Unit 5: Transformation
Introduction
Transformation is the process of uptaking foreign plasmid into cell. Transformation can occur in nature in certain types of bacteria. In molecular biology, transformation is artificially reproduced in the lab via the inducing pores in bacterial cell membranes. Then, antibiotic is used to select the successfully transformed cells with desired plasmid (successful transformants). DNA can be transformed into all kinds of organisms. There are two kinds of transformation, via heat shock or electroporation. Bacterial transformation via heat shock is highlighted in this unit.
Basic concept of transformation
http://www.biology.arizona.edu/molecular_bio/problem_sets/recombinant_dna_technology/05t.htmlBacterial transformation
Preparation of competent cells
For maximum efficiency, competent cells at optimum growth rate is prepared.
A single fresh colony of the desired strain is transferred from an agar plate to liquid medium for a starter culture. This starter culture is subsequently carefully monitored for active growth by continually measuring optical density at 600 nm (OD600). It is crucial that cell should be in the mid-log phase at the time of harvest—which generally occurs at OD600 between 0.4 and 0.9, with the optimal value depending on the culture volume, strain, and protocol. Note that using sterile tools and labware, media, and reagents in transformation is a mandatory to prevent contamination and spoil the results. We recommend that once the cells are harvested for further processing, all samples, reagents, and equipment be kept at 0–4°C in order to improve cell viability and maintain competency.
2. Transformation
To further improve competency, competent cells are chemically prepared by incubating the cells in calcium chloride (CaCl2) before heat shock. The positive charges of Ca2+neutralize the negative charges on both the plasmid and the bacterial cell wall which contribute to the electrostatic repulsion, and weaken the cell wall, making it more permeable.
By heat shock, a pressure difference between the outside and the inside of the cell in created. It induces the formation of pores in the bacterial cell wall so that supercoiled plasmid DNA can enter. At this stage, cells can be mixed by gentle shaking, tapping, or pipetting, but vortexing should be avoided because cells are weak.
3. Recovery
After returning the cells to a more normal temperature, the cell wall will self-heal. Transformed cells are cultured in antibiotic-free S.O.C. medium for a short period to allow expression of antibiotic resistance gene from the acquired plasmid to begin. This step improves cell viability and cloning efficiency. S.O.C. medium, which contains glucose and MgCl2, is recommended to maximize transformation efficiency.
4. Cell plating
After growing in S.O.C. medium, the cells are plated on agar plate with appropriate antibiotic for selection and recovery of successful transformants. Before cell plating, the plates should be prewarmed to a 37℃ and be free of condensation to prevent contamination.
Spread the transformants with culture medium on the agar plate. Incubate the plate for 24 hours. Avoid prolonged incubation as it would often produce fused colonies.
Selection
This step ensures all the cells cultured on agar plate are successful transformants. Each recombinant plasmid contains an antibiotic resistance gene as a selective marker. The antibiotic resistance gene encodes the resistance against an antibiotic. Transformants are cultured on an agar plate that contains antibiotics. Only the transformants with antibiotic resistance can survive on the plate. Therefore the survived cells are successful transformants which have successfully acquired the recombinant plasmid. For example, a recombinant plasmid contains an antibiotic resistance gene for Ampicillin. The recombinant plasmid is transformed into an E.coli. The successful transformants E.coli are now Ampicillin resistant. Only successful transformants survive on agar plate containing Ampicillin. Unsuccessful transformants however, do not survive. E.coli that survive on agar plate are successful transformants.
Transformation efficiency
Transformation efficiency is the efficiency which cells can take up foreign DNA and express genes it. The ideal transformation efficiency is 1108CFU/μL, in order to facilitate future cloning steps. The following is the equation for calculation of transformation efficiency.
Here is an example of calculation of transformation efficiency
50 ng of DNA is ligated in a 20 μL reaction solution. After ligation, the mixture is diluted 2-fold and 5 μL of the diluted ligation mixture is added to 1000 μL of competent cells for transformation.
After transformation, the cell suspension not diluted and 20 µL of the diluted cells are plated. 900 colonies are formed after overnight incubation.
Transforming GM Plants
Below is a brief overview of how plants can be genetically modified. Since plant cell is highly different from bacteria as plant cells have a cell wall and a nucleus membrane. Therefore, to induce a foreign piece of DNA into plant cell, one of the most common methods used is known as Agrobacterium-mediated transformation, which utilizes a bacterium that is carrying the gene of interest to infect and transfer the gene to the plant cell. This involves 2 steps of transformation: transforming the agrobacterium, then using the transformed agrobacterium to infect the plant cell.
Firstly, obtain both the desirable gene and the plasmid from the agrobacterium that will be used. Via restriction digestion, cut open both the gene of interest and the plasmid. This creates an overhang that is complementary with each other.
Secondly, ligate them together with ligase.
Thirdly, transform the ligated plasmid into the agrobacterium and cultivate it at its optimal temperature in nutrient medium to support its rapid growth.
Fourthly, use the transformed bacteria to infect the plant cells. This is done by cutting the plant tissue (eg. leaves) into small pieces and soak in a fluid containing suspended Agrobacterium. The bacteria will attach to many of the plant cells exposed by the cut. The agrobacterium will export its protein and its Transfer DNA (containing the gene of interest) into the plant cell via a structure called pilus. The plant cell will incorporate these genes into its own genome.
Lastly, the plant cell will be differentiated and grow into a complete plant.
Outline of producing a GM plant
Transforming GM Animal
The transformation for animal cell is known as transfection, which is usually done by the following methods: viral transduction methods and non-viral methods. This process is usually done when the organism is still in it’s zygote state so that the entire organism will be transfected as it grow and differentiate. It is usually done while the cell is in its embryo state or early blastocyst state so that the embryo will multiply and differentiate into a fully mature animal later.
Transduction methods utilizes a harmless strain of virus as a vector that carries the gene of interest and infect the targeted animal cell. Virus has the ability to infect other animal cell and incorporate its gene into animals cell’s genome. Scientists exploited this ability of virus to insert genes to transform the cell.
Non-viral method includes multiple methods, from using chemical methods (using calcium phosphate buffer eg.HeBS) to physical method (eg. gene gun, electroporation and heat shock). Microinjection is another common technique used in delivering liquids into the cell, which can be protein or DNA. The exact mechanism for organisms to pick up these foreign DNA is still unknown and remain as one of the hot topics in the field.
Troubleshooting
iGEM has created a page illustrating the possible troubles faced by experimenters during transformation, and helping them to pinpoint the problem and tackling them. You may want to take a look at the page, or look for solutions there in the future. iGEM troubleshooting transformation page http://2015.igem.org/Troubleshooting/Transformation.