Part:BBa_K3628000

T7 RNA Polymerase N (aar. 1~179)

Description

This part that expresses T7 RNA Polymerase N (aar. 1~179) is able to combine with, T7 RNA Polymerase C (aar. 180~883)， Spacer between Photoswitches, lac operator GS linker 1, GS linker 2 , and, photoswitches to take effects. This lead to active T7RNAP.

Usage and Biology

Photoswitches efficiency test

Experiments & Results

Experimental setup

- In this experiment, we combined regulate system and fermentation system. 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.

2、Light-regulated L-dopa production

In this experiment we combined [[Part:BBa_K3628023|J23106-RBS-T7 RNA polymerase N 1~179-pMagFast2-RBS-nMagHigh1-T7 RNA polymerase C 180~883] or [[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] with HpaBC respectively, to test whether the light-regulated HpaBC synthesis can be achieved. If light-regulated HpaBC synthesis is accomplished, light-regulated L-dopa production can then be realized.

Experimental setup

- In this experiment, we transform J23106-RBS-T7 RNA polymerase N 1~179-pMag-RBS-nMag-T7 RNA polymerase C 180~883 and plasmid contains T7 promoter-RBS-HpaB SMS-RBS-HpaC SMS simultaneously into DH5α. We culture the strain overnight to get bacteria culture.
- The monoclonals are later overnight cultivated, and is then inoculated into fresh LB medium with a proportion of 1:200.
- Samples are taken for 1mL each time at several points: 8h, 16h, 24h, 28h, 32h, 44h.
- We applied a levodopa measurement for each sample.

Results

[[Part:BBa_K3628024|J23106-RBS-T7 RNA polymerase N 1~564-Vvd-RBS-Vvd-T7 RNA polymerase C 565~883] and [[Part:BBa_K3628023|J23106-RBS-T7 RNA polymerase N 1~179-pMagFast2-RBS-nMagHigh1-T7 RNA polymerase C 180~883] are efficient and picked. In the following graph, we illustrate the yielding condition of the two photoswitches under different culture conditions. In this graph, we can see our engineered E. coli produce levodopa at a relatively high rate in the Light group, but lower in the Dark group.

L-dopa production under light-regultaion

From the figure, we find we successfully achieve yielding levodopa under the regulation of light. Vvd and pMagFast2-nMagHigh1 have similar regulation efficiency.