The overall goal of this procedure is to knock down genes of interest in a growing population of E.coli. Gene knockdowns are often used to explore basic questions of gene function. These methods may also have application in synthetic biology.
For example, in silencing unwanted genes like virulence factors. The main advantage of this technique is that it can be performed in batch cultures of E.coli without prior genetic modification, with results observable just after a few hours. To begin, using a plasmid containing an sRNA expression cassette designed according to the text protocol, carry out site-directed mutagenesis on the sequence by first identifying the 24-basepair guide sequence in the expression cassette.
Design and synthesize forward and reverse primers with short regions of homology to the existing sRNA cassette, flanking the new 24-basepair guide sequence. Prepare a five milliliter culture of E.coli in LB and antibiotics carrying the template sRNA expression phagemid. Grow the cells overnight at 37 degrees Celsius with shaking.
After extracting and purifying the template sRNA phagemid from the culture, prepare two PCR reactions, using 10 to 50 times the recommended amount of DNA template for the sRNA phagemid and high fidelity polymerase. Prepare one reaction with the forward and one with the reverse primer. After using standard PCR conditions to run the reactions, combine the two reactions in a microcentrifuge tube.
Then anneal the products by heating to 98 degrees Celsius in a boiling waterbath. Immediately turn off the heat source and allow the bath to slowly return to room temperature over the next one to two hours. To eliminate the unmutated template sRNA, add one microliter of DpnI restriction enzyme to the mixture, and incubate at 37 degrees Celsius for one hour, or for the time recommended by the manufacturer for complete digestion.
Next, transform chemically competent E.coli cells with one to five microliters of the annealed PCR product. Then isolate single colonies by selective plating on LB plates with antibiotics. To verify the incorporation of the correct guide sequence with colony PCR.
use a 200 microliter pipet tip to collect a small quantity of cells from a single transformed colony. Add the collected cells to 50 microliters of nuclease-free water in a microcentrifuge tube and mix by pipetting. Then with a benchtop thermocycler, or a boiling waterbath, lyse the cells by heating to 95 degrees Celsius for two minutes.
Using one microliter of the lysed cells as the DNA template, carry out PCR according to the conditions shown here. After verifying the correct guide sequence, inoculate a five milliliter culture with the correct sRNA clone, and grow the cells overnight. The following day, prepare a glycerol stock by combining 750 microliters of the overnight culture with 250 microliters of 60%glycerol in a screwcap cryotube.
To prepare M13-packaged phagemid stocks, prepare a five milliliter overnight culture of E.coli, carrying the sRNA expression phagemid. Also, prepare a five milliliter overnight culture of E.coli, carrying the M13KO7 helper plasmid. The following day, after purifying the DNA, co-transform chemically competent E.coli with one microliter each of the sRNA expression phagemid and helper plasmid.
Plate on LB-agar with antibiotics to select for both constructs. Phagemid expression is a large fitness burden for the cells and may result in lowered transformation efficiency. It may be necessary to introduce the plasmid in two sequential transformation steps.
After incubating the transformed cells overnight at 37 degrees Celsius, prepare a 10 milliliter culture from a single colony of the co-transformed strain. The following morning, centrifuge the culture at 3300 x g for 10 minutes. Collect the supernatant and filter through a 0.2 micron filter.
Store the packaged phagemid filtrate at four degrees Celsius. After preparing F+target cells, according to the text protocol, inoculate a single colony into a five milliliter culture of LB medium with antibiotics, and grow overnight at 37 degrees Celsius with shaking. The following day, use five milliliters of selective LB medium to dilute the F+target cells one to one hundred, and continue incubating.
Culture the cells for about two hours until an OD600 of 0.3 is obtained, indicating log phase growth. Cells targeted for silencing should be in exponential phase when expression of the F pilus silence for efficient phagemid infection. Add a one to one hundred ratio of M13-packaged phagemids to the target cells to achieve a nearly 99%infection of the target population.
Allow the infection to proceed at 37 degrees Celsius with shaking for 30 to 60 minutes. Then assay the sRNA silencing phenotype as desired. Following the protocol demonstrated in this video, the sRNA silencing cassette of plasmid pAB.
001 was altered to target to mKate2. This figure demonstrates that the E.coli strain infected with the anti-mKate2 phagemid showed no detectable mKate2 fluorescence over background. This strain did, however, express GFP, indicating successful uptake of the phagemid.
In this experiment, an E.coli strain with a chromosomally integrated chloramphenicol acetyltransferase, or CAT gene, was infected with a phagemid targeting CAT, and sRNA silencing of chloramphenicol resistance was tested. Cells in which the phagemid targeted CAT showed reduced survival at low chloramphenicol concentrations, and nearly 99%killing at higher concentrations. On the other hand, uninfected cells, or cells infected with sRNA that silenced mKate2, were resistant to chloramphenicol at all concentrations tested.
As shown here, the addition of ampicillin, used to select for only bacteria carrying the phagemid, reduced chloramphenicol survival to undetectable levels. Changing the sRNA target gene and producing infected phagemid can be done in five days. Don't forget that working with phages can be extremely hazardous for other experiments in the lab, and precautions, such as using dedicated labware, should always be taken while performing this procedure to avoid unwanted contamination.
After watching this video, you should have a good understanding of how to design, clone and produce infected phagemids, bearing an sRNA to knock down any gene of interest in an actively growing population of E.coli.
