The overall goal of the following experiment is to integrate a desired DNA construct into the bacterial chromosome at a predetermined locus. This is achieved by a mating protocol involving four engineered bacterial strains. The recipient strain has a landing pad sequence consisting of a spec mycin resistance gene and the e coli beta glucuronidase gene flanked by ATP sites integrated into its chromosome.
The P JH one 10 donor strain carries a plasmid containing at B sites flanking a stuffer red fluorescent protein gene, and an antibiotic resistance gene. The PJC two donor strain carries a plasmid containing the site-specific integrase from the streptomyces phage C 31 under the control of the LAC promoter. The fourth strain MT 616 provides the transfer genes required for conjugation the transfer of plasmids from cell to cell, results in the creation of trans through the recipient strains.
Acquisition of the two plasmids required for integrase mediated cassette exchange. The exchange of the donor cassette from the PJ H one 10 plasmid with the markers of the landing pad cassette on the chromosome results in the loss of the landing pad markers, SPR and UIDA and the maintenance of the donor cassette RFP and GMR in the resulting trans immigrant. The main advantage of this technique over other methods like homologous recombination is the ease and efficiency of the protocol.
It is also transferable to a diverse range of bacteria. The acceptor strain used in this video is an S milli latte strain two days prior to the mating procedure for creating trans integrins inoculate from a single colony into five milliliters of TY media with spect mycin incubate the S milli strain at 30 degrees Celsius for two days with shaking on the day before the mating procedure inoculate each of the following e coli strains into five milliliters of LB liquid media supplemented with the appropriate antibiotic DH five alpha containing the integrase expression plasmid into media with tetracycline MT 616 the mobilizer intermedia with chloramphenicol and DH five alpha containing the donor cassette plasmid into media with Gentamycin incubate the three e Coli strains overnight at 37 degrees Celsius with constant shaking to prepare the cells for the mating procedure. Transfer 1.5 milliliters of each of the four cultures into a tube and centrifuge at 17, 000 times G for 30 seconds to collect the cell pellet, discard the media and resuspend each cell pellet in one milliliter of sterile 0.85%sodium chloride centrifuge the tubes again after discarding the supernatant Resus suspend each pellet in 100 microliters of sterile 0.85%sodium chloride using a pipette individually spot 10 microliters of washed culture for each strain on a plain TY agar plate.
These spots are control spots. Next, add 40 microliters of each strain, excluding the e coli DH five alpha containing Pjc two in a sterile tube and mixed by pipetting up and down spot 120 microliters of this mixture on the plain TY agar plate. This spot is the no integrase negative control.
Finally, add 40 microliters of each of the four strains in a sterile tube mix and spot. 160 microliters of this mixture on the same TDY agar plate. This spot is the integrase mediated cassette exchange mating spot.
Place the plate in a laminar flow hood and allow the spots to dry for about 20 minutes after that. Seal the plate and incubate at 30 degrees Celsius overnight the day after the mating protocol streak. The two control spots and the IMCE mating spot onto TY agar plates supplemented with streptomycin and gentamycin.
The S milli acceptor strain carries a mutation conferring high level resistance to streptomycin for calculation of the IMCE efficiency Resus suspend approximately one quarter of the IMCE mating spot in 500 microliters of sterile 0.85%Sodium chloride make tenfold serial dilutions to 10 to the negative seventh plate Dilutions tend to the negative two through 10 to the negative five on TY agar plates supplemented with streptomycin, gentamycin, and xgl to select for trans immigrants. Plate dilutions tend to the negative three through 10 to the negative seven on TY agar plates, supplemented with streptomycin and xgl to select for both trans immigrants and potential recipients. IE total recipients incubate plates at 30 degrees Celsius for three days.
View RFP trans under green light and through a red filter calculate the percent IMCE efficiency as CFU of trans immigrants compared to the CFU of total recipients, approximately half of trans will have undergone true exchange making them white and insensitive after three days of incubation on Ty supplemented with streptomycin and Gentamycin, there should be no growth in the following Control strains, no integrase control s milli latte UW 2 27 control e coli MT 616 control e coli DH five alpha containing P JH one 10 control and e coli DH five alpha containing PJC two control. In contrast, the IMCE streak from the mating spot should have confluent growth on the head streak and many colonies on the second streak. These next two images show resus suspensions of a 10 to the negative two dilution of the mating spot on TY streptomycin xgl agar, and on TY streptomycin Gentamycin xgl agar on the TY streptomycin Gentamycin xgl agar.
The blue colonies are single recombinants. While the white colonies are trans integrins that have undergone true cassette exchange when viewed under green light and through a red filter, the tend to the negative two dilution of mating spot resus suspension on TY strept cin xgl agar lacks fluorescence. In contrast, the 10 to the minus two dilution of mating spot resus suspension on Ty streptomycin Gentamycin Xgl Agar shows two levels of fluorescence.
The brighter colonies correspond to blue colonies and have higher RFP expression, presumably due to promot a read through from the LAC promoter in the vector. The less bright colonies correspond to white colonies, which have undergone true cassette exchange and contain RFP with only its immediate promoter and no read through from the LAC promoter. These trends immigrants should also be specks mycin sensitive.
The efficiency of trans integration expressed as the percentage of trans immigrants to total recipients should be in the range of 0.5%After watching this video, you should have a good understanding of how to use bacterial conjugation using our engineered strains to insert DNA constructs into the bacterial chromosome.
