Team:Brown/Notebook Protocols/bacterialbasics

= Bacterial Basics =

 Bacteria Structure 

Bacteria are prokaryotic organisms. They are surrounded by a firm cell wall that helps maintain their structure. Bacteria have no nucleus; their genomic DNA is contained in the nucleoid. Bacteria also contain extrachromosomal DNA, called a plasmid. Bacteria reproduce asexually.

''' Different Forms of DNA Transfer:

- Transformation

- Conjugation

- Transduction '''

 Bacterial Transformation 

Bacterial Transformation is the process by which bacteria uptakes DNA from the media (liquid) surrounding it. The DNA can be from cells from the same species or from different species. Once taken up by bacteria, the DNA will integrate into the bacteria’s chromosome. As seen in the diagram at the right, the DNA binding complex that the free DNA passes through is contained within the bacteria’s cell wall. This process is commonly used in genetic engineering.

 Bacterial Conjugation 

During conjugation, DNA from one bacteria cell (donor) is transferred to another bacteria cell (recipient). The two bacteria cells must be in physical contact; the donor bacterium extends its pili to transfer its DNA to the recipient. Plating bacteria on different media is a common way to see whether any mutants (bacteria that have received new DNA) survived. Auxotrophic mutants will not grow if there are no supplements in the media, whereas prototrophic mutants can grow without supplements.

 Bacterial Transduction 

A phage (virus) attaches to a donor bacterium and injects its DNA. It uses the bacterium as a “factory” to make more phages. The newly made phages then break out of the cell and attach to recipient bacterium. They insert their DNA, which then recombines into the bacterium’s DNA. The recipient bacterium is considered transduced and contains the genetic information to synthesize the phage.

 More Details of Chemical Bacterial Transformation 

Although the mechanism by which calcium chloride-mediated bacterial transformation is not completely understood, some basics are known.

During early logarithmic growth, the cell membrane of E. Coli bacteria develops many pores called adhesion zones. These zones differ from the surrounding lipid bilayer membrane by the existence of lipopolysaccharide (LPS) molecules, which can bind foreign DNA. The LPS molecules alone, however, are not enough to bring the foreign DNA into the cell due to the electrostatic repulsion between the DNA’s sugar phosphate backbone and the polar lipids of the lipid membrane. Calcium cations from a calcium chloride solution should theoretically be able to create an electrostatically neutral situation. Cooling the bacteria while in this solution congeals the lipid membrane and shields the ionic charges effectively. Heat-shocking the bacteria to 42ºC creates a heat gradient, through which the foreign DNA along with outside water can enter the cell (this causes the cells to swell).

Cells that can take up foreign DNA from a nutrient-rich external solution are termed chemically competent. The foreign DNA is incorporated into the bacterial genome in one of the three aforementioned ways. If these cells recognize the origin of the foreign DNA, they will replicate it along with the rest of their genome during cell division.

If the exogenous DNA is tagged with an antibiotic resistance gene, only cells that incorporated the correct foreign DNA can be selected by the application of the antibiotic.

Chemical Transformation Procedure

Based on Invitrogen’s One Shot® TOP10 Competent Cells

1. Choose ligation DNA and make sure that it is tagged with an antibiotic resistance gene (usually ampicillin)

2. Centrifuge the vial(s) containing the ligation reaction(s) [foreign DNA] briefly and place on ice.

3. Thaw, on ice, one 50 µl vial of One Shot® cells [chemically competent cells] for each ligation/transformation.

4. Pipette 1 to 5 µl of each ligation reaction directly into the vial of competent cells and mix by tapping gently. Do not mix by pipetting up and down. The remaining ligation mixture(s) can be stored at -20°C.

5. Incubate the vial(s) on ice for 30 minutes.

6. Incubate for exactly 30 seconds in the 42°C water bath. Do not mix or shake.

7. Remove vial(s) from the 42°C bath and place them on ice.

8. Add 250 µl of pre-warmed S.O.C medium to each vial. S.O.C is a rich medium; sterile technique must be practiced to avoid contamination.

9. Place the vial(s) in a microcentrifuge rack on its side and secure with tape to avoid loss of the vial(s). Shake the vial(s) at 37°C for exactly 1 hour at 225 rpm in a shaking incubator.

10. Spread 20 µl to 200 µl from each transformation vial on separate, labeled LB agar plates (+ antibiotic – usually ampicillin). The remaining transformation mix may be stored at +4°C and plated out the next day, if desired.

11. Invert the plate(s) and incubate at 37°C overnight.

12. Select colonies and analyze by plasmid isolation, PCR, or sequencing.

Sources: http://www.dnai.org/text/mediashowcase/index2.html?id=1009

http://www.genome.ou.edu/protocol_book/protocol_adxF.html

Griffiths, Anthony J. F. Introduction to Genetic Analysis, 9th Edition. W.H. Freeman and Company: New York, 2008.

Panja et al. "Plasmid DNA Binds to the Core Oligosaccharide Domain of LPS Molecules of E. coli Cell Surface in the CaCl2-Mediated Transformation Process." Biomacromolecules 9(2008): 2501–2509.