Team:UANL Mty-Mexico/Notebook/Integration

This procedure is described in the Protocols section. We started from the JT2 strain, provided by Dr. Jeff Tabor, as its genotype has already been modified to be properly light-induced.

1. Take one aliquot of the electrocompetent cells and add 500 ug of pTKRED 
2. Incubate for one minute in ice
3. Place the aliquot on the electroporator and  adjust the device at 2.5 kV, 25 mF and 200 Ohms
4. Give the pulse
5. Remove the cells of the electroporator device and add 1 mL of SOC medium
6. Incubate for 1 hour
7. Growing cells (500 μL) in LB agar plates supplemented with tetracycline (5 μg/mL) and incubate overnight at 30 ^oC.
8. Inoculate one colony in 5 mL of LB medium with tetracycline, extract the plasmid DNA (Miniprep) and characterize using EcoRV.

1. Using pTKS/CS as a PCR template, amplify landing pad fragments using the landing pad regions as standardize priming sites, following the next conditions:
1. 95 ^oC for 30s
2. 98 ^oC for 35 cycles of 15s
3. 55 ^oC for 15s
4. 72 ^oC for 30s
2. The resulting PCR reaction should be digested with 1 mL DpnI per 50 mL PCR 2 hours at 37 ^oC
3. Using the above protocol, generate electrocompetent  TKRED  cells
4. Take one aliquot of the electrocompetent TKRED cells and add 100 μg of amplified landing pad fragment 
5. Incubate for one minute in ice
6. Place the aliquot on the electroporator and  adjust the device at 2.5 kV, 25 mF and 200 Ohms
7. Give the pulse
8. Remove the cells of the electroporator device and add 1 mL of SOC medium
9. Incubate for 1 hour
10. Growing cells (500 μL) in LB agar plates supplemented with tetracycline (10 μg/mL), spectinomycin (100 μg/mL) y glucose 0.5%
11. The other 500 μL of the electroporated should be allow to sit overnight and grown in LB the next day.
12. Potential integrants were picked onto LB plates supplemented with 34 mg/ml chloramphenicol to screen against colonies which had been transformed with undigested pTKS/CS plasmid.
13. The colonies grown must have integrated the landing pad fragment

1. Inoculated individual colonies electroporated with the pTKIP plasmid (following the protocol said before) in 5 mL of M9 minimal medium with IPTG 2 mM and L-arabinose  0.2 % w/v.
1. The M9 minimal medium prepared according to the following specifications:
1. To make M9 Salts aliquot 800ml H2O and add:
1. 64g Na2HPO4-7H2O
2. 15g KH2PO4
3. 2.5g NaCl
4. 5.0g NH4Cl
5. Stir until dissolved
6. Adjust to 1000ml with distilled H2O
7. Sterilize by autoclaving
2. Measure ~420 ml of distilled H2O (sterile)
3. Add 500 ml of M9 salts (While it’s still hot)
4. Add 20 ml of 0.1M MgSO4 (sterile)
5. Add 10 ml of 40% glycerol (or other carbon source)
6. Add 200 μl of 0.5M CaCl2 (sterile)
7. Adjust to 1000ml with distilled H2O
2. Incubate for 1 hour at 37  °C in a Shaking Water Bath
3. Add 100 μg/mL of spectiomycin
4. Incubate for 4 hours at 30 oC in a Shaking Water Bath
5. Add 100 μg/mL of hygromicin and incubate overnight
6. Dilute the sample to 10⁵ and plated 500 ml on LB plates with hygromycin (100 μg/ml) and grown at 37 ° C overnight
7. Potential integrants are picked and screened on LB plates containing 100 μg/ml ampicillin or 10 μg/ml tetracycline to verify the loss of the landing pad and donor plasmid.