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

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.

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:

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.

 

• 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.