Brown BIOL1220:Notebook/SynBio in Theory and Practice/Bacterial Basics

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 on 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.

Bacterial Basics

Protocols

Bacterial Transformation

1. Grow a 5mL overnight culture of XL1-Blue in LB broth at 37°C with vigorous shaking.

2. The next morning, add 50μL of this culture to a fresh tube of 5mL LB. Use the plastic 15mL snap top tubes. Again incubate at 37°C with vigorous shaking, and grow to A600 = 0.6-0.7. This takes a couple of hours, and the suspension will begin to take on a silky appearance. Do not let this culture overgrow - the health of the cells declines with confluence. An alternative procedure is to start a culture of XL1-Blue cells from a recently streaked plate, (streaked on LB/tetracycline) and to grow for several hours.

3. Cool cells on ice for 10 min., then take the tops off the tubes and while using the green tube adapters, spin in Sorvall SS-34 rotor for 5 minutes, 4°C, at 3,000 rpm.

4. Discard the supernatant and quickly put cells back on ice. [From here on, keep cells on ice as best you can, even when resuspending them in fresh buffers.].

5. Resuspend the cells in 2mLs of ice cold (sterile) CaCl2 (50mM) using your P-1000 Pipettman. Leave on ice for 15 minutes.

6. Centrifuge the cells as in #3 above. Discard the supernatant and resuspend cells in 500 μL CaCl2. Use immediately for transformation or store at -70 °C. If cells used for transformation are frozen, remove tube from freezer and immediately put on ice. Allow these cells to thaw on ice, which takes about 30 minutes.

7. To transform competent cells, put 100-200μL in a precooled 13 x 100mm disposable glass culture tube. Add DNA ligation mixture to the cells, mixing gently on ice. [For a positive control of transformation, use 1 μL of supercoiled plasmid pBS DNA from any miniprep.] Cover the tube with parafilm and incubate for 30-40 minutes on ice. Prepare a beaker of water (tap water is fine) at 42°C. Heat shock the cells by placing the glass culture tube at 42°C for 2 minutes, and then back on ice. Add 1mL ice cold LB media and then incubate cells in the 37°C water bath for 1 hour, making sure the tube is capped with Parafilm.

8. Melt by microwaving the bottle of LB (or NZCYM is fine) top agar, and aliquot 3 mLs for each transformation into glass 13 x 100mm culture tubes at 48°C in the dry bath. Allow top agar to cool to 48°C before using (about 10-15 minutes, but the top agar will not solidify while in the temperature block).

9. Prewarm an LB/Amp (100μg/mL) bactoplate in the 37°C white incubator.

10. [If you are using blue/white color selection, get X-gal (2%) and IPTG (100 mM) ready.] Mix cells and top agar [and 50 μL X-gal, 20 μL IPTG] together to a uniform suspension, pour onto bactoplate and tip plate so that agar covers entire surface. Be careful to not introduce bubbles and work quickly so that the agar does not solidify before pouring.

11. Let agar solidify at room temperature, and then invert in 37°C white incubator for overnight.

For 50 mM CaCl2 use 0.73 gms of CaCl2 - 2H2O per 100 mls of water, and autoclave.

Sources

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.

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

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