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Revision as of 14:40, 16 October 2018

Notebook

Protocol

Carotene Extraction Protocol.pdf
Carotene Identify Protocol.pdf
Carotene Production Protocol.pdf
Molecular Cloning Plasmid extraction DNA electrophoresis DNA clean.pdf
Molecular Cloning Protocol DNA recombanation.pdf
Cloning Protocol E.coli transformation.pdf
Molecular Cloning Protocol PCR.pdf
Yeast Transformation Protocol.pdf

Diary
0

2018.5.13

Confirmed project plan.
Prepared experiment equipment and reagents.

1

2018.5.14-2018.5.20

Molecular cloning test.
Yeast transformation test.

2

2018.5.21-2018.5.27

Modeling beginning.

3

2018.5.28-2018.6.3

Plasmid backbone propagation：
E. coli expression: pET-28a
yeast expression: pNH603, pNH604, pNH605, pNH606
Required gene segment synthesis beginning.

4

2018.6.4-2018.6.10

Gibson assembly, golden gate assembly, and other seamless cloning test.
IPTG induced expression test.

5

2018.6.11-2018.6.17

Restriction recombination test.
Began to construct SUMO-SIM system.

6

2018.6.18-2018.6.24

Construction E. coli expression plasmid：
pET-28a-FKBP-yEGFP-HOTag3
pET-28a-Frb-yEGFP-HOTag6

7

2018.6.25-2018.7.1

Fluorescence observation test.

8

2018.7.2-2018.7.8

Joined iGEM conference at Nankai University held by NKU_CHINA.

9

2018.7.9-2018.7.15

Construction yeast transformation plasmid of carotene synthesis:
pNH603-crtE/crtI/crtYB
pNH604-crtE/crtI/crtYB
pNH605-crtE/crtI/crtYB
pNH606-crtE/crtI/crtYB

10

2018.7.16-7.22

Completed Interlab tasks.
Meeting up with Fudan-CHINA.
Insert 4 kinds of promoters PDH3, TEF1, Ura3, Tet07 into 4 kinds of yeast transformation plasmids pNH603, pNH604, pNH605, pNH606.

11

2018.7.23-2018.7.29

Construction yeast transformation plasmids of SPOT:
pNH604-PDH3/TEF1/Ura3/Tet07-FKBP-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-yEGFP-HOTag6
pNH604-PDH3/TEF1/Ura3/Tet07-SUMO-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-SIM-yEGFP-HOTag6

12

2018.7.30-2018.8.5

Transferred FKBP-Frb SPOT system and SUMO-SIM SPOT system into yeast.
Nucleic localization test.
Fluorescence microscope observation and flow cytometry measurement of transformed yeast cell lines.

13

2018.8.6-2018.8.12

Rapamycin induction concentration test of FKBP-Frb SPOT system.

Construction yeast transformation plasmids of carotene production:
pNH603-PDH3/TEF1/Ura3/Tet07-Frb-crtI-HOTag6
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-crtE-HOTag6
pNH606-PDH3/TEF1/Ura3/Tet07-Frb-crtYB-HOTag6

Construction yeast transformation plasmids of light induced SPOT system:

pNH604-PDH3/TEF1/Ura3/Tet07-phyB-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-PIF-yEGFP-HOTag6
pNH604-PDH3/TEF1/Ura3/Tet07-VVDn-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-VVDp-yEGFP-HOTag6

14

2018.8.13-2018.8.19

Carotene production and extraction test.

Construction yeast transformation plasmids of carotene production with SPOT:
pNH603--PDH3/TEF1/Ura3/Tet07-Frb-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-Frb-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-Frb-crtYB-HOTag6
pNH603--PDH3/TEF1/Ura3/Tet07-SIM-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-SIM-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-SIM-crtYB-HOTag6

15

2018.8.20-2018.8.26

Carotene production and extraction test.

Construction yeast transformation plasmids of carotene production with SPOT:
pNH603--PDH3/TEF1/Ura3/Tet07-Frb-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-Frb-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-Frb-crtYB-HOTag6
pNH603--PDH3/TEF1/Ura3/Tet07-SIM-crtI-HOTag6
pNH605--PDH3/TEF1/Ura3/Tet07-SIM-crtE-HOTag6
pNH606--PDH3/TEF1/Ura3/Tet07-SIM-crtYB-HOTag6

16

2018.8.27-2018.9.2

Carotene production and extraction test.

Construction the control group plasmids of SPOT:

pNH604-PDH3/TEF1/Ura3/Tet07-FKBP-yEGFP
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-yEGFP
pNH604-PDH3/TEF1/Ura3/Tet07-SUMO-yEGFP
pNH605-PDH3/TEF1/Ura3/Tet07-SIM-yEGFP
pNH604-PDH3/TEF1/Ura3/Tet07-FKBP-mCherry
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-mCherry
pNH604-PDH3/TEF1/Ura3/Tet07-SUMO-mCherry
pNH605-PDH3/TEF1/Ura3/Tet07-SIM-mCherry
pNH603-H2A-CFP

Joined the 5th CCiC at ShanghaiTech.

17

2018.9.3-2018.9.9

Growth curve measurement of the SPOT system yeast.
Characterization of the carotene production by SPOT system.

18

2018.9.10-2018.9.16

The ability of phase separation induced by rapamycin in FKBP-Frb SPOT system test and longer time observation.
Yeast fermentation test.

19

2018.9.17-2018.9.23

Completed the two kinds of fluorescence repeat in both FKBP-Frb and SUMO-SIM SPOT system.
Carotene production and quantification test.

Construction yeast transformation plasmids of ABA induced SPOT system:
pNH604-PDH3/TEF1/Ura3/Tet07-ABI-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-PYL1-yEGFP-HOTag6

Construction yeast transformation plasmids of GA induced SPOT system:
pNH604-PDH3/TEF1/Ura3/Tet07-GAI-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-GID-yEGFP-HOTag6

20

2018.9.24-2018.9.30

FRAP test of FKBP-Frb SPOT system.
BioBrick submitted bioparts construction.

21

2018.10.1-2018.10.7

Construction yeast transformation plasmids of GFP nanobody SPOT system:
pNH605-PDH3/TEF1/Ura3/Tet07-Frb-GFPnanobody-HOTag3

Construction yeast transformation plasmids of NAD sensor SPOT system:
pNH604-PDH3/TEF1/Ura3/Tet07-NAD1A-yEGFP-HOTag3
pNH605-PDH3/TEF1/Ura3/Tet07-NAD1B-yEGFP-HOTag6

22

2018.10.8-2018.10.14

Data collection and modeling processing.
HPLC quantification of carotene.

23

2018.10.15-17

WIKI and poster making.
Presentation preparation.