Team:Warwick/Experiments

Experiments

PCR

PCR was performed using Phusion and Q5 High-Fidelity polymerases (NEB) and MyTaq polymerase (Bioline). Reactions were set up on ice and incubated in a thermocycler as per the manufacturers' published protocols. Cycling conditions were adjusted to accomodate the polymerase used as well as template length and primer T_m values (for NEB enzymes, found via the NEB T_m Calculator).

Agarose Gel Electrophoresis

To make a 1% agarose gel, 0.5 g agarose powder was added to 50 ml 1 X TAE buffer and the mixture heated in a microwave until the agarose was completely dissolved. When the mixture had cooled but not yet set, 5μl of SYBR SAFE DNA Gel Stain (NEB) was added and the gel poured into a casette. Once set, the gel was placed in a tank and covered with 1 X TAE buffer. After removal of the comb and loading, a constant voltage of 120 V was applied for 40 minutes. Alternatively, SYBR SAFE was omitted and gels were incubated in ethidium bromide solution for 5-10 minutes after running. Gels were then imaged with UV light.

Plasmid Purification

Plasmids were purified from transformed E. coli DH5α cells using the GeneJET Plasmid Miniprep Kit (Thermo Scientific) according to the manufacturer's protocol.

PCR Cleanup

Following PCR, products were cleaned up using the QIAquick PCR Purification Kit as per the manufacturer's recommendations.

DNA Gel Extraction

DNA was excised from agarose gels and purified using the GeneJET Gel Extraction Kit (Thermo Scientific) as per the manufacturer's published protocol.

Measuring DNA Concentration

DNA concentrations were determined using a NanoDrop instrument (Thermo Scientific). Before measurements were taken, 1 μl distilled water or buffer (as appropriate) was used to blank the device. 1 μl DNA solution was then measured and DNA concentration as well as sample quality (evident from shape of the curve) noted.

Gibson Assembly

Following addition of overhangs for Gibson assembly via PCR, fragments were assembled using Gibson Assembly Master Mix (NEB) as per the manufacturer's recommendations.

TOPO Cloning

For our second round of cloning, constructs were assembled using the Champion pET151 Directional TOPO Expression Kit with BL21 Star(DE3) One Shot Chemically Competent E. coli (Thermo Scientific) as per the manufacturer's protocol.

Chemical Transformation

After thawing, 2 μl of a chilled construct was added to chemically competent E. coli DH5α cells (NEB/made by Corre group) on ice. Tubes were gently flicked to mix and incubated on ice for 30 minutes. Cells were then heat-shocked in a water bath at 42 °C for 30 seconds and transferred to ice for 2 minutes. Following addition of 950 μl SOC media (NEB), tubes were transferred to a shaking incubator at 37 °C and 250 rpm for 60 minutes. 100 μl cells was then plated onto warm selection plates and incubated overnight at 37 °C.

Restriction Digest

As well as using DpnI (NEB) to digest methylated template DNA following PCR, we used PstI and HindIII (NEB) digests to verify the identity of DNA fragments on an agarose gel. 1 μl restriction enzyme was added to each PCR tube on ice and incubated at 37 °C for 1 hour in a thermocycler. Tubes were subsequently incubated at 80 °C to heat inactivate enzymes.

Colony PCR

Following transformation of DH5α cells and overnight growth on a selective plate, 100 μl LB was inoculated with a single colony. A PCR reaction was then set up on ice using MyTaq polymerase (Bioline) according to the manufacturer's instructions but with 1 μl cell suspension in place of template DNA. Cycling conditions were as usual for MyTaq with a 10-minute initial denaturation for cell lysis and annealing temperature and extension time adjusted according to the primers and DNA fragment of interest respectively. Products were analysed on an agarose gel.

Oil Media Experiment

Oil media was prepared by addition of palmitic oil to LB media containing ampicillin at 0%, 0.5%, 1%, 2%, 5% and 10% oil concentrations. Bottles were made up to the same final volume with PBS. 100 ml oil media was then inoculated with 2 ml overnight culture of transformed E. coli DH5α cells. Samples were taken every 30 minutes for 8 hours and serial dilutions made up to 10^-12 for each sample. 5 μl of each of the final four dilutions were plated onto LB ampicillin plates so that each plate was spotted with 16 samples. Plates were incubated at 37 °C overnight and CFUs counted.

MicrobeMeter Experiment

Oil media was prepared by addition of olive oil to LB media containing ampicillin at 0.5% as informed by the oil media experiment and model. In a MicrobeMeter (Humane Technologies), media was inoculated with 100 μl of overnight culture of both transformed and untransformed BL21 E. coli cells. Following growth for 5 hours, expression of lipases under the T7 promoter was induced with 10 μl 1 mM IPTG. Cell density was monitored by the MicrobeMeter, through OD₆₀₀ measurements at 5-minute intervals for 24 hours.

Tributyrin Lipase Assay (Qualitative)

1% w/v neutral tributyrin was added to melted tributyrin agar (TBA) in a 50 ml falcon tube. After vortexing until proper emulsification of tributyrin was acheived (solution cloudy and homogenous), plates were poured and allowed to solidify. Bacteria were then streaked directly onto a third of the plate. In the other two thirds, 20 μl of liquid culture or 20 μl cell-free supernatant was pipetted into cut-out wells, enabling detection of secreted lipases. Following incubation at 37 °C for 48 hours, lipase activity was identified by presence of clear halos on agar around colonies and wells.

P-Nitrophenol Lipase Assay (Quantitative)

Lipase activity was monitored spectrophotometrically via the hydrolysis of p-nitrophenol octanoate to octanoic acid and p-nitrophenol, which absorbs light at 400 nm. The assay was performed as detailed by Gupta et al. (2002) and used to determine k_M, k_cat and V_max.