Team:HZAU-China/Results
Results
The function of dCas9 and gRNA
The function of dCas9 and gRNA We first construct the gRNA targeting at OriC and test if it could work as we expected.
Through OD measurement we observed an inhibition of growth when dCas9 and gRNA are all expressed.
For further description of this synchronization method, we quantifying the number of oriC versus terC by using qPCR.
The results confirm that the initiation of chromosome replication is inhibited.
The flow cytometric is also used to conduct the run-out experiment to verify the synchronization.
According to our results, we can conclude that the dCas9 and gRNA(oriC) can be used to synchronize the cell cycle
of
E.coli
The light induced CRISPR/dCas9 system at transcription level
An optimized CcaS-CcaR system is constructed under the design of our advisor, the performance of optimized system
is as following.
We next insert the sequence of gRNA(OirC) after the Pcpcg2, and measure the OD performance under the green light
or red light separately.
The result shows a difference in growth rate under green light and red light. Similarly, next we performed the qPCR
to further demonstrate the synchronization.
At last, we test if this system can make reverse regulate, which is essential for the concept of computer control, please view DEMONSTRATION. The light induced CRISPR/dCas9 system at protein level After getting DNA from IDT, we first constructed and tested the function of pMag and nMag through Luciferase assay. The result verifies the function of pMag and nMag. We next test the expression of split dCas9 to check if it is hydrolysis by SDS-PAGE.
After inserting it into pET plasmid, the results turnout that protein is not hydrolysis.
The last reason we doubt is that if it is because the split method that makes the split dCas9 can’t complement.
We replaced the pMag and nMag with a more reliable chemical induced dimerization protein, FKBP & FRB. The result
shows that no inhibition of growth is observed after adding rapamycin, which induces the dimerization of FKBP
and FRB.
In summary, in this project we achieved controlling cell in the level of DNA replication, proved the feasibility
of using CRISPR to regulate cell cycle, and further regulate it with light. A reverse regulation of our system
is demonstrated by OD measurement and qPCR. At the same time, a corresponding software and hardware are developed
to achieve the computer-controlled cell cycle. The software is used to predict the internal replication procession
and control the hardware to regulate cell by light. For more information, please view
Demonstrate
NotesWhen using the CcaS-CcaR system please note that the atc concentration is different from the former concentration
200ng/ml. After transformation of two plasmids related to CcaS-CcaR system into bacteria, it becomes more
sensitive to aTc. A final concentration of 200ng/ml will be toxic to the bacteria, and a working concentration
as 0.1~1ng/ml is recommended.
In a light-control system, the exist of light sensing molecule is very important, but it is also very neglectable.
The aTc may lose its inducing function if added into a relatively high concentration culture (OD~0.6) when you
use CcaS-CcaR system.
Future plan
The future plan including two aspects. One is further developing the system. First we plan to replace the inducible
promoter with a constitutive promoter J23115. We plan to developing CcaS-CcaR CRISPRi system by knocking it into
genome to make it more significant. At the same time, we hope that we can make the pMag-nMag system work because
this system is smaller and less effect on bacterial metabolism. The other aspect is using this system for several
certain applications, like characterize the stability of genome gene expression after synchronizer, and so on.
We plan to knock the sfGFP into the genome and measure the fluorescence per cell to determine the noise between
each cell and characterize whether synchronization can make the gene on genome more stable.
For further description of this synchronization method, we quantifying the number of oriC versus terC by using qPCR.
The results confirm that the initiation of chromosome replication is inhibited.
The flow cytometric is also used to conduct the run-out experiment to verify the synchronization.
We next insert the sequence of gRNA(OirC) after the Pcpcg2, and measure the OD performance under the green light
or red light separately.
The result shows a difference in growth rate under green light and red light. Similarly, next we performed the qPCR
to further demonstrate the synchronization.
At last, we test if this system can make reverse regulate, which is essential for the concept of computer control, please view DEMONSTRATION. The light induced CRISPR/dCas9 system at protein level After getting DNA from IDT, we first constructed and tested the function of pMag and nMag through Luciferase assay. The result verifies the function of pMag and nMag. We next test the expression of split dCas9 to check if it is hydrolysis by SDS-PAGE.
After inserting it into pET plasmid, the results turnout that protein is not hydrolysis.
The last reason we doubt is that if it is because the split method that makes the split dCas9 can’t complement.
We replaced the pMag and nMag with a more reliable chemical induced dimerization protein, FKBP & FRB. The result
shows that no inhibition of growth is observed after adding rapamycin, which induces the dimerization of FKBP
and FRB.
In summary, in this project we achieved controlling cell in the level of DNA replication, proved the feasibility
of using CRISPR to regulate cell cycle, and further regulate it with light. A reverse regulation of our system
is demonstrated by OD measurement and qPCR. At the same time, a corresponding software and hardware are developed
to achieve the computer-controlled cell cycle. The software is used to predict the internal replication procession
and control the hardware to regulate cell by light. For more information, please view
DemonstrateNotes
