IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/30

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Working on LovTAP.Reporter systems
In order to construct the reporter system regulated by LovTAP, I and Zepeda are working together to fuse the promoter trpL with a reporter gene. As we are interested in characterize LovTAP activator and repressor activity. We have designed the following constructions.


 * LovTAP repressor activity: Reporter system

trpL promoter fused to GFP protein:Part:BBa_E0240


 * LovTAP activator activity: Reporter system 

trpL promoter fused to lambda Repressor cI: Part:BBa_P0451 + Part:BBa_K098991 regulating GFP protein:Part:BBa_E0240

Both constructions will be inserted in plasmid pSB3K3 for measurements.

Experimental Setup
1.Insert the trpL promoter through a PCR reaction to the plasmid pSB3K3.

-trpL promoter sequence

tggcaaatattctgaaatgagctgttgacaattaatcatcgaactagttaactagtacgcAagttcacgtaaaaagggtat

Primers designed:

-Primer_trpL_reverse (5'->3'): SpeI site + trpL promoter + XbaI site + EcoRI site GGACTAGTCCCTTGCGTACTAGTTAACTAGTTCGATGATTAATTGTCAACAGCCTCTAGAAGCGGCCGCGAATTC

-Second Primer_trpL_reverse (5'->3'): NheI site + a region of the trpL promoter TTGCTAGCTGAACTTGCGTACTAGTTAACTAGTTCGATG

-Primer forward: Suffix (5'->3'):TAC TAG TAG CGG CCG CTG CAG

-Template: Plasmid pSB3K3


 * LovTAP repressor activity: Reporter system

2.Transform and isolate the plasmid pSB1A2 harboring the part BBa_E0240 (RBS+GFP protein).

3.Fuse the trpL-pSB3K3 construction to the part BBa_E0240.


 * LovTAP activator activity: Reporter system 

4.Transform and isolate the plasmids pSB1AK3 and pSB1A2, harboring the parts BBa_P0451 (RBS+cI repressor) and BBa_K098991(cI regulated promoter+RBS+GFP) respectively. Fuse them to construct the whole cI inverter regulating GFP protein. Then join the inverter to the trpL-pSB3K3 construction.

5. In order to have a positive control of GFP constituve expresion, we decided to use Part:BBa_I20260(Promoter J23101+RBS+GFP). I have to transform this part.

6. Isolate plasmid pSB3K3 for characterization, pSB3K3 backbone will be isolated from part Part:BBa_J04450.

In the next table are all the Parts that will be transformed.

Parts Transformations: Results
In order to test the above transformations I did colony PCRs. Using the same previously described protocol.

Primer Forward: Prefix

Primer Reverse: Suffix

The transformed colonies that were selected to make the colony PCR for each Part were the following:



The sizes of the PCR products are around the expected lenght for each part, but J4450 colony 4 and K098991 colony 7 don´t have the expected parts.

So, at least one colony for each part was correctly transformed. I will extract the plasmids using the previously described protocol.


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