fermentation

Tuesday, November 6, 2007

How GFP gene is incorporate in E.Coli?

Bacterial Transformation Experiment

Transformation of E. coli with GFP gene

Transformation experiments are an important tool in recombinant DNA technology, where the phenotype of cells can be modified through the addition of new genes.
The most common laboratory teaching exercises will be the transformation of plasmid DNA into E. coli followed by selection of transformants containing an antibiotic resistance marker. Although these experiments are successful and instructive, they lack the wonderment that is the essence of genetic engineering.

Research on the regulation of bioluminescence in Vibrio fischeri has provided exciting teaching materials that can be used in a series of molecular biology experiments, including transformation. Bioluminescence transformation experiments show students the excitement and power of recombinant DNA technology.

Seven genes are required to produce bioluminescence in E. coli. These seven lux genes from Vibrio fischeri are cloned into two plasmids where the plasmid pHK724 contains the lux R gene whose gene product is a transcription regulatory protein, while the plasmid pHK555 (lux ICDABE) contains the structural genes required to make light. The lux R gene of pHK555 is inactive because of the insertion of phage DNA. When pHK724 is transformed into E. coli containing pHK555, the resultant colonies grow on selective media will bioluminescence.

(Pictures taken from: http://departments.kings.edu/biology/lux/bacterial.html)

Part 1: Preparation of Competent Cells of E. coli

	1. Culture desired bacterial strain overnight in LB broth with chloramphenicol (Cm) added to a final concentration of 50 mg/ml. Note: Cm must be added to the growth medium to apply selective pressure on E. coli to maintain the plasmid pHK555 which contains a chloramphenicol resistance gene.
	2. Inoculate 200 µl of overnight culture into 50 ml of LB with antibiotic Cm to final concentration of 50 mg/ml LB in an Erlenmeyer flask plugged with cotton or capped with an inverted breaker.
	3. Incubate at 37ºC with agitation and monitor culture density with photoelectric colorimeter Note: Examples of the types of photoelectric colorimeter is Spectronic 21 and Klett-Summerson (Baxter Diagnostics, Inc.).
	4. Allow the culture to reach an optical density of 0.35 to 0.50 at a wavelength of 550 nm. Note: This is mid-log phase of growth and generally takes 2.5 hours. This is equivalent to 40-80 Klett units when using a Klett colorimeter.
	5. Pipet 1 ml culture to 1.5-ml Eppendorf tube and microfuge for 2 minutes before pouring off the supernatant. Add a second 1 ml and repeat microfuge and pouring of supernatant. Note: If a microfuge is not available, any centrifuge can be used to pellet the E. coli cells.
	6. Resuspend pellet in 200 ml of frozen storage buffer (FSB).
	7. Incubate on ice for 15 minutes.
	8. Microfuge 2 minutes and pour off the FSB.
	9. Resuspend pellet in 200 ml FSB.
	10. Bacteria are now "competent" and ready for transformation. Note: Competent cells can only be stored in the refrigerator for up to 24 hours or for up to 6 months if stored in a -70°C freezer. The FSB (CaCl2 solution) should be stored at 4°C and made fresh every 6 months.

Part 2: Plasmid Preparation

Numerous techniques are available for extracting plasmids from E. coli. The procedure does not require a microfuge. At the end of the extraction, the plasmid is resuspended in 100 µl of 10 mM Tris/EDTA buffer, pH 8.3-8.5.

Part 3: Transformation of E. coli

	1. Add plasmid pHK724 DNA (5µl) to 50-200µl of competent cells.
	2. Incubate on ice 15 minutes.
	3. Place tubes in 42°C water bath for 90 seconds (heat shock).
	4. Incubate at room temperature for 5 minutes.
	5. Add 1 ml of LB and incubate broth at 37°C for 1 hour.
	6. Add 100 µl to selective solid medium. Spread with glass spreader.
	7. If transforming for luminescence, incubate agar plates at 30°C or room temperature for 30-48 hours.
	8. The bioluminescent colonies are viewed in a dark room. Note: It takes about 5 minutes for one's eyes to "dark adapt."

Something extra:
To find out the influence of temperature on microbial growth, selective agar plates can be incubate at both 30°C and 37°C. E. coli will grow at both temperatures but will grow faster at 37°C since its optimal growth temperature is 37°C (the temperature of the human gut).

However, the genes for bioluminescence are from the marine bacterium, Vibrio fischeri. Vibrio fischeri has adapted to a cooler environment (the ocean) and, therefore, the proteins for bioluminescence are heat denatured at 37°C.

(All pictures taken from: http://departments.kings.edu/biology/lux/bacterial.html)

7:34 AM

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