Part:BBa_K3628024

J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883

Description

This part contains Spacer between Photoswitches, lac operator, GS linker 1, GS linker 2, and Vvd. This lead to active T7RNAP.

Usage and Biology

Photoswitches efficiency test

Experiments & Results

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.

Measurement on photoswitches' efficiency

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.

Yucai_SZ 2021

Authors iGEM Yucai_SZ 2021, Haixin Wang. We inserted this part into a pSB4C5 backbone, and transformed it into the Escherichia coli DH5α host cell which had harbored a pSB1A3 plasmid containing a pT7-amilCP transcriptional unit. Then test it in tubes.

• Inoculate a colony into LB medium and culture overnight in dark (200 rpm, 37 ℃).
• Overnight cultures are inoculated 1:100 into two tubes with fresh LB medium. When cultured in shaking table, one tube is exposed to 480 nm blue light, and the other one is covered by tin foil, to protect it from light
• After 12 hours of shaking culture, 500 μL culture were sampled and transferred into 1.5 mL EP tube.
• Centrifugate the sample tubes at 10 000 g for 30 seconds and observe the colors of bacterial precipitations.

The left was cultured under blue light, and the right was in dark

This system can work under blue light.

Actually, We tested four photosensitive T7RNAP systems,BBa_K3628023, BBa_K3628022, BBa_K3628024 and BBa_K4055001, with the same structure under the same conditions:

There are four groups of blue light systems, from left to right are BBa_K3628023、BBa_K3628022、BBa_K3628024 ，BBa_K4055001 .Each group had blue material on the left that was light exposed and white on the right that was protected from light.