Difference between revisions of "Team:IIT Delhi/protocol"

Revision as of 21:16, 28 October 2017

iGEM IIT Delhi

Abstract

Lack of digital responses in Synthetic Biology have inhibited the diverse potential that accompanies the digitization of biological circuits. This year we aim to develop synthetic modules for signal processing in biological systems, in the form of elements of specialized logic gates based on transcriptional regulation. We move from developing near digital logic gates with sharp responses, to more specialized collapsible and reconfigurable circuits which can perform various operations like developing square pulses. Further, to realize this aim of making a square wave generator, we engineered a five node repression based ring network to give digital oscillations. Quantitative computational modelling would be used to tailor the cellular environment and observe period, steepness, noise and amplitude variations. Our project poses to be an integral element in genetic networks intended to solve scientific challenges for years to come, ranging from making light sensitive frequency modulators and bacterial memory storage systems.

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LAB PROTOCOLS

Accurate Experimenting for Accurate Results !!

Preparation of competent cells:-

Dilute an overnight culture of E. coli 1:200 with LB broth.

Incubate at 37°C with shaking (at 200 rpm) until the cells reach early log phase (OD600 = 0.25-0.4).

We already have 1X TSS in 4 deg fridge(old). Use it without dilution or thawing. Keep it inside the icebox just after taking out from the fridge.
OR
(if the above 1X TSS is not available)
While cells are growing, thaw 2X TSS on ice and dilute an appropriate amount 1:1 with sterile distilled water (100µl of diluted TSS will be needed for each ml of cells). Chill on ice.

Place 2-ml aliquots of early log-phase cells into sterile 2-ml micro-centrifuge tubes and pellet the cells by centrifugation at 4°C at 3000g for 10 min.(6-8 mins for taking part from Igem kit)

Remove the supernatant and discard. Add 0.2 ml of the ice-cold 1X TSS and place the tubes on ice.

Gently suspend the cells by pipetting.

Proceed with the transformation protocol below (Step 2), or immediately freeze cells by immersion in liquid nitrogen or a dry ice/ethanol bath. Store the frozen cells at –70°C.

Transformation:-

Thaw frozen TSS-competent cells slowly on ice(if stored at -70°C).

Add 100 pg -200 ng (2.5 to 4 ul)(15ul for ligation product)of DNA to each tube of competent cells.
Note: Addition of more than 10ng of DNA may significantly decrease transformation efficiencies.

Flick the tubes to mix the cells and DNA and incubate the cells on ice for 30 minutes.

Transfer the tubes to water bath/dry bath(42°C) for 90 seconds.

Transfer the tubes to ice and incubate for an additional 10 minutes.

Add 800 ul (total 1 mL)of LB broth and incubate the cells at 37°C for up to 1 hour with shaking (at 200 rpm).

Centrifuge the cells at 3000g for ~ 6min (10 mins after ligation)at 4deg(in temperature control centrifuge).

Aspirate the tubes to leave the pellets with 1/4 broth .(keep ~300ul)

Plate the cells on-to the appropriate selective or differential medium and incubate overnight at 37°C.Check the procedure for antibiotic.

For Ampicillin: 12ul Amp + 188 ul MQ. In MCT spread it on the culture plate before adding the DNA.
For Chloramphenicol: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.
For Kanamycin: 1:1000 volume ratio of antibiotic : culture broth. Directly suspend into the culture broth and spread it on the plate.

Incubate on ice for 30 minutes. Expect a 2-fold loss in TE for every 10 minutes you shorten this step.

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Contact Us	Address
	E-mail: iitd.igem@gmail.com Undergraduate Laboratory Department of Biotechnology and Biochemical Engineering, IIT Delhi