Bacteroides mutagenesis and genomic insertions

The modified suicide plasmid, pExchange tdk, containing a knockout fragment were transformed into S17 λ pir E.coli cells, referred to as the “donor” strain. B. ovatus tdk- or B. thetaiotaomicron tdk- is the “recipient” strain. The donor and recipient strains were cultured (5 ml) to roughly equivalent cell densities in LB broth and TYG media respectively (Figure 2.2a). Cells were harvested by

centrifugation and washed in TYG medium. Equal sized cell pellets were then re-suspended in 1 mL TYG medium and spread evenly on the surface of BHI plates with no antibiotic. These plates were incubated agar side down and grown for 16-24 hours until a thick lawn has formed; E.coli should grow first, creating an anaerobic environment underneath this growth in which Bacteroides can thrive, providing the necessary conditions for plasmid conjugation from the donor to the recipient strains (Figure 2.2b). This biomass was scraped from the plate and re-suspended in 5 ml TYG

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medium. Then 100 µl of this solution, along with three serial dilutions (1:10, 1:100, 1:1000) were plated onto BHI + gentamycin (200 µg/ml) + erythromycin (25 µg/ml) plates. These antibiotics select for the recipient strain and the pExchange tdk plasmid, thus colonies represent single recombinant where the pExchange tdk has recombined with the genomic DNA via one of the flanks. These plates were incubated anaerobically for up to 2 days or until colonies formed, then 10 colonies were picked and re-streaked onto fresh BHI + gentamycin + erythromycin plates to minimise wild type

contamination (Figure 2.2c). Then 10 colonies were cultured overnight in TYG medium, 1 ml of each culture was taken and a pooled stock created. A glycerol stock can be made at this stage for

safekeeping (Figure 2.2d).

The pooled stock alongside three serial dilutions (1:10, 1:100, 1:1000) was plated upon BHI + FUdR (200 µg/ml) and allowed to grow anaerobically for 2 days or until colonies appeared. FUdR is toxic to strains able to synthesise thymidine. The recipient strain lacks the tdk gene, but this has been complemented within the pExchange tdk plasmid, in this manner FUdR selects for the second recombination event, whereby the second flank incorporates into the genome and the pExchange tdk sequence is eliminated. Following this growth, 10 FUdR resistant colonies were re-streaked onto fresh BHI + FUdR plates to minimise wild-type contamination (Figure 2.2e). 10 resistant colonies were picked and cultured in 5 ml of TYG so that genomic DNA could be extracted and glycerol stocks could be made (Figure 2.2f).

Isolated DNA was screened for successful knockout mutations using PCR. The downstream and upstream primers used to create the plasmid (primer 1 & primer 4, Figure 2.1) were used to amplify the clones, using wild-type Bacteroides as a control; the wild-type strain will produce a fragment which is the length of the target gene (500-2000 bp), plus the length of both flanks (1,000 bp each). Any successful knockouts will lack the target gene, yielding a fragment of 2,000 bp. Clones which appeared successful after screening were then sequenced to ensure the correct mutation had taken place.

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Figure 2.2 Generating Knockout Strains of Bacteroides ovatus and Bacteroides thetaiotaomicron. (a)

The donor and recipient strains were cultured in 5 ml of LB and TYG media respectively. (b) Equal size cell pellets were harvested by centrifugation, washed in TYG, combined and re-suspended in 5 ml TYG and plated onto BHI plates containing no antibiotics (yellow). These plates were not inverted during growth. (c) The plates were scraped and the biomass re-suspended in 5 ml TYG. This was plated onto BHI plates containing gentamycin (200 µg/ml) and erthyromycin (25 µg/ml) (green). Resistant colonies were re-streaked onto fresh plates to minimise wild-type contamination. (d) 10 colonies (these represent the first recombination event) were picked and cultured overnight in TYG. (e) The cultures were pooled into one stock, which was plated onto BHI containing FUdR (200 µg/ml) (blue) to select for the second recombination event, as before these are re-streaked. (f) 10 resistant colonies are cultured overnight in TYG. Glycerol stocks are prepared and DNA extracted for analysis. Inset: A visualisation of the donor and recipient DNA, and the first and second recombination events. Figure taken from Shapiro (2015).

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In a similar method, unique sequences of DNA referred to here as Tags can be inserted into one of two ATT sites of Bacteroides to allow for differentiation of species or mutants within a co-culture. The tags are carried on modified suicide vectors, pNBU2-tag11 and pNBU2-tag1, which were used to transform CC118 E. coli competent cells which are plated on LB-ampicillin plate overnight. Overnight cultures were then made from the resulting colonies and from glycerol stocks of the Bacteroides in LB and TYG, respectively. The overnight cultures were then used to inoculate sterile media for a 4 h outgrowth. The 4 h cultures were centrifuged (6000 x g, 15 min) and resuspended in 1 ml TYG together. This resulting E. coli-Bacteroides culture was plated onto BHI-agar without antibiotic. There plates were then incubated aerobically for 24-30 h and the resulting biomass was removed and resuspended in fresh TYG (5 ml). The resulting cell suspension was diluted 10-1_{, 10}-2 _{and 10}-3 _and

plated onto BHI-agar with gentamycin (200 µg/ml) and tetracycline (2 µg/ml). The plates are incubated anaerobically for 2 days, then 10 colonies were selected and re streaked onto fresh BHI- agar with gentamycin ant tetracycline which were incubated anaerobically for a further 3 days. Single colonies were selected and grown in TYG overnight. These cultures were subjected to genomic DNA extraction and PCR was performed to check for tag insertion. A second PCR was performed to show which site has been destroyed by insertion of the tag. PCR controls are used with wild type genomic DNA.