Genomic Insertion Protocol
You will need two plasmids: pG80ko and pInt80-649. pG80ko can be found in the freezer stocks in box 4-1, position 55. It contains a stuffer insert that is about 700 bps and should be replaced with the part you want to insert. pInt80-649 can be found in the freezer stocks in box 4-1, position 51. pG80ko's sequence can be found here. pIn80-649's sequence can be found here.
This plasmid maps are shown below:
pG80ko contains the R6K origin of replication, which is only active in the presence of Pir protein. This plasmid will eventually be inserted entirely into the genome of E. coli. The helper plasmid, pInt80-649, can only be replicated inside E. coli l at temperatures below 43C because the CI857 protein, which is necessary for replication, is inactivated at high temperatures. In order to perform a genomic insertion, the DNA to be inserted is placed on pG80ko using an EcoRI/PstI digestion. This plasmid is transformed into cells that already contain pInt80-649 (and thus express Pir). PG80ko carries an phi80 attP site that allows for recombination with the phi80 attB site in the E. coli genome. This recombination event is aided by the integrase which is expressed by the helper plasmid, pInt80-649. Cells that have undergone a recombination event can be selected by growth on gentamicin plates at 43C. High temperatures cause the cells to stop replication of the helper plasmid and consequently stop production of Pir too. Without Pir, pG80ko is incapable of replication unless the plasmid has been inserted into the genome.
With that background, the steps for performing this insertion are listed below:
- Perform an EcoRI/PstI digestion on the pG80ko plasmid with a stuffer insert and gel purify the vector. Also perform an EcoRI/PstI digestion and gel purification of the part to be inserted. This part is probably contained in an BioBrick standard vector.
- Ligate pG80ko (cut with E/P) together your part (cut with E/P).
- Transform into Ec100D::pir+ cells which are frozen down in the freezer (Box 4-1, position 54). These cells express pir consitiuively to allow for replication of the R6K origin. You will need to do a TSS transformation.
- Plate the transformation on gentamicin plates at 15ug/ml
- Grown up a colony from the transformation, miniprep it, and perform a digestion to verify successful ligation. Save the miniprep!
- You need to pick a cell strain now. I found that MC4100 cells worked, while HB101 cells did not (I was not able to determine why). I would recommend using MC4100 cells if you can.
- Do a TSS transformation of pInt80-649 into the cell strain of your choosing.
- Plate on an Amp plate (50-100 ug/mL) at 30C. This plasmid is temperature sensitive, so never expose the cells to high temperatures (including your 1 hour rescue).
- Pick a colony for growth and do another TSS Transformation with the PG80ko plasmid that you constructed and miniprepped earlier
- Plate this transformation on a gentamicin plate (15ug/mL) at 37C.
- Grow a cell to saturation at 37C in LB+gentamicin (15ug/mL).
- Restreak from the liquid culture on to a gentamicin plate at 43C. These cells should be your integrants!
- Confirm using primers attPhi80-1 and attPhi80-4 (sequence given below - oligos already purchased in the Campbell Lab) to sequence the genomic DNA.
You can also perform PCR on the integrant colonies using the 4 primers shown below. attPhi80-1 and attPhi80-4 bind to the phi80 site in the genome. attPhi80-2 and attPhi80-3 bind to the attB site on the pG80ko plasmid. The expected results are given in the CRIM paper, and shown in the table below. I found that this procedure gave some unexpected results (see gel below) for my controls and would recommend sequencing instead of PCR for confirmation of the integrants.
|sequence Temp (°C)||No integrant with 1 and 4||Single integrant with 1 and 2, 3 and 4||Multiple integrant with 1 and 2, 3 and 2, 3 and 4|
|63||546||409, 732||409, 595, 732|
My PCR results using these primers to confirm integration of BBa_K091206 are shown below. All bands were expected except for the bands in lanes 9-11. These bands are difficult to explain and are the reason that I would recommend sequencing to confirm the integration in the future.