Genomic Insertion Protocol

This genomic insertion protocol utilizes CRIM technology and was modified by the Anderson Lab at UC Berkeley. For more information on the technology, you can read the article on CRIM technology.

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 containing BBa_K091206 for insertion. This is where your part will be.

pInt80-649.

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:

1. 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.
2. Ligate pG80ko (cut with E/P) together your part (cut with E/P).
3. 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.
4. Plate the transformation on gentamicin plates at 15ug/ml
5. Grown up a colony from the transformation, miniprep it, and perform a digestion to verify successful ligation. Save the miniprep!

1. Y

Note that you'll need a pir strain for replication of pG80 plasmids. You can drop your biobrick into the Eco/pst sites of pG80ko, transform the pir116 cells. Grow up, miniprep, and map a single colony. Make competent cells of pInt80-649 in your target strain (plate them on Amp). It is temperature sensitive, so do all growth manipulations at 30 degrees. Transform in your pG80 derivative, plate on 15ug/mL gentamicin plates at 37 degrees. Grow a single colony to saturation at 37 in LB+15ug/mL gentamicin, then restreak on a gentamicin plate at 43 degrees. You can use the oligos below to PCR amplify the phi80 locus for confirmation of integration and sequencing.

attPhi80-1: CTGCTTGTGGTGGTGAAT

attPhi80-2: ACTTAACGGCTGACATGG

attPhi80-3: ACGAGTATCGAGATGGCA

attPhi80-4: TAAGGCAAGACGATCAGG

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