Chapter IV: Investigation of repair protein expression levels and DNA-mediated
4.2 Materials and Methods
P1 phage transduction was used in order to generate InvA∆nth and InvA∆muty for the InvA growth assay. The donor strain of either ∆nth or ∆muty was grown overnight with antibiotic selection. This overnight was then diluted 1:100 into 2mL of fresh LB with 20mM MgCl2, 5mM CaCl2, and 0.1% glucose. After ~2 hours of growth at 37C, 40µL of P1 phage was added to the mixture and the mixture
was allowed to grow until all cells had been lysed by the phage ( 3 hours). 50uL of chloroform was then added to the lysate. Following brief vortex, the lysate was spun down in small 1.5 mL centrifuge tubes at 14000 rpm for 2 minutes. The supernatant was preserved and a few more drops of chloroform was added to the mixture for sterilization. The recipient strain InvA was grown overnight in the appropriate antibiotics. The cells were then pelleted at 6000rpm for 2min followed by resuspension in 300µL LB with 10mM MgSO4 and 5mM CaCl2. 100µL of p1 lysate was added into 100µL of recipient cells and incubated for 30 minutes at 37C.
200µL of 1M Na-Citrate at pH 5.5 and 1mL of pure LB was added and the culture was shaken for 1 hour to allow for antibiotic resistance expression. Cells are then spun down at 6000rpm for 5 minutes, decanted, and resuspended with 100µL of LB with 100mM Na-Citrate. This cell aliquot was then spread on an LB-plate with the correct antibiotics. InvA∆muty strain creation was performed with Dr. Michael Grodick. Colony PCR was performed on InvA to verify rrnA operon inversion and gene knockout using primers listed in Table B.2.
Next generation sequencing of InvA∆muty
Putative InvA∆muty was processed with a blood and tissue DNA extraction kit (Qiagen). The extracted genome of InvA∆muty was subject to Illumina sequencing with a read length of 100bp for 8 million reads to ensure adequate coverage of the entire genome. The acquired fastq files were aligned using bowtie-2 software against the reference genome of K-12 E. coli NC_008253 and converted, merged, sorted, and indexed into the appropriate genome file using samtools. Sequence alignments were visualized using the Integrative Genomics Viewer (IGV).
InvA growth assay
Overnight cultures of the E. coli InvA∆nth containing various rescue plasmids were prepared by inoculating single colonies in 3 mL of LB media containing 50 µg/mL ampicillin. After overnight growth of ~20 hours, 50µL of the culture was placed into a 10mL multichannel pipette tray with 10mL of LB-amp along with inducer at varying concentrations if applicable. Strains were then put into columns in the microplate depending on rescue plasmid genotype. The microplates were then subject to shaking at 37C for 12 hours taking an OD600 or 630 reading every 10 minutes. Plate reader data was saved to a.csvand data analysis was performed using scripts written inpython 3.6.5usingmatplotlib, seaborn, andbokehfor data visualization [14–16].
Creation of pBAD, pPro, pLac plasmids for EndoIII expression
In order to tune protein expression, propionate, lactose, and arabinose based inducible systems were used and are designated as pPro, pLac, and pBAD respec- tively. Plasmids were assembled via Gibson assembly using vectors acquired from Addgene. pLac plasmid backbone is from pBbS5a-RFP (Plasmid# 35283 Addgene), pPro plasmid backbone is from pPro30 (Plasmid# 17809 Addgene), and pBAD plas- mid backbone is pBbS8a-RFP (Plasmid# 35274 Addgene) [8, 17]. RFP insert was taken from pBbS5a-RFP or pBbS8a-RFP (both are identical) and and EndoIII inserts extracted from either theE.coligenome using colony PCR or from the lab EndoIII overexpression plasmid. RFP was used either as a control or, in some cases, fused to the N or C-terminus of nth for future experiments to quantify EndoIII expression.
Primers used are listed in Table B.3.
UV sensitivity colony counting assay
Overnight cultures of the E. coli ∆nth or ∆muty containing various pBAD rescue plasmids were prepared by inoculating single colonies in 5 mL of LB media containing 100 µg/mL ampicillin and 50 µg/mL kanamycin. 200µM or 50µM arabinose was added during this step for some assays. After overnight shaking at 37C, the cultures were diluted 1:500 into 10 mL of fresh LB media also containing antibiotics. Cultures were then treated with either 50µM of arabinose if not induced during the overnight step. Cultures were then incubated at 37C for 3 hours. An OD600 measurement was taken and cell concentrations were adjusted to match optical density using phosphate buffered saline (PBS) to an OD600of 0.4 and then diluted 20200-fold and placed into a transparent petri dish. These dishes were placed inside the UVP-crosslinker and subject to one treatment of UV radiation at 30J/m2. 10µL of the solution of cells was then taken from the petri dishes and placed into patterns on a plate as droplets. The plate was then tilted to allow for the cell droplets to spread and grow on a bigger surface area and thus allowing for easier counting.
Plates were then incubated at 37C overnight. Resultant plates are imaged using a BioRad gel imager on the Coomassie blue setting. Colonies are counted up with the assistance of OpenCFU software (Figure 4.2) [18]. Regions of interests can be manually drawn out and the software will count automatically.
Genome integration of pBAD-nth
Integration of WT, D138, and Y82A EndoIII genenthwas accomplished using the clonetegration technique [13]. The nth gene insert under an arabinose promoter
Figure 4.2: OpenCFU interface for drawing regions of interest and counting colonies.
including araC operators was cloned into the cloning module using Gibson assembly.
The parent insert contains a toxic ccdB gene to prevent parent plasmid transformed strains from proliferating. Additionally, pOSIP contains a R6Kγorigin of replication which can only replicate in strains that are pir+. This means that during integration of other strains, the integration plasmid will not replicate and thus only one copy is allowed to integrate. Integration occurs via the lambda integrase which takes the attP site on pOSIP and finds a matching attB site inside the genome ofE.coli. Thus, integration only occurs at a specific place inside the strain. Additionally, the flaking terminators integrated into the genome ensure that transcription of the gene insert is exclusive to itself and is unaffected and will not affect endogenous transcription.
Primers used for creation of clonetegration plasmids are listed in Table B.1. Colony PCR performed to confirm genome integration.
Plate-reader UV-sensitivity assay
Overnight starter cultures of∆araC(pRFP) and∆nth transformed or genome integrated with WT, D138A, or Y82A EndoIII were cultured similarly to the above protocol. After OD600 adjustment of all strains to the same OD, a 16-fold dilution into PBS was performed on each strain into a transparent petri-dish for 254nmUV- irradiation at 90J/m2. After treatment, the cell solution was diluted 16-fold into
Figure 4.3: Clonetegration genome integration module developed by Endy et al.
Adapted with permission from St-Pierre, F. et al. ACS Synth. Biol. 2013, 2, 537.
Copyright 2013 American Chemical Society.
7mL LB with 100µg ampicillin. 200µL of each genotype was placed into a unique column in a 96-well microplate and placed into a plate-reader (BioTek) for OD600 readings every 10 minutes over 670 minutes in total. Plate reader data was saved to a.csv and data analysis was performed using scripts written inpython 3.6.5using matplotlib,seaborn, andbokehfor data visualization.