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./p>

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

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.

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: