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
Confirmed project plan.
Prepared experiment equipment and reagents.
Molecular cloning test.
Yeast transformation test.
Modeling beginning.
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.
2018.6.4-2018.6.10
Gibson assembly, golden gate assembly, and other seamless cloning test.
IPTG induced expression test.
2018.6.11-2018.6.17
Restriction recombination test.
Began to construct SUMO-SIM system.
2018.6.18-2018.6.24
Construction E. coli expression plasmid：
pET-28a-FKBP-yEGFP-HOTag3
pET-28a-Frb-yEGFP-HOTag6
2018.6.25-2018.7.1
Fluorescence observation test.
2018.7.2-2018.7.8
Joined iGEM conference at Nankai University held by NKU_CHINA.
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
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.
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
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.
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
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
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
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.
2018.9.3-2018.9.9
Growth curve measurement of the SPOT system yeast.
Characterization of the carotene production by SPOT system.
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.
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
2018.9.24-2018.9.30
FRAP test of FKBP-Frb SPOT system.
BioBrick submitted bioparts construction.
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
Restriction recombination test.
Began to construct SUMO-SIM system.
As the model predicts, the concentration of components and the interaction strength affect the kinetics of phase separation. First we controlled the expression levels of components by using several stable or inducible promoters and observe the system's behavior. We found that the formation of organelles happened in specific promoter combinations and can be controlled by inducible promoters. The analysis result does not only fit well with the simulation, but provides potential methods to control the organelles in applications.
Figure2 (a) Phase diagram of a phase separation system with three components(simulation). To fit our system, the x-axis and the y-axis stands for the two components in the granules. The asymmetry comes from the assumption that the two components have different interactions with water. (b) Fluorescence movies of different promoter combinations of FKBP-Frb mediated system after adding rapamycin. Only in specific combinations, synthetic organelles can be formed by phase separation. (c) The formation process of SUMO-SIM mediated synthetic organelles can be controlled by inducible promoters. While the expression of Tet07-SIM-mCherry-HoTag6 is induced by dox gradually, the granules will occur abruptly in some time.
The strength of interaction modules can be also controlled. In the rapamycin-induced organelle system, changing the concentration of rapamycin will affect the apparent value of K, a parameter reflecting the interaction strength in our model. In a gradient rapamycin-inducing experiment, the delay time from adding inducer to granules formation was found to be shorter when concentration of rapamycin increases. So we have confirmed the influence of two parameters in models and increased the flexibility of our synthetic organelles.
Figure3 (a) A simulation of organelle formation process in different interaction strength of components. (b) The speed of FKBP-Frb mediated organelle formation increases with the increasing concentration of rapamycin.
Since SPOT can form in the cell and be controlled, we go further to consider the functions of SPOT. The functions of SPOT can be descripted in three catalogs: Spatial segmentation, Sensor and metabolic regulation. We verified the spatial segmentation with the condensation of substrates, also we can load the protein we want by fusing it with nanobody. We then verified the sensor with detecting rapamycin and ABA, which shows strong relativity between the concentration and the proportion of yeasts with SPOT. To find the law behind metabolism in the SPOT, we fuse the enzymes that can produce β-carotene into SPOT and measure the difference between with or without SPOT in produce of β-carotene.
SPOT has been well verified and has various functions. And in the future, this modular system will have great potential in science and practice using. SPOT can change the modules to gain more different properties like diverse inducing method, we can also use it as a platform and then load other protein with some interactions like the interaction between nanobody and GFP. What’s more, we might have the ability to form differernt SPOTs in the cell and regulate them respectively. The functions of SPOT can also diverse. We can build a real time sensor for molecule in living cells to monitoring the concentration changing in environment or in cells. More metabolism pathway can be test in SPOT and we will find some laws of the function of regulate the metabolism. To be summary, more achievement is coming true with SPOT.