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Wet lab

In the laboratory we wee working in the construction of the circuit. In the very beggining we didn't have no equipment to use in molecular biology. The principal helped us with the equipment required to work in the laboratory and we started working in March. In a general way here are explained the steps we followed.

1. The first step is to hydrate all the biobricks of the list.
2. Then they must be transformed into bacteria.
3. After that, we add the bacteria into Petri dishes with agar-based growth medium.
4. The next step was to resemble in test tubes.
5. Do MiniPreparation of plasmid DNA with all the biobricks
6. Check the quality of the DNAs by running

Then the biobricks will follow a different process divided by sections: Stand-by, High concentration, Low concentration

In order to build the section of Stand-by the following steps must be followed:

1. Cut the pieces 1-6M with EcoRI and SpeI, and the part 3-12M with XbaI and PstI.
2. Ligate them into the plasmid pSB1T3 (1-7A).

In order to build the section of High concentration the following steps must be followed:

1. Cut the part 1-6E with EcoRI and SpeI, and the part 1-10E with XbaI and PstI.
2. Ligate them into the plasmid PBB1K3 (1-5A) and we obtain the part pBAD-RBS
3. Cut the part 1-14N with EcoRI and SpeI , and the part 1-2M
4. Ligate them into the plasmid PSB1T3 (1-7A) and we obtain the part YFP-cI434.
5. Cut the part pBAD-RBS with EcoRI and SpeI and the part YFP-cI434 with XbaI and PstI.
6. Ligate them into the the plasmid PSB1A3 (1-1G).



In order to build the section of High concentration the following steps must be followed:

1. Cut the part 2-11J with EcoRI and SpeI, and the part 3-12O with XbaI and PstI.
2. Ligate them into the plasmid PSB1T3 (1-7A) and we obtain the part PRM-RFP.
3. Cut the part 2-14A with EcoRI and SpeI, and the part 1-10C with XbaI and PstI.
4. Ligate them into the plasmid PSB1K3 (1-5A) and we obtain the part pLL-cI.
5. Cut the part 2-12G with EcoRI and SpeI, and the part 1-10G with XbaI and PstI.
6. Ligate them into the plasmid PSB1K3 (1-5A) and we obtain the part phiR73-c22.
7. Cut the part pLL-cI with EcoRI and SpeI, and the part pRM-RFPwith XbaI and PstI.
8. Ligate them into the plasmid PSB1A3 (1-1G) and we obtain the part pLL-cI-pRM-RFP.
9. Cut the part 1-14N with EcoRI and SpeI, and the part phiR73-c22 with XbaI and PstI.
10. Ligate them into the plasmid PSB1T3 (1-7A) and we obtain the part pBad-phiR73-c22.
11. Cut the part pBad-phiR73-c22 with EcoRI and SpeI, and the part pLL-cI-pRM-RFP with XbaI and PstI.
12. Ligate them into the plasmid PSB1K3 (1-5A) and we obtain the low concentration circuit.

 

Until now

• We had some problems transforming the part 1-14N. We ran out of DNA of this biobrick so we contacted with the IGEM team from the Tecnológico de Monterrey and they gave us some DNA. But it didn’t work when we try to transform it.
• We have DNA in tubes stored and others in the refrigerator of the lab to use them continuously.
• We have cut all the biobricks but after certain time they tend to degradate so we have to do it continuously.
• The Ligation 3 is already done. This is composed by the parts: 2-12G and 1-10G.

 

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iGEM High School Division, 2012 edition - CIDEB-UANL_Mexico team