Difference between revisions of "Part:BBa K2381014"

 
 
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<partinfo>BBa_K2381014 short</partinfo>
 
<partinfo>BBa_K2381014 short</partinfo>
  
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It is a fusion protein fused N fragment of split luciferase  with light-induced dimerization domain. It is used to prove that our light-induced dimerization domain is working.
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<body>
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<p>
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this part can response to blue light (470nm), rapidly bind to the corresponding parts, BBa_K2381013, due to eletrostatic interaction and dimerize. Its cofactor for light sensing is flavin adenine dinucleotide (FAD). After dimerization the split luciferase will complement to recover its abillity to generate fluorescenece.
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</p>
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<img src="https://static.igem.org/mediawiki/parts/d/df/HZAU_2017_MagLuc.png" width="700px"/>
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</br>
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<b>Fig.1 </b>Prove the function of pMag and nMag. n=1. n=1
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</br>
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</br>
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2381014 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2381014 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2381014 parameters</partinfo>
 
<partinfo>BBa_K2381014 parameters</partinfo>
 
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=SMS_Shenzhen improved this part in 2020=
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SMS_Shenzhen combines this part with [[Part:BBa_K3628004|nMag]], [[Part:BBa_K3628016|Spacer between Photoswitches]], [[Part:BBa_K3628017|lac operator]], [[Part:BBa_K3628020|GS linker 1]], [[Part:BBa_K3628021|GS linker 2]], and [[Part:BBa_K3628008|Vvd]], forming the [[Part:BBa_K3628022|J23106-RBS-T7 RNA polymerase N 1~179-pMag-RBS-nMag-T7 RNA polymerase C 180~883]]. This lead to active T7RNAP.
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==Experiments & Results==
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===1、Photoswitches efficiency test===
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===Experimental setup===
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- In this experiment, we transform[[Part:BBa_K3628022|J23106-RBS-T7 RNA polymerase N 1~179-pMag-RBS-nMag-T7 RNA polymerase C 180~883]] and plasmid contains GFP regulated by T7 promoter simultaneously into DH5α. We culture the strain overnight to get bacteria culture.<br>
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- 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>
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===Results===
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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>
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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>
 +
===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 [[Part:BBa_K3628022|J23106-RBS-T7 RNA polymerase N 1~179-pMag-RBS-nMag-T7 RNA polymerase C 180~883]] and plasmid contains  [[Part:BBa_K3628018|T7 promoter-RBS-HpaB SMS-RBS-HpaC SMS]] simultaneously into DH5α. We culture the strain overnight to get bacteria culture.<br>
 +
-  The monoclonals are later overnight cultivated, and is then inoculated into fresh LB medium with a proportion of 1:200.<br>
 +
-  Samples are taken for 1mL each time at several points: 8h, 16h, 24h, 28h, 32h, 44h. <br>
 +
- We applied a levodopa measurement for each sample.<br>
 +
===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.  <br>
 +
[[File:T--SMS_Shenzhen--5.png|600px|thumb|center|L-dopa production under light-regultaion]]<br>From the figure, we find we successfully achieve yielding levodopa under the regulation of light. Vvd and pMagFast2-nMagHigh1 have similar regulation efficiency.  <br>

Latest revision as of 23:51, 27 October 2020


LucN-pMag

this part can response to blue light (470nm), rapidly bind to the corresponding parts, BBa_K2381013, due to eletrostatic interaction and dimerize. Its cofactor for light sensing is flavin adenine dinucleotide (FAD). After dimerization the split luciferase will complement to recover its abillity to generate fluorescenece.


Fig.1 Prove the function of pMag and nMag. n=1. n=1



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1362
  • 1000
    COMPATIBLE WITH RFC[1000]



SMS_Shenzhen improved this part in 2020

SMS_Shenzhen combines this part with nMag, Spacer between Photoswitches, lac operator, GS linker 1, GS linker 2, and Vvd, forming the J23106-RBS-T7 RNA polymerase N 1~179-pMag-RBS-nMag-T7 RNA polymerase C 180~883. This lead to active T7RNAP.

Experiments & Results

1、Photoswitches efficiency test

Experimental setup

- In this experiment, we transformJ23106-RBS-T7 RNA polymerase N 1~179-pMag-RBS-nMag-T7 RNA polymerase C 180~883 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.

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.