardization.

Week 11
23/09/2018 - 29/09/2018
Transformation in vivo for Plasmid ALL, Plasmid H1, Plasmid H2 but encounter with obstacles.
Debug and find new way to solve them.

Week 12
30/09/2018 - 06/10/2018
Transform again in vivo.
Successfully obtain assembled Plasmid ALL and Plasmid H2 within yeast strain(H1 fail), after verification of verifying primers.

Week 13
07/10/2018 - 13/10/2018
Try to amplify genomic fragments in E.coli( strain EPI300) by electrotransformation but failed.
Successfully amplify fragment H2 (19kb) by PCR.
Debug transformation(Too much anti-biotic), and re-transform successfully by chemical transformation.

Week 14
14/10/2018 - Present
Try to amplify Plasmid ALL by multicopy of pGF skeleton(OriV)

We have compiled an account of everything that has happened in the lab over the summer. We are excited to show you how busy we have been in iGEM 2018! We believe that sharing our work flow will convey the passion and dedication of iGEM students all around the world, as we continuously push our limits to achieve high quality results.

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