Team:UANL Mty-Mexico/wetlab/mini project

From 2014.igem.org

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<p>The last year our team participated with the project “Thermo coli” (<a href="https://2013.igem.org/Team:UANL_Mty-Mexico">Team: UANL_Mty-Mexico</a>). The main biobrick (K1140006) that we used was composed for the fluorescent protein mCherry (E1010) regulated by the ptet promoter (R0040) and a 37°C thermometer. This year we decided to use that biobrick part to develop a mini-project. We want to proveif it is possible to enhance the repression of a gene by a combined use of the repressor protein TetR (transcriptional regulation) and a RNA thermometer (post-transcriptional regulation).</p>
<p>The last year our team participated with the project “Thermo coli” (<a href="https://2013.igem.org/Team:UANL_Mty-Mexico">Team: UANL_Mty-Mexico</a>). The main biobrick (K1140006) that we used was composed for the fluorescent protein mCherry (E1010) regulated by the ptet promoter (R0040) and a 37°C thermometer. This year we decided to use that biobrick part to develop a mini-project. We want to proveif it is possible to enhance the repression of a gene by a combined use of the repressor protein TetR (transcriptional regulation) and a RNA thermometer (post-transcriptional regulation).</p>
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<p align="center"><b>Constructions</b></p><br>
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<p align="center"><b>Constructions and Parts</b></p><br>
<p>The fluorescent part was synthetized based on the promoter ptet (R0040), a RBS fused to a RNA thermometer which was obtained on a previous work (Neupertet al, 2008), the mCherry (E1010) and the transcriptional terminator T7 (B0010-B0012). </p>
<p>The fluorescent part was synthetized based on the promoter ptet (R0040), a RBS fused to a RNA thermometer which was obtained on a previous work (Neupertet al, 2008), the mCherry (E1010) and the transcriptional terminator T7 (B0010-B0012). </p>
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<figure> <center>
<figure> <center>
   <img src="https://static.igem.org/mediawiki/2014/c/ce/Miniprojectuanl_1.jpg" width=400px>
   <img src="https://static.igem.org/mediawiki/2014/c/ce/Miniprojectuanl_1.jpg" width=400px>
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   <figcaption><span class="text-muted"><font size="2">Figure 1. Here is shown the structure of the part (K1140006).
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   <figcaption><span class="text-muted"><font size="2">Figure 1. This part (K1140006) changes its behavior in response to temperature variations above and below 37°C. This part is available in pUC57, pSB1C3 and pSB2K3.
  </span></font> <br></figcaption>
  </span></font> <br></figcaption>
</figure> </center>
</figure> </center>
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<p> Also, it was constructed a part that does not have a thermometer, so that we could see its effect on the fluorescence. This part was constructed using the same promoter (R0040) with the RFP/mCherry protein generator (K081014). </p>
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<figure> <center>
 +
  <img src="https://static.igem.org/mediawiki/2014/2/20/Miniprojectuanl_2.jpg" width=400px>
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  <figcaption><span class="text-muted"><font size="2">Figure 2. This part does not change its behavior in response to temperature. The biobrick part is avai
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lable in pSB1C3 and pSB2K3.
 +
</span></font> <br></figcaption>
 +
</figure> </center>
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 +
<p>We constructed TetR generatorswith different constitutive promoters. We used the promoters J23109, J23106 and J23111 with the relative force 106, 1185 and 1487, respectively.These parts have a RBS (B0034), the CDS for the TetR repressor (C0040) and the T7 transcriptional terminator (B0010-B0012). Moreover, we used the biobrick part K145201 as a positive control to expression of TetR, because it is a generator with promoter force 396. Also the RBS and transcriptional terminator are both the same. </p>
 +
 +
<figure> <center>
 +
  <img src="https://static.igem.org/mediawiki/2014/d/db/Miniprojectuanl_3.jpg" width=400px>
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  <figcaption><span class="text-muted"><font size="2">Figure 3. The names of constructed parts are BBa_K1480003, K1480004 and K1480005. Their respective promoters are J23109, J23106 and J23111. Actually, those parts are just available in pBca.
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</span></font> <br></figcaption>
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</figure> </center>
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 +
<p align="center"><b>Measurements</b></p><br>
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<p>For the measurements, we made constructions in order to have the biobrick parts in compatible plasmids. It means that those which codifies to mCherry are in pSB2K3 and the tetR genes are in pSB1A2.<br>
 +
The bacteria E. coli Top10 were transformed to have the next 7 cultures: </p>

Revision as of 17:39, 17 October 2014

Miniproject

Introduction


It is know that TetR (like others transcriptional repressors) can allow a basal expression. Because of that, it would be a good idea to reduce that basal expression using a molecular tool which does not cause noise to circuits and systems.

The last year our team participated with the project “Thermo coli” (Team: UANL_Mty-Mexico). The main biobrick (K1140006) that we used was composed for the fluorescent protein mCherry (E1010) regulated by the ptet promoter (R0040) and a 37°C thermometer. This year we decided to use that biobrick part to develop a mini-project. We want to proveif it is possible to enhance the repression of a gene by a combined use of the repressor protein TetR (transcriptional regulation) and a RNA thermometer (post-transcriptional regulation).

Constructions and Parts


The fluorescent part was synthetized based on the promoter ptet (R0040), a RBS fused to a RNA thermometer which was obtained on a previous work (Neupertet al, 2008), the mCherry (E1010) and the transcriptional terminator T7 (B0010-B0012).

Figure 1. This part (K1140006) changes its behavior in response to temperature variations above and below 37°C. This part is available in pUC57, pSB1C3 and pSB2K3.

Also, it was constructed a part that does not have a thermometer, so that we could see its effect on the fluorescence. This part was constructed using the same promoter (R0040) with the RFP/mCherry protein generator (K081014).

Figure 2. This part does not change its behavior in response to temperature. The biobrick part is avai lable in pSB1C3 and pSB2K3.

We constructed TetR generatorswith different constitutive promoters. We used the promoters J23109, J23106 and J23111 with the relative force 106, 1185 and 1487, respectively.These parts have a RBS (B0034), the CDS for the TetR repressor (C0040) and the T7 transcriptional terminator (B0010-B0012). Moreover, we used the biobrick part K145201 as a positive control to expression of TetR, because it is a generator with promoter force 396. Also the RBS and transcriptional terminator are both the same.

Figure 3. The names of constructed parts are BBa_K1480003, K1480004 and K1480005. Their respective promoters are J23109, J23106 and J23111. Actually, those parts are just available in pBca.

Measurements


For the measurements, we made constructions in order to have the biobrick parts in compatible plasmids. It means that those which codifies to mCherry are in pSB2K3 and the tetR genes are in pSB1A2.
The bacteria E. coli Top10 were transformed to have the next 7 cultures:

The game was made with the program “Game Maker” which allows you, the programmer, to make its own game without the necessity of a specific programming language.

GALLERY

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