Difference between revisions of "Part:BBa K3628008"
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<partinfo>BBa_K3628008 short</partinfo> | <partinfo>BBa_K3628008 short</partinfo> | ||
| − | This | + | =Description= |
| − | + | This part that expresses Vvd is able to combine with [[Part:BBa_K3628016|Spacer between Photoswitches]], [[Part:BBa_K3628017|lac operator]], [[Part:BBa_K3628020|GS linker 1]], [[Part:BBa_K3628021|GS linker 2]] to take effects. This lead to active T7RNAP. | |
| − | + | =Usage and Biology= | |
| − | + | Photoswitches efficiency test | |
| − | === | + | ==Experiments & Results== |
| − | + | ===1、Photoswitches efficiency test=== | |
| − | + | ===Experimental setup=== | |
| − | < | + | - In this experiment, we transform [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] and plasmid contains GFP regulated by T7 promoter simultaneously into DH5α. We culture the strain overnight to get bacteria culture.<br> |
| − | < | + | - Bacteria cultures overnight are inoculated 1:200 in fresh LB medium for two times each. One is exposed to blue light, and the other one is covered by tin foil, to protect it from light.<br> |
| − | + | - Take 1mL cell culture for each measurement.<br> | |
| − | + | - harvest the cells by centrifuge. Discard the supernatant and add 1mL PBS. Blow the bacteria to dissolve.<br> | |
| − | + | - Repeat step 2 for three times, to exclude the deviations influenced by the medium. <br> | |
| − | === | + | - Take 100μL cell culture and mix it with 900μL PBS. Sufficiently mix. |
| − | < | + | - Transferred 200μL of it to a 96 well plate.<br> |
| − | < | + | - Measure absorbance under OD 600 nm and the fluorescent intensity by a microplate reader. The excitation light is 485nm, and emission light is 535nm.<br> |
| + | ===Results=== | ||
| + | Test photoswitch efficiency. Here is the result of the photoswitches efficiency test, which is what we have mentioned before in the experiment part. After we have harvested the cells, they are applied the fluorescence intensity measurement. <br> | ||
| + | We divide fluorescence intensity by OD 600nm and get a relative GFP expression condition. The values are applied to view the photoswitches efficiencies by ratio. To convey the result more directly, we illustrate the following column diagram. A truncation is made by us to put all data in one graph. <br> | ||
| + | [[File:T--SMS_Shenzhen--6.png|600px|thumb|center|Measurement on photoswitches' efficiency]]<br> | ||
| + | Only effective photoswitches are presented in the graph. In these groups, blue bars (Light) are higher than the black bars (Dark). <br>This means GFP is expressed in a larger amount in Light groups. This indicates a successful construction of photoswitches.<br> | ||
| + | From the Figure, we find pMag-nMag cannot achieve light regulation. Therefore, we choose [[Part:BBa_K3628023|J23106-RBS-T7 RNA polymerase N 1~179-pMagFast2-RBS-nMagHigh1-T7 RNA polymerase C 180~883] and [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] to regulate levodopa yielding in the next experiment.<br> | ||
Latest revision as of 19:43, 27 October 2020
Vvd
Description
This part that expresses Vvd is able to combine with Spacer between Photoswitches, lac operator, GS linker 1, GS linker 2 to take effects. This lead to active T7RNAP.
Usage and Biology
Photoswitches efficiency test
Experiments & Results
1、Photoswitches efficiency test
Experimental setup
- In this experiment, we transform [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] and plasmid contains GFP regulated by T7 promoter simultaneously into DH5α. We culture the strain overnight to get bacteria culture.
- Bacteria cultures overnight are inoculated 1:200 in fresh LB medium for two times each. One is exposed to blue light, and the other one is covered by tin foil, to protect it from light.
- Take 1mL cell culture for each measurement.
- harvest the cells by centrifuge. Discard the supernatant and add 1mL PBS. Blow the bacteria to dissolve.
- Repeat step 2 for three times, to exclude the deviations influenced by the medium.
- Take 100μL cell culture and mix it with 900μL PBS. Sufficiently mix.
- Transferred 200μL of it to a 96 well plate.
- Measure absorbance under OD 600 nm and the fluorescent intensity by a microplate reader. The excitation light is 485nm, and emission light is 535nm.
Results
Test photoswitch efficiency. Here is the result of the photoswitches efficiency test, which is what we have mentioned before in the experiment part. After we have harvested the cells, they are applied the fluorescence intensity measurement.
We divide fluorescence intensity by OD 600nm and get a relative GFP expression condition. The values are applied to view the photoswitches efficiencies by ratio. To convey the result more directly, we illustrate the following column diagram. A truncation is made by us to put all data in one graph.
Only effective photoswitches are presented in the graph. In these groups, blue bars (Light) are higher than the black bars (Dark).
This means GFP is expressed in a larger amount in Light groups. This indicates a successful construction of photoswitches.
From the Figure, we find pMag-nMag cannot achieve light regulation. Therefore, we choose [[Part:BBa_K3628023|J23106-RBS-T7 RNA polymerase N 1~179-pMagFast2-RBS-nMagHigh1-T7 RNA polymerase C 180~883] and [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] to regulate levodopa yielding in the next experiment.