IGEM:UNAM/2009/Notebook/Modeling logbook Claudia/2010/09/03

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Working on LovTAP fused to promoters:Ligations to backbone pSB3K3 ans pSB1C3

Once the plasmid pSB3K3 was correctly digested with the enzymes EcoRI and PstI, I have started the dephosphatation reaction in order to prepare it for ligation with promoters-LovTAP and BBa_K098991(cI regulated promoter+RBS+GFP) constructions.

  • Dephosphatation mixture
Reactive Quantity
DNA 15μL
Buffer 10X 3μL
Dephosphatase Enzyme 1.5μL
HPLC Up to a final volume of 30 μL of the mixture (10.5μL)


Procedure

1.Incubate the samples at 37°C during 20 min.

2.Incubate the samples at 65°C during 10 min.

Ligation Procedure:LovTAP-Promoters and BBa_K098991 to plasmid pSB3K3 and pSB1C3

1. Prepare the ligation mixture taking into account the quantity of the DNA inserts -LovTAP+Promoters and BBa_K098991- and the receiver DNA -plasmid pSB3K3/pSB1C3-. This can be check in the following gel.

Ligations LovTAP+promoters and BBa_K098991 to plasmid pSB3K3..Lane1:Ladder.Lane2:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated replica 1.Lane3:Plasmid pSB3K3 (EcoRI/PstI)dephosphatated replica 2.Lane4: Plasmid pSB1C3 (EcoRI/PstI)dephosphatated .Lane5:Plasmid LovTAP+J23102 (EcoRI/PstI).Lane8: Plasmid LovTAP+J23105 (EcoRI/PstI).Lane9:Plasmid LovTAP+J23114 (EcoRI/PstI).Lane10:Plasmid LovTAP+J23117 (EcoRI/PstI).Lane11:LovTAP Mr.Gene PCR amplified product (EcoRI/PstI). Lane12:BBa_K098991 (EcoRI/PstI.
  • Ligation mixture using plasmid pSB3K3
Reactive Quantity
DNA insert (LovTAP-J23117, LovTAP-J23105, LovTAP-J23114, LovTAP-J23102 and K098991) 6μL
DNA plasmid (pSB3K3) 3μL
Buffer 10X T4 Ligase 2μL
Enzyme T4 Ligase 1μL
HPLC Up to a final volume of 20 μL of the mixture (8μL)
  • Ligation mixture using plasmid pSB1C3
Reactive Quantity
DNA insert (LovTAP PCR amplified product from Mr.Gene) 6μL
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 (8μL)


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 LovTAP ligated to each promoter and BBa_K098991, if the ligation was correctly done.

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 EcoRI and SpeI and K098991 (cI regulated promoter+RBS+GFP) 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. The plasmid harboring J23101 promoter will be digested with SpeI and PstI.

Part Lenght
K098991 (cI regulated promoter+RBS+GFP) 933 bp
P0451(RBS+cI repressor) 930bp
promoter J23101 38 bp
  • Restriction enzymes EcoRI and SpeI

Plasmid digested: Plasmid harboring P0451 isolated from colony 6.


  • Restriction enzymes XbaI and PstI

Plasmid digested: Plasmid harboring K098991 isolated from colony 8.


  • Restriction enzymes SpeI and PstI

Plasmid digested: Plasmid harboring promoter J23101.

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


The reactions were incubated at 37°C overnight.

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

Results restriction enzyme assays

Quantity loaded 3μL

Restriction enzyme assays. Lane1:Ladder. Lane2:Plasmid pS1C3. Lane3:Plasmid harboring promoter J23101 (SpeI/PstI).Lane4:P0451 colony 6 (EcoRI/SpeI) .Lane5: K098991 colony 8 (XbaI/PstI).

The sizes of the digested products are around the expected lenght for each part.

Working on Lumazine synthesized from Mr.Gene

I am going to do PCR reactions in order to amplify the Lumazine construction synthesized.

The product obtained will be purified using the High Pure PCR Product Purification kit from Roche.


Primers:

Forward:Preffix primer

Reverse:Suffix primer


PCR template:

Plasmid harboring Lumazine from Mr.Gene

  • Reaction 1
Reactive Quantity
MgCl2 3μL
Buffer 3.3X 6μL
dNTP's 3μL (0.4mM)
Primer Forward 2.5μL (5pmol/μL)
Primer Reverse 2.5μL (5pmol/μL)
DNA template(Mr.Gene Plasmid Lumazine 1μL + 9μL HPLC) 1μL
HPLC Up to a final volume of 30 μL of the mixture


  • Reaction 2
Reactive Quantity
Buffer 3.3X 9μL
RTTH enzyme 0.5μL
HPLC Up to a final volume of 20 μL of the mixture


  • 35 PCR programmed cycles:

The reaction starts at 95°C during 5 min. After this step, the reaction 2 is loaded to the reaction 1 sample.

Each cycle is programmed as follows:

Temperature Time
94°C 45s
60°C 45s
72°C 1 min

After the cycle 35, the temperature changes to 72°C during 10 min and ends at 4°C.

Results: Lumazine PCR from Mr. Gene

Lumazine construction was well amplified. The PCR product from each replica was around the expected size 805nt.

Lumazine Mr.Gene PCRs. Lane1:Ladder. Lane4: Lumazine PCR. Lane5:Lumazine PCR. The other lanes are samples from other experiments