Plate and Media Preparation
LB agar plates were prepared according to manufacturer directions and autoclaved for sterilization. Appropriate antibiotic solutions were prepared with the following final concentrations:
- Amp 50 ug/ul
- Kan 50 ug/ul
- Clrm 50 ug/ul
Arabinose was added to the appropriate plates and Broth media when needed to induce expression of one of the parts.
Dh5 alpha competent cells were used to express our constructs. Transformation was done as below:
- Cells were incubated with 5 ug of plasmid DNA for 30 mins on ice.
- Heat shock was performed at 42 degrees for 45 secs.
- Immediate incubation on ice for 2 mins.
- Recovery in 750 ul of SOC media was performed and tubes incubated at 37 degrees for one hour.
- 100 ul of transformed cells were placed on appropriate selective plates and incubated overnight at 37 degrees.
Single colonies were picked from successfully transformed plates and suspended in 10 ml of LB-broth complemented with appropriate antibiotics and inducers where needed. Tubes were incubated at 37 degrees for 16 hours with a rotation speed of 220 rpms.
Inoculations were subsequently pelleted by centrifugation for 2 mins at maximum speed and pellet recovered. Miniprep of plasmid DNA was performed according to the QIAGEN miniprep kit. Briefly, pellet was first resuspended in 250 ul of P1 buffer and transferred to microcentrifuge tube. 250 ul of P2 buffer was then added and tubes inverted 4-6 times for mixing and lysis. Mix was then neutralized with 350 ul of N3 buffer and mixed. Tubes were centrifuged at 13000 rpm for 10 mins and 800 ul of supernatant then was transferred to qiaprep columns, centrifuged for 60 secs and flow through discarded. Columns were then washed with PE buffer and centrifuged for 60 secs. A dry spin was performed to get rid of residual wash buffer and DNA was eluted with 50 ul of EB buffer in a sterile micro-centrifuge tube.
RNA isolation was performed using the QIAGEN RNAeasy plus kit according to manufacturer’s protocol. You can view the detailed protocol here
RT-PCR was performed on RNA isolated from the E. coli cells transformed with the arabinose inducible Gb3 synthase coding device. A non-induced control was also used for negative control and RNA isolated from transformed cells with no inducer in media.
The Invitrogen “first strand Synthesis system” (Cat No.18080-051) was used to perform RT-PCR. Total RNA previously isolated was used and a step by step protocol is as follows:
- Heat RNA-dT-mix for 2 min at 70 C, cool to ambient temperature; keep the tube at room temperature
- Add half of the RNA mix to each of the two reaction tubes, and mix the resulting solution
- Incubate at room temp for 5 min, 10 min at 37, and 60 min at 42 C
- 30 minutes at 55 C.
- Terminate the RT reaction by incubating for 10 min at 85 C
- Store the reaction at -30
PCR amplification of the Gb3 Synthase gene was performed using 3 different sets of primers. KOD polymerase was used to perform PCR and protocol is as follows:
Master mix was prepared for 8 reactions and each primer set was used with both + and – inducer RT-PCR samples as follows
PCR was performed in 8 strip tubes capped with thermal cycler setup as below with heated lid option active:
- Polymerase activation: 1 x: 95 °C 5’
- Amplification cycles (30 X):
- 95 °C for 30”
- 57 °C for 30”
- 70 °C for 60’’
- Final extension: 1x 70 °C for 5’
- Post PCR: 8 °C indefinite
- Amplification cycles (30 X):
Agarose gel electrophoresis
1% agarose gels were prepared and casted and 10 ul of each PCR sample were mixed with 2 ul of premixed loading dye with 1:1000 syber green dye. Gel was run at 135V for 25 mins and visualized under blue light for detection. 100 bp ladder (Biorad) was used for reference.
PAGE gel electrophoresis
Ready-made mini-Protean precast Biorad gels were used to perform PAGE in 1x TBE buffer. 10 ul of samples were loaded in each well and electrophoresis carried out at 200v for 30 mins. Gels were then stained using Pierce™ Zinc Reversible Stain Kit (ThermoFisher cat #24582) according to manufacturer’s protocol.
For Indole detection, Indole, Kovac's Reagent was used according to manufacturer’s protocol.
E. coli cells were transformed with a previously submitted part and cultured for downstream validation and characterization. (Part: Part:BBa_K1867024)