IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/28

{| width="800"
 * style="background-color: #EEE"|[[Image:TeamLogo.jpg|350px]] UNAM-Genomics-Mexico team
 * style="background-color: #F2F2F2" align="center"|  |Main project page
 * style="background-color: #F2F2F2" align="center"|  |Main project page


 * colspan="2"|
 * colspan="2"|

Working on cI inverter construction and fusion to pSB3K3 backbone. LovTAP repressor activity reporter system
Once the plasmids harboring the parts showed in the next table were correctly isolated,  I am going to digest P0451(RBS+cI repressor) with SpeI and PstI, this will be ligated to the Part K098991 (cI regulated promoter+RBS+GFP)  digested with XbaI and PstI in order to fuse them to construct the whole cI inverter. Then it will be ligated to trpL and J23101 promoters.

Plasmid digested: Plasmid harboring P0451 isolated from colony 6.
 * Restriction enzymes SpeI and PstI

The reactions were incubated at 37°C overnight.

Note: Inactivate the enzymes at 80°C during 20 min.

Repeated experiment:Working on cI inverter construction and fusion to pSB1C3 backbone. LovTAP repressor activity:reporter system
Due to the first attempt to ligate  K098991 (cI regulated promoter+RBS+GFP) and P0451(RBS+cI repressor) inside plasmid pSB1C3 failed, I am going to repeat experiment.

Ligation Procedure: P0451 + K098991 to plasmid pSB1C3
1. Prepare the ligation mixture taking into account the quantity of the DNA insertS -K098991 and P0451- and the receiver DNA -plasmid pSB1C3-. This can be check in the following gel. Quantity loaded 3μL.






 * Ligation mixture 1

2. Incubate the sample at 16°C overnight.

3. Transform the cells. Click here for the protocol.

4. Culture the cells in the proper selective medium, in our case LB + Kanamycin.

5. Incubate the petri dishes at 37°C overnight.

6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight.

7. Analyze the colonies with Colony PCR  to confirm that they contain the correct ligation

The primers that I am using are:

Forward (5'->3'): Preffix primer.

Reverse:(5'->3'): Suffix primer.

These primers would amplify the whole cI inverter, if the ligation was correctly done.

Repeated experiment:Working on J23102 promoter: Ligation to P0451/Lumazine/LuxY
Due to the first attempt to ligate P0451, Lumazine and LuxY to promoter J23102 failed, I am going to repeat the experiment.

Ligation Procedure:Promoter J23102 with P0451 (RBS+cI repressor)/Lumazine and LuxY
1. Prepare the ligation mixture taking into account the quantity of the DNA insert -P0451 (RBS+cI repressor),LuxY and Lumazine- and the receiver DNA -plasmid harboring the promoter J23102-. This can be check in the following gel.






 * Ligation mixture: P0451(RBS+cI repressor)


 * Ligation mixture: Lumazine/LuxY

2. Incubate the sample at 16°C overnight.

3. Transform the cells. Click here for the protocol.

4. Culture the cells in the proper selective medium, in our case LB + Kanamycin.

5. Incubate the petri dishes at 37°C overnight.

6. Re-culture the resultant colonies in the proper selective medium; incubate them at 37°C overnight.

7. Analyze the colonies with Colony PCR  to confirm that they contain the correct ligation

The primers that I am using are:

Forward (5'->3'): Preffix primer.

Reverse:(5'->3'): Suffix primer.

These primers would amplify LuxY, Lumazine and P0451:RBS+cI repressor plus promoter J23101, if the ligation was correctly done.


 * }