Team:SYSU-China/file/Project/Result/B2H.html

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<h1>B2H</h1>
<h1>B2H</h1>
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<p>
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So as to test if our B2H (signal converter) work, we designed another plasmid to replace the M13 bacteriophage vector before the M13 part is integrated in our machine. This plasmid is pTRG.
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</p>
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<p>
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The target plasmid pTRG carries a low-copy ColE1 replication origin and confers tetracycline resistance. It expresses the target protein fused to the N-terminal domain of the protein rpoA. We have constructed two plasmids under this category. One is called pTRG-R, fusing rpoA with RFP, and the other one named pTGR-G is a fusion protein including rpoA and GAL11P (a mutant of GAL11 protein).
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</p>
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<p>
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pBT is constructed as the final form in our machine. We have designed three kinds of fusion proteins in all. The first one called pBT-M fuses λcI with multiple cloning sites (MCS). And the second one is a fusion protein named pBT-R containing λcI and RFP. Then we fuse λcI with LGF2 (the GAL4 dimerization domain) to create pBT-L.
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</p>
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<p>
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pRPR is also built as the final form except that the reporter gene is GFP in the following test experiments.
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</p>
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<p>
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LGF2 and GAL11P are proved to have strong interaction with each other and are often used as positive control in general two-hybrid system.  (reference) RFP is BBa_J04450.
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</p>
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<h2>1. Test for three antibiotics</h2>
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<p>
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In order to guarantee the coexistence of pBT, pTRG and pRPR in the bacteria, we decided to use antibiotics as a means of stress screening. The basic concentrations we applied for each antibiotic were 35mg/mL for Ampicillin, 50mg/mL for Chloramphenicol and 12.5mg/mL for Tetracycline. We transformed the plasmid as the following groups:
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</p>
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/a/a4/Sysuchina_32.png"><img src="https://static.igem.org/mediawiki/2014/a/a4/Sysuchina_32.png" alt="" /></a>
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<p>
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S for pRPR-RFP (Amp), B for pBT-LGF( Cmr), T for pTRG-Gal11P (Tet). Use BL21 to perform the transformation. 50μL for each group.
 +
After the transformation, all groups of bacteria were cultured in liquid LB containing three kinds of antibiotics for 72 hours. ⨹SBT and △SBT were respectively cultured in LB with 1 and 1/2 of each basic antibiotic concentration mentioned above. SBT, BT, ST, SB, B, S, T and control were cultured in LB with 1/3 of each basic antibiotic concentration.
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</p>
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<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/e/e8/Sysuchina_10.png"><img src="https://static.igem.org/mediawiki/2014/e/e8/Sysuchina_10.png" alt="" /></a>
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<p1>
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Fig.1 The result of three plasmids coexistence experiment
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</p1>
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<p>
 +
According to the results, these three plasmids can coexist in the bacteria. We chose LB with 1/4 of each basic antibiotic concentration to screen for the coexistence of three plasmids after transformation and LB with 1/3 of each basic antibiotic concentration to culture the bacteria.
 +
</p>
 +
 +
<h2>2. Test for B2H</h2>
 +
<p>
 +
We transformed the plasmid as the following groups:
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</p>
 +
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/a/a9/Sysuchina_33.PNG"><img src="https://static.igem.org/mediawiki/2014/a/a9/Sysuchina_33.PNG" alt="" /></a>
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<p>
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Then the bacteria of these groups were tested for the expression level of reporter gene-GFP. The excitation wavelength was 510nm, while the absorption wavelength was 550nm.
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</p>
 +
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/b/b1/Sysuchina_12.PNG"><img src="https://static.igem.org/mediawiki/2014/b/b1/Sysuchina_12.PNG" alt="" /></a>
 +
<p>
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The resulting figure showed that there was a significant difference between the positive control of LG and the other experimental groups. The fluorescence intensity of the positive control was nearly 5 times larger than the ones of the other groups. RFP didn’t have any influence on the expression of GFP. The independent expression of LGF2 and GALP11P also affected the expression of GFP without conspicuousness.
 +
</p>
 +
 +
<h2>3. Test for different degrees of interaction and corresponding gene expression</h2>
 +
<p>
 +
We wanted to know if different degrees of interaction between bait and target could be detected by the expression of reporter gene, so we conducted a test for it. According to Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain (Patricia et al.2001), we used the technique of Site-Directed Mutagenesis (SDM) to get three mutants of LGF2. They are E75A (the corresponding plasmid is marked as L), D78A (M) and R74A (H). And they respectively have low, medium and high level of interactions with GAL11P. 
 +
</p>
 +
<p>
 +
We transformed the plasmid as the following
 +
groups:
 +
</p>
 +
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/a/a3/Sysuchina_34.png"><img src="https://static.igem.org/mediawiki/2014/a/a3/Sysuchina_34.png" alt="" /></a>
 +
<p>
 +
Then the bacteria of these groups were tested for the expression level of reporter gene-GFP. The excitation wavelength was 510nm, while the absorption wavelength was 550nm.
 +
</p>
 +
<a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2014/8/81/Sysuchina_35.png"><img src="https://static.igem.org/mediawiki/2014/8/81/Sysuchina_35.png" alt="" /></a>
 +
<p>
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The result indicated that there were considerable differences among the expression of GFP activated by L, M and H. The fluorescence intensity of H group was much larger than that of M group and M was also larger than L. This was consistent with the result in the reference and confirmed that our signal converter could distinguish the different degrees of protein-protein interaction.
 +
</p>
<!--SYSUCHINA!-->
<!--SYSUCHINA!-->

Revision as of 14:51, 17 October 2014

Contents

B2H

So as to test if our B2H (signal converter) work, we designed another plasmid to replace the M13 bacteriophage vector before the M13 part is integrated in our machine. This plasmid is pTRG.

The target plasmid pTRG carries a low-copy ColE1 replication origin and confers tetracycline resistance. It expresses the target protein fused to the N-terminal domain of the protein rpoA. We have constructed two plasmids under this category. One is called pTRG-R, fusing rpoA with RFP, and the other one named pTGR-G is a fusion protein including rpoA and GAL11P (a mutant of GAL11 protein).

pBT is constructed as the final form in our machine. We have designed three kinds of fusion proteins in all. The first one called pBT-M fuses λcI with multiple cloning sites (MCS). And the second one is a fusion protein named pBT-R containing λcI and RFP. Then we fuse λcI with LGF2 (the GAL4 dimerization domain) to create pBT-L.

pRPR is also built as the final form except that the reporter gene is GFP in the following test experiments.

LGF2 and GAL11P are proved to have strong interaction with each other and are often used as positive control in general two-hybrid system. (reference) RFP is BBa_J04450.

1. Test for three antibiotics

In order to guarantee the coexistence of pBT, pTRG and pRPR in the bacteria, we decided to use antibiotics as a means of stress screening. The basic concentrations we applied for each antibiotic were 35mg/mL for Ampicillin, 50mg/mL for Chloramphenicol and 12.5mg/mL for Tetracycline. We transformed the plasmid as the following groups:

<a class="fancybox" rel="group" href="Sysuchina_32.png"><img src="Sysuchina_32.png" alt="" /></a>

S for pRPR-RFP (Amp), B for pBT-LGF( Cmr), T for pTRG-Gal11P (Tet). Use BL21 to perform the transformation. 50μL for each group. After the transformation, all groups of bacteria were cultured in liquid LB containing three kinds of antibiotics for 72 hours. ⨹SBT and △SBT were respectively cultured in LB with 1 and 1/2 of each basic antibiotic concentration mentioned above. SBT, BT, ST, SB, B, S, T and control were cultured in LB with 1/3 of each basic antibiotic concentration.

<a class="fancybox" rel="group" href="Sysuchina_10.png"><img src="Sysuchina_10.png" alt="" /></a> <p1> Fig.1 The result of three plasmids coexistence experiment </p1>

According to the results, these three plasmids can coexist in the bacteria. We chose LB with 1/4 of each basic antibiotic concentration to screen for the coexistence of three plasmids after transformation and LB with 1/3 of each basic antibiotic concentration to culture the bacteria.

2. Test for B2H

We transformed the plasmid as the following groups:

<a class="fancybox" rel="group" href="Sysuchina_33.PNG"><img src="Sysuchina_33.PNG" alt="" /></a>

Then the bacteria of these groups were tested for the expression level of reporter gene-GFP. The excitation wavelength was 510nm, while the absorption wavelength was 550nm.

<a class="fancybox" rel="group" href="Sysuchina_12.PNG"><img src="Sysuchina_12.PNG" alt="" /></a>

The resulting figure showed that there was a significant difference between the positive control of LG and the other experimental groups. The fluorescence intensity of the positive control was nearly 5 times larger than the ones of the other groups. RFP didn’t have any influence on the expression of GFP. The independent expression of LGF2 and GALP11P also affected the expression of GFP without conspicuousness.

3. Test for different degrees of interaction and corresponding gene expression

We wanted to know if different degrees of interaction between bait and target could be detected by the expression of reporter gene, so we conducted a test for it. According to Recruitment of the transcriptional machinery through GAL11P: structure and interactions of the GAL4 dimerization domain (Patricia et al.2001), we used the technique of Site-Directed Mutagenesis (SDM) to get three mutants of LGF2. They are E75A (the corresponding plasmid is marked as L), D78A (M) and R74A (H). And they respectively have low, medium and high level of interactions with GAL11P.

We transformed the plasmid as the following groups:

<a class="fancybox" rel="group" href="Sysuchina_34.png"><img src="Sysuchina_34.png" alt="" /></a>

Then the bacteria of these groups were tested for the expression level of reporter gene-GFP. The excitation wavelength was 510nm, while the absorption wavelength was 550nm.

<a class="fancybox" rel="group" href="Sysuchina_35.png"><img src="Sysuchina_35.png" alt="" /></a>

The result indicated that there were considerable differences among the expression of GFP activated by L, M and H. The fluorescence intensity of H group was much larger than that of M group and M was also larger than L. This was consistent with the result in the reference and confirmed that our signal converter could distinguish the different degrees of protein-protein interaction.

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