Team:IIT Madras/Protocol

1. Transformation:

(a) Added 50-100µg of DNA to competent cell.
(b) Kept on ice for 30 minutes.
(c) Heat shock was given at 42°C for 90 sec.
(d) Kept on ice for 5 min.
(e) Added 400µl of LB media.
(f) Incubated at 37°C for 1h in shaker.
(g) Plating was done with 250µl of culture.
(h) Plates were incubated overnight at 37°C.

2. Mini Prep: DNA Isolation using HiPura Silica Kit

(a) Single isolated colonies were picked and inoculated in 3ml media.
(b) Overnight incubation was done at 37°C.
(c) 1.5ml of culture was taken and centrifuged at 12,000 rpm for 2 min.
(d) Supernatant was discarded and pellet was kept on Ice.
(e) The pellet was re-suspended with 100µl of chilled TGE buffer (25mM Tris pH 8.0, 50mM Glucose, 10mM EDTA) treated with RNAse (0.25 µl / 200 µl TGE buffer) was added to it.
(f) 200µl of Sol II containing 0.2N NaOH and 1% SDS was added and the solution was mixed gently and kept on ice for 3-5 min.
(g) 150µl of cold potassium acetate solution (3M Potassium and 5M acetate) was added and the solution was mixed gently and finally kept on ice for 3-5 min.
(h) Mixture was centrifuged at 14,000 rpm for 5 min.
(i) The supernatant was transferred to a clean centrifuge tube.
(j) 3 volumes of Chaotropic Salt Solution was added.
(i) 10µl of Glass Powder Suspension was added and then kept for mixing in rotospin for 30 min.
(j) Centrifugation was done at 14,000 rpm for 2 min and the supernatant was discarded.
(k) The glass powder pellet was re-suspended in 500µl of diluted wash solution (1 volume of wash Solution as provided with the HiPura Silica kit added to 9 volume of Milli-Q and 10 volume of absolute ethanol.)
(l) Centrifugation was done at 12,000 rpm for 2 min and the supernatant was discarded.
(m) Step (k) & (l) were repeated twice. After the last wash, the supernatant was removed completely and the pellet was air dried for 5 min.
(n) Glass Powder Pellet was re-suspended into 20µl of autoclaved double distilled water (or TE buffer, pH 8.0 for long term storage).
(o) Incubation at 55°C for 20 min was done.
(p) Centrifugation at 14,000 rpm was done for 2 min and the supernatant was carefully taken out and was stored in -20°C
(q) Confirmation was done by running 2µl of isolated plasmid DNA on agarose gel against a standard marker.

3. Restriction Digestion

(a) 20µl of restriction digestion reaction was set up on ice.
(b) 2µl of NEB buffer 4 (CutSmart) was used for the High Fidelity enzymes PstI and SpeI.
(c) 1µg of DNA was added to it.
(d) 0.2µl each of restriction enzymes were added to the mixture.
(e) Autoclaved double distilled water was used to make up the volume to 20µl.
(f) The reaction mixture was kept at 37°C for 8-12h.
(g) The restriction digestion was confirmed by loading on agarose gel against a standard marker.

4. Elution of Plasmid DNA using HiPura Silica Kit.

(a) Digested sample was run on agarose gel.
(b) The required size band was excised under UV light and the gel pieces were loaded into a vial.
(c) 3 volume of Chaotropic Salt Solution was added to it.
(d) Incubated at 55°C till agarose melted completely.
(e) 10µl of Glass Powder Suspension was added to it.
(f) The mixture was mixed and incubated at room temperature for 15-20 min in the rotospin.
(g) The mixture was centrifuged at 12,000 rpm for 2 minutes and the supernatant was discarded.
(h) 500µl of diluted Wash Solution (1 volume of wash Solution as provided with the HiPura Silica kit added to 9 volume of Milli-Q and 10 volume of absolute Ethanol.) was added to the pellet.
(i) Centrifugation was done at 12,000 rpm for 2 min and the supernatant was discarded.
(j) Step (h) & (i) were repeated twice. After the last wash, the supernatant was removed completely and the pellet was air dried for 5 min.
(k) Glass Powder Pellet was re-suspended into 20µl of autoclaved double distilled water (or TE buffer, pH 8.0 for long term storage).
(l) Incubation at 55°C for 20 min was done.
(m) Centrifugation at 14,000 rpm was done for 2 min and the supernatant was carefully taken out and was stored in -20°C
(n) Confirmation was done by running 2µl of isolated plasmid DNA on agarose gel against a standard marker.

5. Ligation

(a) 10µl of reaction was set up.
(b) Purified Vector and Insert were taken in the ratio of 1:3.
(c) 1µl of 10X Ligase buffer was added to it.
(d) 1µl of T4 Ligase was added to the mixture.
(e) Finally, the volume was made up to 10µl by adding autoclaved double distilled water.
(f) Ligation reaction mixture was kept overnight at gradient temperature over the range of 8-22°C.

6. Indole Test

(a) Inoculate the tryptophan broth with the test organism and incubate at 37°C for 24-28 h.

(b) Add 0.5ml of Kovac’s reagent to the culture and gently agitate.

(c ) Indole positive bacterial culture produces a red colour, while indole negative bacterial culture produces a yellow colour.

7. AHL induction for Protein Synthesis

(a) Pick a single isolated colony and innoculate 3ml LB media (primary culture) and grow at 37°C for 12-16 h.

(b) Use 0.5ml (1%) of primary culture to innoculate 50ml LB media (secondary culture).

(c ) Incubate this culture at 37°C till the OD600 reaches 0.6.

(d) Induce the culture with 1mM of AHL and grow at 37°C for 3 h.

(e) Keep the culture on ice for 20 min to stop growth.

(f) Spin the culture down at 6000 rpm for 10 mins at 4°C, collect the supernatant in a autoclaved flask and store the bacterial pellet for downstream processing.

8. Analytical Reverse Phase HPLC

(a) Take the supernatant from induced culture and lyophilize the solution till powdered form is obtained.

(b) Resuspend the powder in 2ml of Milli-Q water and use a pipette to mix properly.

(c) Pass the solution through a 0.22 µm membrane filter using a syringe to get rid of aggregates.

(d) Pass this solution through a reverse phase HPLC machine with TFA (tri-fluoro acetate) as the stationary phase and use Acetonitrile as the mobile phase.

(e) As acetonitrile concentration is increased on a gradient, different eluted fractions of the protein mixture can be observed as peaks.