Team:Nanjing High School/Results

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Result

Summary of Achievements in Our Experiments:

  1. We have succeeded in inserting the gene we want into plasmid.
  2. We have transformed the plasmid to DH5α cells.
  3. We have extracted plasmid from the DH5α cells and transformed these plasmid into BL21-AI cells for protein expression.
  4. Our experiment with blue light show the result that we expected.

Construct the cells that are needed for the blue light

Goal:

Cell ①: BL21AI cells with pCDF-T7-Cas1Cas2
Cell ②: BL21AI cells with pSB1C3-fixk2-Cas1Cas2 and pCDF-T7-Blue Sensor
Cell ③: BL21AI cells with pET28a-Fixk2-TfusCas3, pCDF-T7-Cas1Cas2, and pACYC-J23100-Blue Sensor

Constructing the Cells:

Plasmids that available at the beginning:

  1. Plasmids with Blue Sensor gene on it (from Shanghai Jiao Tong University）
  2. Plasmids with fixk2 gene on it (from Shanghai Jiao Tong University)
  3. MG1655 genome of Escherichia coli
  4. Plasmids with Thermobifida fusca Cas3 on it (from Cornell University)

Polymerase chain reaction (PCR) is a method widely used in molecular biology to copy certain DNA segments. We used a system of total 25 μl for PCR to get the DNA segments that we wanted.
Firstly, we successfully constructed the plasmids we need. Our primary gaol was to construct FixK2-Cas1Cas2, pCDF-T7-BlueSensor, pET28a-FixK2-TfusCas3, and pACYC-J23100-BlueSensor. Here is our constructing results.


Homologous Recombination

The linear gene and vector segments copied by PCR were combined and formed integrated plasmids.
The plasmids that we got from this process:
pSB1C3-fixk2-Cas1Cas2



pCDF-T7-Blue Sensor



pCDF-T7-Cas1Cas2


pET28a-fixk2-TfusCas3



pACYC-J23100-Blue Sensor


Bacterial Transformation

As long as we got all the plasmids we need, they are transformed into DH5α cells from plasmid duplication. Then, the plasmids are extracted from the DH5α cells and transformed into BL21 AI cells for DNA expression.

Light Experiment:

The cells that we used in the light experiment were:
① T1:
pCDF-T7-Cas1Cas2

② F1:
pSB1C3-Fixk2-Cas1Cas2
pCDF-T7-Blue Sensor

③ F3:
pET28a-Fixk2-TfusCas3
pCDF-T7-Cas1Cas2
pACYC-J23100-Blue Sensor

T1 was the control group which was not affected by blue light.
F1 was growing without the presence of blue light. It was supposed to have DNA lengthening.
DF1 was growing with the presence of blue light. It was supposed to have little or no DNA lengthening.
F3 was growing without the presence of blue light. It was supposed to have little or no DNA lengthening.
DF3 It was supposed to have little or no DNA lengthening. It was supposed to have DNA lengthening.
(See Design for details about how they function, click Link)

Result:
During our project, we have designed a new part to record the time E.coli is exposed to blue light. Cas1-Cas2 is a special protein that can catch fragments. In our experiment, we transferred the plasmid with this protein to the micro-organism so that when the micro-organism is placed under blue light, the promoter will function and the protein will then begin to catch fragments. After a certain amount of time, gel analysis we did to examine the result showed that samples under blue light had a shorter strip, which meant Cas1-Cas2 effectively caught and stored the fragments.



Significant length growth in T1 cells are found.
No length growth in DF1 and F1 cells.
No length growth in DF3 cells. Significant length growth in F3 cells.

Discussion:

① Significant length growth in T1 cells indicated that our CRISPR system work well.
② As more experiments were done, F3 cells, with or without blue light presence, never had length growth.   We considered that original F3 cells had already been contaminated.
③ The experiment result on F3 were completely opposite of what we expected.

  1. Promoter efficiency might be poor.
  2. The wavelength of the blue light that were bought online were not tested, so there might be problems exist with the light.
  3. The stability of plasmids were not good. There might be situations where the cells that were used for light experiment lost their plasmids over time.
  4. The rate of protein degradation in cells had not been measured, so the measurement of DNA capture efficiencymight be affected.

 

Another experiment result







Future Design:

Repeat the experiment and wait for the result.