IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/08/16

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Working on LovTAP synthesized plasmid from Mr. Gene

Restriction enzyme assay:Test

After finishing the isolation of LovTAP plasmids from Mr.Gene that were transformed, I have started the restriction enzyme assay in order to test if the plasmids obtained are correct .

  • Restriction enzymes Mixture:

Restriction enzymes: EcoRI and PstI.

Reactive Quantity
Plasmid DNA 5μL
Buffer 2 2μL
BSA 1μL
Enzymes 1μL for each one.
HPLC Up to a final volume of 20 μL of the mixture

According to the next image, the band between 750bp and 1000bp observed in Lane 6 and 7 is around the expected size of LovTAP that should be 889bp, considering the cutting pattern using EcoRI and PstI enzymes. The other two bands around 1500bp and 650bp could correspond to fragments of the plasmid sequence from Mr.Gene, which size is 2262bp, thus suggesting that inside the plasmid there is a restriction site for EcoRI or PstI.

PCR colony trpR- and LovTAP restrictions. Lane1:Ladder. Lane2:E.coli k12 wt. Lane3:trpR- colony 2. Lane4: trpR- colony 3. Lane5: trpR- colony 9. Lane6:LovTAP colony 1. Lane7:LovTAP colony 5.Lane8:LovTAP colony 9.
PCR colony trpR- and LovTAP restrictions. Lane1:Ladder. Lane2:E.coli k12 wt. Lane3:trpR- colony 2. Lane4: trpR- colony 3. Lane5: trpR- colony 9. Lane6:LovTAP colony 1. Lane7:LovTAP colony 5.Lane8:LovTAP colony 9.

Restriction enzyme assay:Preparation for LovTAP Ligation with promoters

The mixture was made by duplicated.

  • Restriction enzymes Mixture:

Restriction enzymes: XbaI and PstI.

Reactive Quantity
Plasmid DNA 15μL
Buffer 2 4μL
BSA 1μL
Enzymes 2μL for each one.
HPLC Up to a final volume of 40 μL of the mixture
LovTAP restriction enzyme assy XbaI and PstI. Lane1:Ladder. Lane2 and Lane3: LovTAP Colony 1. The other lanes are samples from other experiments
LovTAP restriction enzyme assy XbaI and PstI. Lane1:Ladder. Lane2 and Lane3: LovTAP Colony 1. The other lanes are samples from other experiments

LovTAP extraction from gel

Taking into account the aformentioned results we have decided to extract from the gel the band corresponding to LovTAP that is around 882nt, and once the band is purified we are going to do the ligation reactions with the respective promoters and plasmid pSB1C3.

Working on LovTAP promoters:Transformation

Continuing working with the promoters for LovTAP, I had to repeat the transformations for J23110 and J23117. As well I decided to choose more medium and weak promoters, they are underline in the next table:

Strength Entry Plate Well
Medium J23105 2010_1 18M
Medium J23110 2009_1 20C
Medium J23106 2010_1 18O
Low J23117 2010_1 20O
Low J23114 2010_1 20I
Low J23116 2010_1 20M

All these promoters are contained inside J61002 plasmid, and control the expression of RFP protein. Besides, the plasmid harbors an ampicillin resistance.

Experimental Setup

1.Get the plasmids harboring each promoter, from the corresponding plates.

2.Transform cells.

3.Grow up the transformed cells in LB medium with ampicillin antibiotic. This is because the plasmids harboring each promoter have a resistance against ampicillin.

Reactive Quantity
Ampicillin 1μL for each LB medium mL

Results:LovTAP promoters

  • Promoter J23105 was successfully transformed. Three colonies were re-cultured and produce RFP protein. I will extract the plasmid from one of them.
  • Promoter J23110 was successfully transformed. Three colonies were re-cultured and produce RFP protein. I will extract the plasmid from one of them.
  • Promoter J23106 was successfully transformed. Three colonies were re-cultured and produce RFP protein. I will extract the plasmid from one of them.
  • Promoter J23117 was successfully transformed but none colony seems to produce RFP protein yet. We think that the production is very low given that is the weaker promoter that was chosen, so that the colonies were re-cultured and incubated at 37º C until the RFP production is detected.
  • Promoter J23114 was successfully transformed. Two colonies were re-cultured and produce weakly RFP protein. I will extract the plasmid from one of them.
  • Promoter J23116 was successfully transformed. Three colonies were re-cultured and produce weakly RFP protein. I will extract the plasmid from one of them.



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