Team:ArtScienceBangalore/Notebook/Bacterial Transformation : The Process

From 2009.igem.org

What are we doing?

We are attempting to alter the genetic composition of a bacteria by adding foreign genetic material to it. For this , the bacteria needs to be conditioned to take foreign material and make it its own. Now it is this process of 'conditioning' that forms the scientific method described below. This, in scientific terms is called 'Transformation'.

How are we doing this?

We start by gathering the materials we need:

1. Eppendorf tubes

The tiny plastic tubes that are used in labs while dealing with solutions that are extremely less in quantity. Unlike regular test tubes, they come with an easily usable lid and a slightly triangular bottm.

2. LB solution

Luria Bertani solution; named after a scientist is a source of required nutrients and enables the bacteria to grow.

3. Dry DNA (from the DNA Distribution Kit )

The foreign DNA that we are adding to the bacteria.

4.Centrifuging Machine

This machine rotates the samples that are put in it.It contains a plate of 18 circular wells that hold the samples. Since it is acted upon by centrifugal force,the samples must be arranged in balance to avoid any disembodiment of the samples in the machine. After centrifuging, the substance in the eppendorf tubes separates intp 2 distict phases-the liquid phase and the solid phase

Here,the liquid phase is called the 'Supernatant' and the solid phase is called the 'pellet'

5 Lysis

6.pTET/PBAD

7.Positive control

Holds the gene that promotes Ampicillin Resistance.

8.Negative control

Hold the gene that does not promote Ampicillin resistance. If any colonies started forming in the negative control batch, that would mean the entire batch is contaminated.

Once we understood what each substance did, we began the scientific step-by-step process that was explained to us by Navneet.

The Process:

1) Four eppendorf tubes were taken and marked lysis, p-tet, +ve and -ve. The positive control e and the negative control did not. # The dry DNA was extracted by mixing each part with 15 micro ml of water using a micro-pipette.

2) 2-3 micro ml of this plasmid solution was then added to 100 micro ml of DH 5-alpha E.Coli cells.

3) The bacteria were incubated in ice for 40 minutes, then given a heat shock for 30 seconds @ 42 degrees Celsius and then put in ice for 2-5 minutes. The heat shock causes tiny pores in the walls of the bacteria to expand, allowing the plasmids to go through.

4) The Bacteria were then mixed into 330 micro ml LB (Luria Bertani) # The bacteria were then left to cultivate for approximately an hour @ 37 degrees Celsius in the shaker.

5) Centrifuged at room temperature for 3 minutes @ 8000 rpm.

6) Discard 900 micro ml of the supernatant and dissolve pellets in remaining 100 micro ml. Spreading helps ensure that you will be able to pick out a single colony.

7) These were then spread on LB Agar plates containing 100 micro gram per ml Ampicillin.

8)The plates were incubated at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics, especially ampicillin, start to break down and un-transformed cells will begin to grow.

Some images from the process:

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