Team:Tsinghua/Results
Experiment & Results
1. Testing qualitatively the blue-light and red-light system.
This experiment was designed to confirm the efficacy of both red-light and blue-light system. For the red light system, we transformed a plasmid containing the entire red light system (Cph8, HO1 and PcyA), along with a promoter, a terminator and an OmpR regulated reporter. Therefore, when bacteria carrying this gene are provided with the red light, the reporter cannot be synthesized. The result supported this prediction because we can observe a distinctive expression pattern between the treated (provided with red light) and untreated (kept in dark) group. On the contrary, we added another cI repressor in front of the response regulator in the blue-light system, so its reporter gene, a GFP, will be expressed when bacteria carrying this plasmid are provided with blue light. As is shown below, on the right we can observe that two tubes exhibit slightly different shades of green.
Figure 1. The test results for blue-light and red-light systems.
2. Determining the relationship between iPTG concentration and optical density (OD) value.
(1) Measurement of standard curve of OD value against relative concentration of bacteria.
(a) Incubate bacteria with liquid LB at 37°C overnight.
(b) Dilute the suspension with 1-9 volumes of liquid LB.
(c) Measure OD600 value of each dilution of the suspension, using liquid LB as blank.
Samples used:
|
Dilution factor |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
Bacteria suspension/μl |
600.0 |
300.0 |
200.0 |
150.0 |
120.0 |
100.0 |
85.7 |
75.0 |
66.7 |
60.0 |
|
Liquid LB/μl |
0.0 |
300.0 |
400.0 |
450.0 |
480.0 |
500.0 |
514.3 |
525.0 |
533.3 |
540.0 |
Table 1. Dilution of bacteria suspension
|
Bacteria/Dilution factor |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
|
|
2.00 |
1.34 |
0.99 |
0.78 |
0.63 |
0.54 |
0.53 |
0.39 |
0.36 |
0.31 |
|
|
2.00 |
1.37 |
1.00 |
0.77 |
0.63 |
0.54 |
0.49 |
0.41 |
0.36 |
0.32 |
|
|
1.10 |
0.61 |
0.43 |
0.32 |
0.27 |
0.24 |
0.18 |
0.17 |
0.15 |
0.15 |
Table 2. OD600 values measured
(2) Measurement of OD600 of bacterias after treatment with iPTG gradient.
(a) Dilute iPTG to 2% (20 g/μl). Add 10 μl 20% iPTG to 90μl ddH20.
(b) Dilute iPTG to 0.1% (1 g/μl). Add 10 μl 2% iPTG to 190μl ddH20.
(c) Establish gradients of iPTG. Add gradients of 0.1% iPTG to bacteria suspension.
|
Final concentration/nmol·μl-1 |
0.00 |
1.40 |
2.80 |
4.20 |
5.60 |
6.99 |
8.39 |
|
0.1% iPTG/μl |
0 |
1 |
2 |
3 |
4 |
5 |
6 |
|
Bacteria suspension/ml |
3.000 |
2.999 |
2.998 |
2.997 |
2.996 |
2.995 |
2.994 |
|
Final concentration/nmol·μl-1 |
9.79 |
11.19 |
12.59 |
13.99 |
15.39 |
16.79 |
18.18 |
|
0.1% iPTG/μl |
7 |
8 |
9 |
10 |
11 |
12 |
13 |
|
Bacteria suspension /ml |
2.993 |
2.992 |
2.991 |
2.990 |
2.989 |
2.988 |
2.987 |
|
Final concentration/nmol·μl-1 |
19.58 |
20.98 |
22.38 |
23.78 |
25.18 |
26.58 |
27.98 |
|
0.1% iPTG/μl |
14 |
15 |
16 |
17 |
18 |
19 |
20 |
|
Bacteria suspension /ml |
2.986 |
2.985 |
2.984 |
2.983 |
2.982 |
2.981 |
2.980 |
|
Final concentration/nmol·μl-1 |
29.37 |
30.77 |
32.17 |
33.57 |
34.97 |
36.37 |
37.77 |
|
0.1% iPTG/μl |
21 |
22 |
23 |
24 |
25 |
26 |
27 |
|
Bacteria suspension /ml |
2.979 |
2.978 |
2.977 |
2.976 |
2.975 |
2.974 |
2.973 |
Table 3. Establishment of iPTG gradient.
(d) Incubate at 37°C for 14 h.
(e) Measure OD600 of each tube.
|
iPTG concentration/nmol·μl-1 |
0.00 |
1.40 |
2.80 |
4.20 |
5.60 |
6.99 |
8.39 |
|
OD600 |
1.71 |
1.69 |
1.69 |
1.79 |
1.57 |
1.61 |
1.56 |
|
iPTG concentration/nmol·μl-1 |
9.79 |
11.19 |
12.59 |
13.99 |
15.39 |
16.79 |
18.18 |
|
OD600 |
1.51 |
1.39 |
1.30 |
1.11 |
1.18 |
1.00 |
0.92 |
|
iPTG concentration/nmol·μl-1 |
19.58 |
20.98 |
22.38 |
23.78 |
25.18 |
26.58 |
27.98 |
|
OD600 |
0.83 |
0.94 |
0.65 |
0.59 |
0.51 |
0.35 |
0.30 |
|
iPTG concentration/nmol·μl-1 |
29.37 |
30.77 |
32.17 |
33.57 |
34.97 |
36.37 |
37.77 |
|
OD600 |
0.14 |
0.11 |
0.07 |
0.05 |
0.04 |
0.04 |
0.02 |
Table 4. Measurement of OD600 of each tube.
Figure 2. The relationship between relative iPTG concentration and OD600.
3. Confirming the bacteria genome knock-out experiment.
In this experiment, we used a recombinase system to insert a Kanamycin gene into the genomic region where we want to target and then utilized other recombinase to remove the Kanamycin gene. Therefore, in order to confirm that we successfully knock-out a gene, colony PCR was done. As can be seen in Figure 3, roughly 1kb bands appear nearly in every lane, supporting that our knock-out system was effective.
Figure 3. The gel results of the knock-out experiment.
4. Fusion protein construction.
In this experiment, we planned to fuse two proteins, a recombinase and a Cas9, together. In addition, we added a roughly 36bp linker between the two in order to increase its flexibility. Eventually, we successfully fused two proteins with the seamless cloning, as is supported by the appearance of 4-5kb bands in nearly all lanes.
Figure 4. The gel results of the fusion protein design.
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