Team:ZJUT China B/Wet collaboration
Wet collaboration
There are two main aspects of our lyophilization experiments for adding water Tardigrade intrinsically disordered proteins (TDPs).
Firstly, in order to provide a storable version of the experiment, we lyophilized the reaction mixture used for Cas13 RNA dependent detection, and combined the detection system with the device.
In order to prevent the contamination of RNase, we not only sealed the chip, but also used RNase free reagents. The lyophilized chip only needs to be treated with water, so that various components can be melted and activated and tested.
As the direct lyophilization may affect the activity of cas13 protein, we searched for a lot of data. We found that the tolerance of cas13 protein to dryness could be improved by adding TDPs, a specific disorder protein of water bear. QHFZ-China team sent us parts which could express TDPs to help us prove our lyphilization improvement concept. At the same time, our results could help them prove that the protein will remain active after lyophilization. But due to time pressure, we didn't succeed before deadline.
Secondly, we helped QHFZ-China team to test the effect of lyophilization on bacteria protection. We extend the lyophilization time, and then test its survival rate, so as to better simulate the actual use and make up for the lack of data from QHFZ-China team.
Culture
First, we transferred plasmids 33020, 106094, 330-106 and GFP mailed from the QHFZ into competent Escherichia coli cells. After culture, 50ul was coated or scribed on the plate with Kanamycin, and the following culture media were added to Kanamycin resistance.
Figure 1 Growth of four colonies on plate
Induction
In order to verify whether the plasmid was transformed successfully, we amplified it by PCR. Then we stored some strains that were successfully transformed so as to facilitate repeated experiments.
Figure 2 PCR results of nucleic acid adhesives
Figure 3 bacteria retaining tube (part)
We added the successfully transformed single colony into 500ul LB medium and expanded it into a shaking table for 5-7 hours until it was turbid.
Figure 4 expanded culture (part of test tube）
Then 3ul bacteria solution was placed in 3ml medium for one hour, then added to 30ul IPTG for induction, and spent overnight in the shaking table to induce the expression of protein.
Figure 5 IPTG induction (partial test tube)
Lyophilization drying
Before lyophilization, we should control the number of cells and check whether GFP expresses fluorescence. Therefore, we take out the strain, suck 500ul into EP tube, centrifugate at 12000r / min for 1 minute, discard the supernatant, and irradiate green fluorescence with blue light under dark conditions, that is, GFP has obvious green fluorescence, indicating that the induction is successful, then the experiment can continue.
Figure 6 Centrifugation
Figure 7 GFP can emit fluorescence.
When dealing with the control group experiments, we found that they tried to use the success of GFP expression to conduct parallel experiments to prove the success of population protein expression. However, according to the team's experience last year, iGEM judge said that "indirect experimental results can show some feasibility, but direct experiment is very important". Therefore, the expression of green fluorescent protein can not 100% prove the expression of the target protein, so it is necessary to carry out SDS-PAGE experiment. Therefore, we suggest that they use SDS page to display the expression of TDPs directly.
Next, we measured the concentration of the induced bacteria. We use LB to adjust the zeros, dilute the strain properly, configure the 200ul system, and measure the OD600 value with the enzyme labelling instrument.
Figure 8 OD value detection result
When doing lyophilization, we take 109 cells in the EP tube, according to experience and dilution formula.
Figure 9 freeze-dried products
(the measured OD value after dilution is linear at 0.1-1;According to the empirical data from QHFZ, when the OD value is 1, the number of cells is 109/ml; for example, when the dilution ratio is 10 times, the measured OD=0.2 is OD=2 when the dilution is not diluted, and the number of cells is =2*109/ml, which should absorb 500 microliter.)
Centrifuge for 3min under the condition of 8000r/min, and then remove the supernatant from the super clean table.
We took 100 μl of precooled 3% glucose solution, put it into EP tube, mixed it well until there was no plaque, and then transferred to the blank test tube. 3% glucose solution was freeze-dried solution, which was sterilized by filtration or heating.
After lyophilization, we put the test tube tightly and put it at room temperature to simulate the real environment for a period of time. The result shows that the lyophilization volume is reduced after placing it for a period of time.
Figure 10 freeze-dried products
In order to test its lyophilization effect, we take one tube to simulate the smear, add 1ml water and cover the lid tightly, then scroll 15s, so that the freeze-dried pieces are hung up and placed at room temperature for 10 minutes, so that the bacteria can absorb water sufficiently. After homogenization, the bacteria were diluted to 106 /ml by gradient, and 100ul coated plates (105 cells) were collected and incubate in incubators of 37 degrees Celsius.
Figure 11 smear plate
On the second day, count the colonies with a counter.
Results:
The results showed that the colony with TDPs still had good activity after lyophilization and preservation for one day, and the number of colonies growing was significantly higher than that of the experimental group without TDPs. The water bear protein can protect the bacteria from lyophilization damage.
Because of the difficulty of protein expression and purification, QHFZ could not verify the lyophilization protective effect of TDPs. We expressed and purified the 33020 protein donated by QHFZ, and then carried out the lyophilization experiment on the obtained protein, and compared with the freeze-dried protein without TDPs, so as to verify the idea of lyophilization and TDPs to preserve cas13 protein, and help QHFZ fill the blank of experimental data of TDPs on protein lyophilization protection.
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