iGEM 2017 — Team HZAU-China

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# Team:HZAU-China/Notebook

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* [Home](https://2017.igem.org/Team%3AHZAU-China)
* Project
  + [Description](https://2017.igem.org/Team%3AHZAU-China/Description)
  + [Design](https://2017.igem.org/Team%3AHZAU-China/Design)
  + [Results](https://2017.igem.org/Team%3AHZAU-China/Results)
  + [Demonstrate](https://2017.igem.org/Team%3AHZAU-China/Demonstrate)
* Wetlab
  + [Experiments](https://2017.igem.org/Team%3AHZAU-China/Experiments)
  + [Improve](https://2017.igem.org/Team%3AHZAU-China/Improve)
  + [InterLab](https://2017.igem.org/Team%3AHZAU-China/InterLab)
* Drylab
  + [Model](https://2017.igem.org/Team%3AHZAU-China/Model)
  + [Software](https://2017.igem.org/Team%3AHZAU-China/Software)
  + [Hardware](https://2017.igem.org/Team%3AHZAU-China/Hardware)
* HP
  + [Silver HP](https://2017.igem.org/Team%3AHZAU-China/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3AHZAU-China/HP/Gold_Integrated)
* Team
  + [Members](https://2017.igem.org/Team%3AHZAU-China/Team)
  + [Collaborations](https://2017.igem.org/Team%3AHZAU-China/Collaborations)
  + [Attributions](https://2017.igem.org/Team%3AHZAU-China/Attributions)
* [Safety](https://2017.igem.org/Team%3AHZAU-China/Safety)
* [Parts](https://2017.igem.org/Team%3AHZAU-China/Parts)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

##### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

##### Inspiration

You can see what others teams have done to organize their notes:

* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)

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# Team:HZAU-China/Experiments

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* [Home](https://2017.igem.org/Team%3AHZAU-China)
* Project
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  + [Design](https://2017.igem.org/Team%3AHZAU-China/Design)
  + [Results](https://2017.igem.org/Team%3AHZAU-China/Results)
  + [Demonstrate](https://2017.igem.org/Team%3AHZAU-China/Demonstrate)
* Wetlab
  + [Experiments](https://2017.igem.org/Team%3AHZAU-China/Experiments)
  + [Improve](https://2017.igem.org/Team%3AHZAU-China/Improve)
  + [InterLab](https://2017.igem.org/Team%3AHZAU-China/InterLab)
* Drylab
  + [Model](https://2017.igem.org/Team%3AHZAU-China/Model)
  + [Software](https://2017.igem.org/Team%3AHZAU-China/Software)
  + [Hardware](https://2017.igem.org/Team%3AHZAU-China/Hardware)
* HP
  + [Silver HP](https://2017.igem.org/Team%3AHZAU-China/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3AHZAU-China/HP/Gold_Integrated)
* Team
  + [Members](https://2017.igem.org/Team%3AHZAU-China/Team)
  + [Collaborations](https://2017.igem.org/Team%3AHZAU-China/Collaborations)
  + [Attributions](https://2017.igem.org/Team%3AHZAU-China/Attributions)
* [Safety](https://2017.igem.org/Team%3AHZAU-China/Safety)
* [Parts](https://2017.igem.org/Team%3AHZAU-China/Parts)

Experiment
Materials and methods
Plasmid construction
The fragment was PCR amplified with pfu(company) or PrimeSTAR(Takara) according to product length. Product is recycled with gel extraction kit from qiagene after electrophoresis. Golden gate is conducted with total 10 liter containing 100 ng template, 5 liter buffer, 5U BsaI, and 10 U T4 ligase. With 50 cycles with 37 5min and 16 10min finished with 55 5min and deactivate at 98 for 5 min. Then transformed into trans5a using commercial component cell (xxx). homologous recombination to conducted using vyzyme xxxxx kit and operate following the guidebook.
The OD measurement
Cell are cultured overnight in LB broth containing corresponding antibiotics, and diluted into 1% fresh LB broth. When OD is reaching 0.6 add aTc of final concentration 200 ng/ ml to induce expression of dCas9 and inject it into 24 cell culture plate. Culture the plate in 37 and 150 rpm. Every hour put it into a plate reader to measure its OD.
Quantitative PCR
The qPCR experiment is conducted using qPCR kit from takara(Code No.RR820). The sample is collected every 30min and freeze at liquid nitrogen after centrifugal. The genome is for qPCR is extracted using Code No.9763(takara).
Flow cytometry
Bacteria shall be cultured in LB until OD is between 0.6 and 1.0 (37℃, 200r/min). Dilute the culture medium with LB to approximately 108 cells mL-1. Add rifampicin (rif) and cephalexin (ceph) to the final concentration is 200μg/mL and 12μg/mL. Culture in the previous condition for 4 hours and the bacteria can be then used for flow cytometry.
All culture samples were centrifuged at 16000g for 1 min and resuspended in ice-cold ethanol to a final concentration of 70%. Samples in 70% ethanol can be stored at 4℃ until they were prepared for flow cytometry. Cells were pelleted by centrifugation at 16000g for 1min at 4℃. Wash in 10mM Tris (pH=8)/ 10mM MgCl2 (buffer A) as before and resuspend in buffer A. Take 0.5μL of the sample. Add it into 10μL DAPI(1μg/mL) diluted with buffer A and dye for 5min in dark. Dilute the sample to 1mL with buffer A and it can be used for flow cytometry analysis (Becton-Dickinson).
The Agarose Pad Assay
To acquire the growth rate of bacteria for establishing the growth function, an agarose pad assay is conducted. Mix the 0.15g agarose and 10ml LB to reach the concentration of 1.5%,then heat it and add needed matter after cooling slightly.The melted agarose is supposed to be pipetted onto a cover glass(The volume is about 2.5ml.).Then put another clean cover glass on it to get a smooth flat surface after about 3 minutes. The next step is to pipette about 2.5 μL E.coli solution of 0.02 OD and get a new cover glass on it till the solution dries.It’s worth noting that maintain the favorable environment is vital.We used parafilm to avoid the evaporation and tinfoil to keep out the light when observed dCas9 4-1 which was induced by aTc. Observe the agarose pad under microscope to find the optimum bacteria, for instance, which are under the fission.The final thing you do to take and save picture 10 minutes per in 8 to 10 hours.
Microfluidics
To better observe the growth of E.coli, we adopted 3D-Microfluidics tech to take real-time observation on monolayer bacteria, which is more visualized and convenient to understand the division of a single cell.

 Our steps of experiment are below：

1. Growing spdCas9/gRNA OriC3 E.coli cultures in LB(100ng/ml ampicillin and 70ng/ml Chloramphenicol) overnight(200rpm, 37℃).

2. Take 1ml bacteria culture and add it into 100ml Erlenmeyer flask which contains 100ml LB（100ng/ml ampicillin and 70ng/ml Chloramphenicol）and cultivate for 2~3h（200rpm, 37℃). When OD get near to 0.6, concentrate 100ml bacteria culture to 5ml.

3. Use 2.5ml sterile syringe to collect 2.5ml concentrated, 5ml sterile syringe for LB(ampicillin and Chloramphenicol) and 5ml LB(ampicillin and Chloramphenicol) containing 200ng/ml aTc.

4. Injecting the bacteria culture through the tiny tube into the microfluidic chip. E.coli flow into the chamber and grow in there.

5. Firstly, inject LB without aTc at the speed of 200ul/h, observe by inverted microscope for 10h. Then inject LB with 200ng/ml aTc at the speed of 200ul/h, observe by inverted microscope for 10h.

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# Team:HZAU-China/Results

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* [Home](https://2017.igem.org/Team%3AHZAU-China)
* Project
  + [Description](https://2017.igem.org/Team%3AHZAU-China/Description)
  + [Design](https://2017.igem.org/Team%3AHZAU-China/Design)
  + [Results](https://2017.igem.org/Team%3AHZAU-China/Results)
  + [Demonstrate](https://2017.igem.org/Team%3AHZAU-China/Demonstrate)
* Wetlab
  + [Experiments](https://2017.igem.org/Team%3AHZAU-China/Experiments)
  + [Improve](https://2017.igem.org/Team%3AHZAU-China/Improve)
  + [InterLab](https://2017.igem.org/Team%3AHZAU-China/InterLab)
* Drylab
  + [Model](https://2017.igem.org/Team%3AHZAU-China/Model)
  + [Software](https://2017.igem.org/Team%3AHZAU-China/Software)
  + [Hardware](https://2017.igem.org/Team%3AHZAU-China/Hardware)
* HP
  + [Silver HP](https://2017.igem.org/Team%3AHZAU-China/HP/Silver)
  + [Integrated and Gold](https://2017.igem.org/Team%3AHZAU-China/HP/Gold_Integrated)
* Team
  + [Members](https://2017.igem.org/Team%3AHZAU-China/Team)
  + [Collaborations](https://2017.igem.org/Team%3AHZAU-China/Collaborations)
  + [Attributions](https://2017.igem.org/Team%3AHZAU-China/Attributions)
* [Safety](https://2017.igem.org/Team%3AHZAU-China/Safety)
* [Parts](https://2017.igem.org/Team%3AHZAU-China/Parts)

Results
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.
![](https://static.igem.org/mediawiki/2017/9/97/T--HZAU-China--Results_Figure1.png)
Figure 1. In this figure, several controls are conducted to demonstrate that it is the function of CRISPR that affect
the growth of culture not the function of gOriC nor the toxicity of dCas9. The purple line represents the experimental
group while the others represent different controls.
For further description of this synchronization method, we quantifying the number of
*oriC*, the origin of chromosome replication, versus
*terC*, the terimination of chromosome replication, by using qPCR. The results confirm that the initiation
of chromosome replication is inhibited.
![](https://static.igem.org/mediawiki/2017/3/31/T--HZAU-China--Results_Figure2.png)
Figure 2. In this figure, the blue column represents the ratio of
*oriC* to
*terC* of cells growing in common condition. The red column represents that of cells suppressed by dCas9-oriC
for 1 hour and the green column for 2 hours. The purple column shows the ratio of oriC to terC of cells treated
with Rifampicin, a widly used chemical agent to block chromosome replication initiation. The results shows that
the efficiency of dCas9-oriC system is relatively comparable to this widly used agent.
Flow cytometry is also used to conduct the run-out experiment to verify the synchronization.
![](https://static.igem.org/mediawiki/2017/a/a0/T--HZAU-China--Results_Figure3.png)
Figure 3. In this figure, different peaks represent different oriC numbers. The untreated sample have 4 peaks while
after treated with dCas9-oriC system the copy number of the same sample has reduced to 2 peaks, which shows that
the oriC number of each cell is reduced.
According to our results, we can conclude that the dCas9 and gRNA(oriC) can be used to synchronize the cell cycles
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.
![](https://static.igem.org/mediawiki/2017/3/36/T--HZAU-China--improve.png)
We next insert the sequence of gRNA(OirC) after the Pcpcg2, and measure the OD performance under the green light
or red light separately.
![](https://static.igem.org/mediawiki/2017/6/68/T--HZAU-China--Results_Figure5.png)
The result shows a difference in growth rate under green light and red light. But simply repression cannot achieve
a computer-controlled system. We further tested if this regulation is reversible. For more information please
click
[DEMONSTRATION.](https://2017.igem.org/Team%3AHZAU-China/Demonstrate)
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.
![](https://static.igem.org/mediawiki/2017/2/25/T--HZAU-China--magluciferase.png)
The result verifies the function of pMag and nMag. We next tested the expression of split dCas9 to check if it is hydrolysis by SDS-PAGE.
![](https://static.igem.org/mediawiki/2017/4/4a/T--HZAU-China--sdspage1.png)
![](https://static.igem.org/mediawiki/2017/3/3b/T--HZAU-China--F1.png)
![](https://static.igem.org/mediawiki/2017/f/fe/T--HZAU-China--sdspage3.png)
![](https://static.igem.org/mediawiki/2017/6/64/T--HZAU-China--F3.png)
After inserting it into pET plasmid, the results turnout that the 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 (data not shown).
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, 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.](https://2017.igem.org/Team%3AHZAU-China/Demonstrate)

Notes
- When 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 existence of light sensing molecule is very important, but it is also very easy to be neglected.
- 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 includes 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-affecting on bacterial metabolism. The other aspect is using this system for several
certain applications, like characterizing the stability of genome gene expression after synchronization, 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.

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