The aim of this procedure is to investigate the functional relationships between bacterial genes and pathways. This is accomplished by first preparing a arrays of ceia coli, single mutant strains that will be conjugated or mated. The second step is to conjugate the single mutants in an array format on plates.
Next, the double mutants are selected. The final step is to image the double mutant plates and quantize double mutant colonies to determine strain fitness. Ultimately, the colony size data are analyzed to calculate genetic interaction scores and identify functionally interacting genes, pathways and processes.
The main advantage of this technique over existing methods like random mutagenesis, is that many individual double mutant strains can be created. Moreover, the interactions between many genes can be assessed simultaneously irrespective of gene size, essentiality, or single gene mutant fitness. This method can help answer key questions in the systems biology field, such as those relating to gene functions and functional crosstalk between pathways and processes.
The implications of this technique extend toward therapy of bacterial infections because knowledge about most functionally central genes and processes, as well as the understanding of which combinations of bacterial genes, pathways and processes when perturbed improve or reduce bacterial fitness will help develop the most effective intervention strategies To begin this protocol. Two-step nested. PCR amplification is employed to replace the target open reading frame with a chloramphenicol resistance marker from the PKD three plasmid as described in the written procedure.
Confirm the expected product and PCR purify the obtained fragment following the manufacturer's protocol. After eluting the PCR product in 30 microliters of sterile distilled water dilute to approximately 50 milligrams per microliter concentration. The purified product is stored at minus 20 degrees Celsius prior to transformation.
Next, prepare the high frequency of recombination or HFR Caval competent cells for donor construction. First inoculate one milliliter of a saturated overnight culture of the HFR Cavalli strain into 70 milliliters of fresh LB medium with 35 microliters of 50 milligrams per milliliter. Ampicillin in a 250 milliliter flask.
Incubate the culture at 32 degrees Celsius by shaking gently at 220 RPM until an optical density of about 0.5 to 0.6 is obtained. Then transfer the culture to a water bath for heat induction of the Lambda Red Recombination system at 42 degrees Celsius for 15 minutes with shaking at 160 RPM to stop the induction. Transfer the culture to a chilled ice slurry water bath for 10 to 20 minutes at 160 RPM.
Make sure to keep the cells cold until after the transformation. After washing and harvesting the cells by centrifugation as described in the written protocol, decant the senna and resuspend the pellet in 500 microliters of ice cold, 10%glycerol Eloqua. The cells in 50 microliter volumes into individual pre chilled 1.5 milliliter micro centri tubes and immediately proceed to transformation.
Add 100 nanograms of purified gene deletion, cassette to the competent cells. Flick the tube and allow the suspension to sit on ice for five minutes. Then transfer the suspension to a pre chilled electroporation vete electro, operate the cell mixture immediately.
Add one milliliter of room temperature SOC medium, and transfer the electroporated cells in medium into a 15 milliliter culture tube. Incubate the cells at 32 degrees Celsius for one hour with orbital shaking at 220 RPM. Following incubation, centrifuge the cells of 4, 400 times G for five minutes.
At room temperature, remove approximately 850 microliters of the supinate and re suspend the cell pellet in the remaining liquid. Spread the cells on LB plates containing 17 micrograms per milliliter, chloramphenicol and incubate at 32 degrees Celsius overnight. The following day streak out two individual transformants on LB chloramphenicol plates for mutant confirmation streak out the same transformants on LB can mycin plates to confirm that the strains are not can mycin resistant.
Next, the DNA is amplified with three different sets of knockout confirmation primers as described in the written protocol. The first primer set consists of a 20 nucleotide flanking forward primer located 200 base pairs upstream of the targeted region, and a reverse primer, which is complimentary to the chloramphenicol resistance cassette. This amplification is expected to produce a 445 nucleotide amplicon.
The second set includes a forward primer, a kneeling to the chloramphenicol resistance cassette sequence, and a reverse flanking confirmation primer, which is designed to ane 200 base pairs downstream of the three prime end of the deleted gene. This amplification reaction is expected to produce a 309 nucleotide amplicon. The third PCR contains both upstream and downstream flanking primers.
This reaction is required to confirm that the selected strain is not a marrow diploid with one gene locus having been replaced by the cassette and another duplicated, but otherwise wild type gene copy still present. This amplification is expected to produce a 1.4 KB product. The e coli ke io single non-essential gene deletion mutant collection is replica pind robotically to 24 384.
Well, microplate containing 80 microliters of liquid LB medium per well. Supplemented with 50 micrograms per milliliter can mycin to make room for the border control strain, which were lack as a positive control and aid in plate normalizations as well as in automated colony quantization. Remove the inoculated media from the outermost wells of each plate and transfer it to new plates, leaving the outermost wells empty.
Likewise create negative control spots by removing any two strains from a different location within each plate and transferring the strains to a new plate. Next, fill the empty wells, including the border control wells and the negative control wells with LV media containing 17 micrograms per milliliter of chloramphenicol. These negative control spots are expected to be empty in the recipient and therefore double mutant plates.
They serve to ensure that there was no processing or plate handling errors when numbering imaging or pinning the plates. After preparing the border positive strain as described in the written procedure, the array strains as well as the border positive strain are grown overnight at 32 degrees Celsius with 190 RPM orbital shaking the following day, fill the border wells with approximately 80 microliters of the overnight culture. The assembled plates may be used for mating as described in the next section.
Alternatively, for long-term storage, each well in the recipient plates is supplemented with 15%glycerol and the media and glycerol are mixed. The plates are then stored at minus 80 degrees Celsius. Begin the mating procedure by growing the HFR donor strain, bearing the query gene deletion marked with chloramphenicol resistance cassette overnight at 32 degrees Celsius in rich LB liquid medium with 17 micrograms per milliliter of chloramphenicol pin.
The TH ordered recipient mutant collection in 3 84 colony density onto solid LB plates. Supplemented with 50 micrograms per milliliter of can mycin. In parallel pin the donor query mutant strain in 3 84 colony density onto the same number of LB plates.
Supplemented with 17 micrograms per milliliter of chloramphenicol. Incubate the plates overnight at 32 degrees Celsius to conjugate the strains. Pin the donor from the 3 84 colony overnight donor plates onto solid LB plates.
Then pin the strains from a single 3 84 colony overnight recipient plate over the freshly pinned donors. Incubate the pinned conjugation plates at 32 degrees Celsius for 16 to 24 hours. Next, pin each 384 density conjugation plate onto a single solid LB plate containing chloramphenicol.
And can mycin repeat this process until all conjugation plates have been pinned? Incubate the freshly pinned first selection plates for 16 to 36 hours at 32 degrees Celsius. Following incubation, re-pin each first selection plate onto a second double drug selection plate in 1536 SPOT format so that each first selection colony is represented by four colonies.
On the second selection plate, incubate the plates for 16 to 36 hours. At 32 degrees oss photograph the final double mutant selection plates to quantitatively measure the growth fitness of the mutants and to analyze the interactions between gene pairs. Since genes in the same pathway display closely correlated GI patterns, functionally related genes can be grouped by clustering them according to the overall similarity of their S score profiles.
The GI profile similarity represents the congruency of the phenotypes upon mutation of two genes and should be indicative of whether the two genes act in the same or overlapping pathway. Shown here is the distribution of correlation coefficients between the GI profiles of gene pairs encoding physically interacting proteins versus randomly drawn gene pairs. An example of a scatterplot of correlated genetic profiles for two transporters that form a heterotrimeric complex required for sperm excretion is shown as with yeast ecoline gene exhibiting.
Alleviating interactions with highly correlated GI profiles tend to encode pathways that are physically associated or that act coherently in a common biochemical pathway. Here a representative example is shown where the components of the functionally redundant ISC and SUF iron sulfur biosynthesis pathways, which jointly participate in an essential process form distinct clusters that are linked together by extensive aggravating interactions. A statistical measure can be used to find any significant enrichment for interactions between specific combinations of pathways, integration of GI networks with physical interactions and other functional association data, such as genomic context.
Relationships can reveal the organization of higher order functional modules that define core biological systems in bacteria. Cluster analysis can also be applied to GI networks to predict the functions of annotated genes. Since clustering algorithms vary.
However, putative functional assignments determined through clustering require independent experimental verification. While attempting this procedure, it is important to remember to include all necessary positive and negative Following this procedure. Other methods like proteomics, biochemical follow-up experiments and chemical genomics can be performed in order to answer additional questions about specific gene functions as well as the nature of functional relationships between genes and pathways.
After watching this video, you should have a good understanding of how to construct double mutant strains, assemble recipient arrays, and perform ESGA screens for investigating the functional relationships between genes and processes in e coli.
