Chapter 2. Design and Construction of Strain ESS
3.3. Materials and methods
3.3.1. Bacterial strains and culture conditions
The strains used are listed in Table 3.1. E. coli MG1655 was used as a parental strain. Bacteria were cultured at 37°C in Luria–Bertani broth (LB) or minimal medium supplemented with 4 g/L cellobiose. Strains carrying temperature-sensitive plasmids were grown at 30°C. Media were supplemented with suitable antibiotics (50 μg/mL kanamycin, 30 μg/mL chloramphenicol, or 100 μg/mL ampicillin). For long-term storage, cells were maintained as 20% glycerol stocks at −80°C. Protein expression was induced by addition of tetracycline (1–5 ng/mL) or sodium propionate (20 mM). M9 minimal medium supplemented with 2 mM MgSO4, 0.1 mM CaCl2, and 4 g/L cellobiose was used to characterize growth of the modified strains. Initial experiments to optimize the inducer concentration were performed in 96-well plates, and cell growth was monitored every hour using a Tecan Infinite F200 PRO plate reader (Tecan-US, Durham, NC). Further validations were performed in 250-mL shake flasks, in which cells were inoculated (1:100) from overnight LB cultures into 50 mL M9 medium supplemented with the test sugar; the cultures were incubated at 37°C with rotation at 200 rpm. Cell growth was monitored by measuring the optical density at 600 nm (OD600) with a Biochrom Libra S22 spectrophotometer.
Table 3.1. E. coli strains and plasmids used in this study
Strains/plasmids Description/genotype Reference/source
Strain
E. coli MG1655 Wild-type 37
OSS MG1655 with CP12chb plus CP12asc 26
ESS OSS adapted in cellobiose for 30 days 26
OSS-C MG1655 with CP12chb 26
OSS-A MG1655 with CP12asc 26
ESS-B ESS with ∆ascB This study
ESS-F ESS with ∆chbF This study
ESS-T ESS with ∆chbBAC This study
Plasmids
pSIM5 λ-Red recombinase expression plasmid and temperature sensitive replication
51
pCP20 Yeast FLP recombinase gene controlled by cI repressor and temperature sensitive replication.
38
22
pKD13 Template plasmid for gene disruption. The resistance gene is flanked by FRT sites. oriR6K-gamma origin requiring the pir + E. coli.
38
pZB Cmr; p15A replicon, PBAD promoter, Pzt1 promoter 52 pZB-G pZB plasmid with pgi, fbaA, gapA and tpiA expressed
from Pzt1
This study pZB-A pZB plasmid with ascB expressed from Pzt1 and with
strong RBS (AAGAAG)
This study pZB-C pZB plasmid with chbF expressed from Pzt1 and with
strong RBS (AAGAAG)
This study pPro-ascB pPro29b plasmid expressing ascB from Ppro This study pPro-chbBAC pPro29b plasmid expressing chbBAC from Ppro This study
3.3.2. Transcriptome analysis
For transcriptome analysis, OSS and ESS were grown on minimal cellobiose medium up to an OD600 of 0.7 as described previously in order to compare the central metabolic changes for strains grown on glucose, acetate or glycerol53. Cells were harvested from 500 mL of the culture and washed with 100 mM sodium phosphate buffer;
the pellet was stored at –80°C until analysis. Transcriptome profiling was performed using the GE microarray, 3*20K (E. coli str K12 substr, MG1655) platform as per the manufacturer’s protocol. Three biological replicates were analyzed for each sample. Genes with signal intensities greater than 100 were considered for further analysis.
Average signal intensity from triplicate readings of each ESS gene was normalized to the corresponding average signal intensity of OSS, and fold-change values were obtained. For functional analysis of the transcriptome, we applied ADGO 54 to the fold-change values. Three categories of Gene Ontology (GO) were chosen simultaneously for functional interpretation, and the Z-statistic method was used to summarize the fold-change values of each GO term. We identified 175 up-regulated and 70 down-regulated GO terms. Representative categories obtained from ADGO are summarized in Table 3.2. A list of genes involved in the CMP (as per the iAF1260 model) 55 was considered for experimental validation. Heat maps were generated using R programming by comparing the relative signal intensity of the selected genes in OSS and ESS.
23 3.3.3. Strain construction
All gene deletions were generated as previously described 51. Briefly, cells carrying the plasmid pSIM5 were induced at 42°C for 15 min, cooled immediately, and made electrocompetent. The competent cells were then transformed with PCR products carrying kanamycin gene and complementary regions upstream and downstream of the gene of interest. Recombinants were selected on LB agar containing kanamycin, and positive clones were verified by PCR and sequencing. Plasmid pZB-G was constructed by a sequence- and ligation-independent cloning (SLIC) method 56. The pgi, fbaA, gapA, and tpiA genes were amplified with primers containing their native RBS sequences and regions homologous to their successive genes. Similarly, plasmid pZB was also amplified by PCR. The PCR products were then independently treated with T4 DNA polymerase for 20 min, and exonuclease activity was terminated by addition of dCTP. The vector and inserts were mixed (1:2 molar ratio) and transformed into electrocompetent cells. pZB-A was constructed by cloning ascB into the PstI and AvrII sites of pZB. pPro- ascB was constructed by cloning ascB into the KpnI and BamHI sites of pPro29b.
3.3.4. Enzyme assay
Phospho-β-glucosidase assays were performed with OSS and ESS grown on minimal cellobiose medium. Cells were collected every 4 h and stored at –20°C. The cell pellet was dissolved in 100 μL of 100 mM sodium phosphate buffer (pH 7.0), 400 μL of 10 mM p-nitrophenyl-β-glucoside was added, and the samples were incubated at 37°C for 40 min. The reaction was arrested by adding 1M sodium carbonate solution. The yellow color developed was quantified by measuring OD at 410 nm and 600 nm.
3.3.5. RT-PCR
Total RNA was isolated using Trizol reagent according to the manufacturer’s procedure. cDNA was synthesized using random hexamers and MuLV reverse transcriptase. Expression of the target genes was quantified using gene-specific primers and Phusion® DNA polymerase. PCR samples (5 μL) were taken after 25, 27, 30, and 32 cycles and analyzed on 0.8% agarose gel. DNA concentration was determined based on the molecular weight marker.
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