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Revision as of 14:37, 16 October 2018

Notebook

Protocol

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.