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

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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.

Part Lenght
K098991 (cI regulated promoter+RBS+GFP) 933 bp
P0451(RBS+cI repressor) 930bp
  • Restriction enzymes SpeI and PstI

Plasmid digested: Plasmid harboring P0451 isolated from colony 6.

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


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 P0451 + K098991 to plasmid pSB1C3.Lane1:Ladder.Lane11:P0451 (EcoRI/SpeI).Lane12: K098991 (XbaI/PstI). The other lanes are samples from other experiments.
Ligation P0451 + K098991 to plasmid pSB1C3.Lane1:Ladder.Lane11:P0451 (EcoRI/SpeI).Lane12: K098991 (XbaI/PstI). The other lanes are samples from other experiments.
Backbones pSB3K3 and pSB1C3..Lane1:Ladder.Lane2 and 3:Plasmid pSB1C3 (EcoRI/PstI)dephosphatated II and 2. The other lanes are samples from other experiments.
Backbones pSB3K3 and pSB1C3..Lane1:Ladder.Lane2 and 3:Plasmid pSB1C3 (EcoRI/PstI)dephosphatated II and 2. The other lanes are samples from other experiments.
  • Ligation mixture 1
Reactive Quantity
DNA inserts (K098991+ P0451) 4μL each
DNA plasmid (pSB1C3) 2μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (11μL)

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.

Gel for ligations: Inserts. Lane1:Ladder. Lane3: Lumazine PCR amplified product from Mr.Gene plasmid (XbaI/PstI). Lane4:P0451 colony 6 (XbaI/PstI). Lane5:LuxY purification from Mr.Gene plasmid (XbaI/PstI).
Gel for ligations: Inserts. Lane1:Ladder. Lane3: Lumazine PCR amplified product from Mr.Gene plasmid (XbaI/PstI). Lane4:P0451 colony 6 (XbaI/PstI). Lane5:LuxY purification from Mr.Gene plasmid (XbaI/PstI).
Gel for ligations: Dephosphatated Backbone J23102. Lane1:Ladder. Lane4: plasmid harboring the promoter J23102 dephosphatated (SpeI/PstI). Lane5: plasmid harboring the promoter J23102 dephosphatated (SpeI/PstI). The other lanes are samples from other experiments
Gel for ligations: Dephosphatated Backbone J23102. Lane1:Ladder. Lane4: plasmid harboring the promoter J23102 dephosphatated (SpeI/PstI). Lane5: plasmid harboring the promoter J23102 dephosphatated (SpeI/PstI). The other lanes are samples from other experiments
  • Ligation mixture: P0451(RBS+cI repressor)
Reactive Quantity
DNA insert (P0451:RBS+cI repressor) 3μL
DNA plasmid (plasmid harboring the promoter J23101) 1μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (13μL)
  • Ligation mixture: Lumazine/LuxY
Reactive Quantity
DNA insert (Lumazine/LuxY) 4μL
DNA plasmid (plasmid harboring the promoter J23101) 1μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (12μL)

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.



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