4.2.1 Recombineering plasmid construction
In order to create a double selection marker and suitable restriction sites for our insertions, we inserted the galK gene from plasmid pDB88 66bp upstream of the SacB selection cassette in pYUB1471, and then inserted an NdeI site at the end of the
downstream target region to create plasmid pNH01 (6,17). 600bp regions encoding the sequences upstream and downstream of mscL in the M. smegmatis MC2155 genome were inserted at either end of the selection cassettes to create pNH02. All reactions were completed using Kapa Hifi polymerase with buffer A. Insertion of galK was completed by amplifying the vector backbone using primers pNH01GK_F and
pNH01GK_R and galK with primers pGK_F and pGK_R. The products were purified
and ligated using the Clontech In-Fusion kit. Upstream and downstream regions of mscL were amplified from MC2155 genomic DNA using primers pMscLU_F and pMscLU_R for the upstream region, and primers pMscLD_F and pMscLD_R for the downstream region. The upstream region was inserted by digestion of pNH01 with NcoI, followed by purification and In-Fusion; thereafter the downstream region was inserted by digestion of pNH01 with NdeI and BamHI followed by purification and In-Fusion.
pNH02 was amplified in E. coli cells, and linearized by restriction enzyme digestion with NdeI and BamHI.
4.2.2 Allelic exchange
100 ng of linearized gene product from pNH02 was electroporated into M.
smegmatis MC2155 expressing Che 60 and 61 proteins in the pJV53 plasmid, and made electrocompetent. Allelic exchange, also known as recombineering, was allowed to proceed for 4 hours at 37 °C. Thereafter, the cells were plated on selective media plates containing hygromycin (HygR 7H9). After 5 days, colonies were streaked onto both HygR and 2-deoxy- galactose + sucrose 7H9 medium (DS 7H9) plates. Colonies which survived on HygR but died on DS 7H9 medium plates were selected for
unmarking. To unmark the cells, they were grown to log phase, OD600 1.0, and infected with bacteriophage pHAe 280 for 72 hours at 37 °C. Cells were then plated on DS 7H9 medium plates. Individual colonies were patched on HygR and DS 7H9 medium plates and colonies that survived on DS 7H9 plates but died on HygR 7H9 medium plates were selected. Unmarking was verified by colony PCR using primers pCPCR_F and
pCPCR_R, and by deep genome sequencing of extracted genomic DNA using the Illumina Hiseq 2500. Sequencing data was analyzed by aligning with Bowtie2, samtools, and visualized using IGV. The verified strain was named MC2155 mscL-.
4.2.3 Protein expression
The inserts, MsMscL and MtMscL, were fused with a GFP marker at the C- terminal domain to allow for rapid protein expression by using fluorescence based detection. We amplified the GFP from a vector used in another study (18). The MtMscL insert was amplified from an existing MtMscL insert in pET19 (19) using primers
pMtMscL_F and pMTMscL_R, and the MsMscL insert was amplified from genomic DNA using primers pMsMscL_F and pMsMscL_R. GFP was amplified using primers
pGFPMtMscL_F, pGFPMsMscL_F and pGFP_R and fused to MsMscL using primers pMsMscL_F and pGFP_R, and to MtMscL using primers pMtMscL_F and pGFP_R.
Plasmid pMyNT (20) was digested with SphI and BamHI to create the insertion site for our insert and put into plasmid PMyNT using the infusion kit from Clontech to create pMsMscL GFP and pMtMscL GFP.
4.2.4 Protein expression of MscL using the acetamide promoter
MC2155 mscL- cells were electroporated with 50 ng of pMsMscL GFP and pMtMscL GFP. After selecting for positive clones on HygR 7H9 plates, the cells were grown at 37 °C in 7H9 medium containing 0.5% glycerol, 0.02 % Tween, and 150 µg/mL hygromycin. After 42 hours, the cells were diluted 1:10000 into 7H9 medium containing the previously mentioned components and 0.2% sodium succinate. Once the cells reached an OD600 nm of 1-1.5, expression of the protein was induced using 0.2 % acetamide for 12 hours at 37 °C. Expressed and non-expressed cells were imaged under a fluorescent light to expose the GFP fluorescence in the cell at a wavelength of 488 nm and detected at 510 nm.
4.2.5 Brightfield and fluorescent microscopy
Protein expression was analyzed by imaging the GFP fluorophore fused with each of our MscL constructs expressed in MC2155 mscL-. Following an expression experiment, 5 µL of cell culture was spread between a microscope slide and coverslip.
Brightfield and fluorescence microscopy was performed on a Nikon eclipse NI-E microscope and images were taken with a Nikon DS Ri2 camera.
4.2.6 Antibiotic resistance assay using mscL- bacteria
Two types of assays were conducted on M. smegmatis, one in liquid culture and another one on agar plates. For both assays, MC2155 and MC2155 mscL- cells were grown in a 3 mL 7H9 medium starter culture at 37 °C. In the liquid culture assay, a suitable starter culture was grown for both M. smegmatis (3 days) and E. coli (12 hours). 50 µL aliquots of these cultures were transferred to individual 3 mL cultures in 7H9 for M. smegmatis. Cultures for M. smegmatis contained a dihydrostreptomycin gradient of 0-25 µM, with concentrations of 0.1, 0.2, 0.4, 0.5, 0.6, and 1, followed by a gradient from 1-25 in steps of 5 µM. The experiments were all done at 37 °C in triplicate.
These cells were grown for 24 hours and the cultures were transferred to a 96 well plate. OD 600 nm was recorded using a Tecan plate reader. For the agar plate method, the OD600 nm of the MC2155 and MC2155 mscL- cultures was recorded and regularized using 7H9 medium. Once regularized, a series of eight serial dilutions was done with 7H9 medium, the dilutions were 1, 1
2, 1 4, 1
8, 1 16, 1
32, 1
64, and 1
128. These dilutions were plated in triplicates onto individual 7H9 agar plates containing 20 µg/mL of spectinomycin, nifuroxazide, and viomycin, and 0.5 µg/mL dihydrostreptomycin. The plates were incubated at 37 °C until colony growth was observed, typically after 3 days.
A series of control experiments using E. coli was done using Frag1 E. coli (E. coli WT) and MJF455 E. coli (E. coli mscL-). E. coli WT and E. coli mscL- starter cultures were grown in 5 mL LB medium for 12 hours at 37 °C. 50 µL aliquots of these cells were transferred to individual 5 mL LB cultures containing 0-70 µM dihydrostreptomycin at 37
°C. The cultures for E. coli contained a gradient of 0-70 µM in steps of 5 µM increasing
concentrations of dihydrostreptomycin. These cells were grown until they reached early exponential phase in the condition without antibiotic, OD600 nm 0.2, and transferred to a 96 well plate. OD600 nm was recorded using a Tecan plate reader.