Team:CIDEB-UANL Mexico/Wet-lab/Planning

From 2012hs.igem.org

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<p> The work in the laboratory is a fundamental component for iGEM, due that in the realization of projects of Synthetic Biology is required to perform experiments and procedures that allow building genetic innovated pieces, able to generate new functions in the microorganisms which are being manipulated.</p>
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    <p>To work in the laboratory we made a plan about how to build the circuit. The principal processes are cut and ligate. To cut the biobricks we need restriction enzymes type II. These enzymes cut in a specific place. Here we hace an example about how to cut and ligate the pieces 1-6M and 3-12M:</p>
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<p>The part 1-6M is in the left side and is cut with the EcoRI and SpeI enzymes./p>
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<p>Genetic manipulation in iGEM is based in the use of BioBricks, which represents the standardization of Genetic Engineering, allowing that for pasting a piece with another, it can be performed by following a simple and reiterative protocol. In this way, the work in the laboratory could be represented by a flow chart with reiterative methodologies in each desired construction. This techniques collection makes the Genetic Engineering a very simple methodology: Synthetic Biology, which can be followed in a very simple way by junior and high school students.</p>
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<p><a href="http://openwetware.org/wiki/Image:3AAssembly.gif">http://openwetware.org/wiki/Image:3AAssembly.gif</a></p>
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<p>The part 3-12M is in the right side and is cut with XbaI and PstI enzymes.</p>
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<p><a href="http://openwetware.org/wiki/Image:3AAssembly.gif">http://openwetware.org/wiki/Image:3AAssembly.gif</a></p>
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<p>To cut a vector we need the EcoRI and PstI enzymes in order to ligate the parts 1-6M and 3-12M in the correct order.</p>
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<p><a href="http://openwetware.org/wiki/Image:3AAssembly.gif">http://openwetware.org/wiki/Image:3AAssembly.gif</a></p>
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<p>In this way, when the 2 parts that were removed  are inserted in a new vector, the part 1 (the left one) will attach to the EcoRI side and the part 2 (the right one) will be attached in the PstI side. XbaI and SpeI take the place in the way that they finish in the initial order but instead of having one biobrick inside, there are 2 biobricks ligated. The enzymes will have this order:</p>
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<p>The project for iGEM-CIDEB 2012 includes the construction of a genetic circuit which allows the detection of different concentrations of a determined contaminant compound and, based in these concentrations, shows a reporter which indicates the present levels in the mentioned molecule in the solutionTo carry out this idea, it’s required to include a genetic circuit with different modules inside the genetic material of E.coli. Being like this, it is planned to use 13 independent genetic pieces (BioBricks) to build each one of the modules of the complete circuit.</p>
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<p>After cutting the parts we attach the two parts  joining them together. For example the parts 1-6M and 3-12M are cut. Then 1-6M has ampiciline resistance and the part 3-12M has ampiciline and kanamicine  resistance. We have to use a vector with a different resistance to see if the bacteria  have the complete new vector when tested. Following this factor is how we chose  the vector that we will use and in this case we chose tetracycline and the vector  with this resistance is the PSB1T3 (1-7A).</p>
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<p>Once the parts are together they are transformed  in bacteria.</p>
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<p>After that we culture in tubes with broth medium and the process is repeated until all the circuit is complete.</p>
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<p>In a general way, the steps followed in the laboratory were:</p>
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<p>In a general way, the steps followed in the laboratory were:</p>
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     <div class="lateral-button"><a href="https://2012hs.igem.org/Team:CIDEB-UANL-Mexico/Wet-lab/Planning#Construction">Construction</a></div>
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     <div class="lateral-button"><a href="https://2012hs.igem.org/Team:CIDEB-UANL_Mexico/Wet-lab/Planning#Construction">Construction</a></div>

Revision as of 00:54, 17 June 2012

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Team: UANL_Mty-Mexico

Team: CIDEB-UANL Mexico

Wet-lab: Planning
Construction maps

To work in the laboratory we made a plan about how to build the circuit. The principal processes are cut and ligate. To cut the biobricks we need restriction enzymes type II. These enzymes cut in a specific place. Here we hace an example about how to cut and ligate the pieces 1-6M and 3-12M:

The part 1-6M is in the left side and is cut with the EcoRI and SpeI enzymes./p>

http://openwetware.org/wiki/Image:3AAssembly.gif

The part 3-12M is in the right side and is cut with XbaI and PstI enzymes.

http://openwetware.org/wiki/Image:3AAssembly.gif

To cut a vector we need the EcoRI and PstI enzymes in order to ligate the parts 1-6M and 3-12M in the correct order.

http://openwetware.org/wiki/Image:3AAssembly.gif

In this way, when the 2 parts that were removed are inserted in a new vector, the part 1 (the left one) will attach to the EcoRI side and the part 2 (the right one) will be attached in the PstI side. XbaI and SpeI take the place in the way that they finish in the initial order but instead of having one biobrick inside, there are 2 biobricks ligated. The enzymes will have this order:

After cutting the parts we attach the two parts joining them together. For example the parts 1-6M and 3-12M are cut. Then 1-6M has ampiciline resistance and the part 3-12M has ampiciline and kanamicine resistance. We have to use a vector with a different resistance to see if the bacteria have the complete new vector when tested. Following this factor is how we chose the vector that we will use and in this case we chose tetracycline and the vector with this resistance is the PSB1T3 (1-7A).

Once the parts are together they are transformed in bacteria.

After that we culture in tubes with broth medium and the process is repeated until all the circuit is complete.

In a general way, the steps followed in the laboratory were:

In a general way, the steps followed in the laboratory were: