Team:UANL Mty-Mexico/wetlab

From 2014.igem.org

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Wet-Lab</div>
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<p align="justify">Here there is a construction map of the circuit:</p>
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<p align="justify"><b>DNA/Program delivery</b></p>
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<p align="justify">We had a lot of problems with the part "PCC1" because there were problems when it was synthesized. When we cut it it only cut in one size couldn't cut it as we expected but we used it in the ligation anyways because all the other parts cut very well except this one. The part "PUC-57" worked very well while cutting with the restriction enzymes and at the ligation. For the Friday 21th, 2013, we got colonies of both ligations. We think the "PCC1" is not a good ligation because it did not released the fragment. But we are going to characterize the part with the "PUC-57" the next week. We cannot be sured right now because it doesn't have a reporter.</p>
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<p align="justify">A P1-based bacteriophage was used as the DNA/program delivery system [1]. Constructed at Anderson's lab in UC Berkeley, this system is very sensitive and its lytic state is only active in the presence of arabinose. If reengineered at the lab, it can be coupled with a new program to substitute the old program or update it. </p>
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<p align="justify">We also uploaded to the Parts Registry the design of our new part.<a href="http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K987000";><font color="blue"> http://parts.igem.org/partsdb/edit_seq.cgi?part=BBa_K987000</font></a></p>
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<p align="justify"><b>DNA/Program digestion system</b></p>
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<p align="justify"> Here is the composite part<a href="http://parts.igem.org/cgi/partsdb/part_info.cgi?part_id=28522";><font color="blue"> http://parts.igem.org/cgi/partsdb/part_info.cgi?part_id=28522</font></a></p>
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<p align="justify">A phagemid-codified TALEN will suppress the previous DNA/program, which strategically contained TALEN sites when used to engineer E. coli since the beginning. TALENs will linearize the DNA, which will be then digested by nucleases in the host organism (linearized DNA is digested by a number of naturally occurring mechanisms in E. coli). The new phagemid contains different TALEN sites which will allow to suppress it in a future (if necessary) using the same strategy with a different TALEN.</p>
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<p align="justify"><b>The whole scheme</b></p>
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<p align="justify"><b><font color="red" size="25px">Conclusion:</font></b><br><br>
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<p align="justify">A DNA delivery system will allow to deliver the new program and the TALEN, and also, reporter proteins can be used to monitor the hacking process. From fluorescent proteins to barcodes in the DNA, the variety of possible reporters will allow us to explore the biology of the whole scheme for further improvement or for its application with different organisms.</p>
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The team focused strongly on the lab work to reach the goal of the project, which is the creation of a bacterium capable of producing a bio-insecticide that helps the potato crop against the plagues of worms. The idea include two central systems working together to produce the insecticide in the best conditions. The lab work focused on the creation of these two parts of the system and for that reason it was necessary the synthetize of two gens which were the Vip3ca3 and the Riboswitch, parts that weren’t found in the parts registry. The lab work started until these two parts were mailed to us from United States. </p>
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<p align="justify">When the parts finally arrived, the work in the lab wasn’t capable of obtain the gen of the Vip3Ca3 in good conditions, only the PUC-57 was growing and its DNA was showed correctly on the gels. Even this we continue with the cuts and ligations to see if we can obtain any special results. After two weeks trying the ligations weren’t growing in the Petri Dishes, so the whole process continue on the last week. </p>
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<p align="justify">The last morning, the Petri’s Dishes showed some colonies which should represent the cuts that were done one weeks before, which means the cuts may be succesful and deeper studies will be done to the sample to characterize if the ligations were actually done. The strongest evidence to support this affirmation is the color of the colonies which relate to the description of the gens. </p>
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<p align="justify"><b>Further applications</b><br>
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The project was thought to be done in a bacterium, but the further plan is to made a transgenic plant that do the same function that the bacteria, to develop its own bio-insecticide, and then to be used in the city crops so the plague can be fight and the potatoes are not affected. Also the idea of the gellan gums can be used in order to maintain a suitable environment for the bacteria. These ideas may be applied or tested in a deeper way in a further work.</p>
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Latest revision as of 03:52, 18 October 2014

Wet-Lab
Results

DNA/Program delivery

A P1-based bacteriophage was used as the DNA/program delivery system [1]. Constructed at Anderson's lab in UC Berkeley, this system is very sensitive and its lytic state is only active in the presence of arabinose. If reengineered at the lab, it can be coupled with a new program to substitute the old program or update it.

DNA/Program digestion system

A phagemid-codified TALEN will suppress the previous DNA/program, which strategically contained TALEN sites when used to engineer E. coli since the beginning. TALENs will linearize the DNA, which will be then digested by nucleases in the host organism (linearized DNA is digested by a number of naturally occurring mechanisms in E. coli). The new phagemid contains different TALEN sites which will allow to suppress it in a future (if necessary) using the same strategy with a different TALEN.

The whole scheme

A DNA delivery system will allow to deliver the new program and the TALEN, and also, reporter proteins can be used to monitor the hacking process. From fluorescent proteins to barcodes in the DNA, the variety of possible reporters will allow us to explore the biology of the whole scheme for further improvement or for its application with different organisms.

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