Biology 2290F/G Study Guide - Final Guide: P200, Biological Hazard, Lysogeny Broth

HELP ASAP;

Please help me understand my lab results, so be detailed to clearly understand . INclude answers to these questions what role does ampicilin (amp) and arabinose (ara) play in the experiment. Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid

GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes).

In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes.

The pGLO plasmid (Fig. 5, not a naturally occurring plasmid) contains the gene for GFP, and also contains the gene for the enzyme β-lactamase (bla), which provides resistance to the antibiotic ampicillin. The β-lactamase protein is produced and secreted by bacteria that contain the plasmid. β-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express β-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

for the first week section of lab

To each of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate.

Transfer this colony to one of the microfuge tubes. Gently vortex the tube until the colony is dispersed. Repeat for the second tube.

Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes.

Incubate both tubes at 42°C for 50 seconds.

Incubate both tubes on ice for 2 minutes.

Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes.

Flick each tube gently. Add 100ul of this transformation mix to the appropriate plates.

Results:

+DNA: LB/amp--> 1 colony, Glow: uncertain

+DNA: LB/amp/ara--> 9 colonies, Glow: yes

-DNA: LB/amp--> 7 colonies, GLow: no

-DNA: LB--> many colonies, Glow: no

HELP ASAP; Please help me understand my lab results, so be detailed to clearly understand

Then , Please include answers to the question below to include in your response

1. what role does ampicilin (amp) and arabinose (ara) play in the experiment.

2) Because GFP was isolated from jelly fisht, what modification to the GFP DNA had to be made to result in expression in E. coli?

BACKGROUND INFORMATION

Transformation of GFP and the pGLO Plasmid GFP is a commonly used reporter protein in research labs, as the fluorescence creates a marker protein that can be used in many types of cell biology and biochemical studies. In basic research, GFP is often fused to a specific target protein of interest, creating a chimeric reporter protein. GFP has been used as a reporter protein to study blood vessel and tumor progression in mice, brain activity in mice, and malaria eradication in mosquitoes for instance (see website mentioned in notes). In the first week you will use a procedure to genetically transform bacteria with the gene that codes for Green Fluorescent Protein. Following the transformation procedure, the bacteria express their newly acquired jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under ultraviolet light. During the second week you will purify the protein away from all of the other proteins produced by the bacteria using chromatography, and during the third week you will evaluate the success of the purification procedure and estimate the size (molecular weight) of GFP by using sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). Fundamental to the process of genetic transformation of bacteria are plasmids. In addition to one large chromosome, bacteria naturally contain one or more small circular pieces of DNA called plasmids. Plasmid DNA usually contains genes for one or more traits that may be beneficial to bacterial survival. In nature, bacteria can transfer plasmids back and forth allowing them to share these beneficial genes. This natural mechanism allows bacteria to adapt to new environments. The occurrence of bacterial resistance to antibiotics is due in large part to the transmission of plasmids which contain antibiotic resistance genes. The pGLO plasmid contains the gene for GFP, and also contains the gene for the enzyme β-lactamase (bla), which provides resistance to the antibiotic ampicillin. The β-lactamase protein is produced and secreted by bacteria that contain the plasmid. β-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express β-lactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. pGLO also incorporates a special gene regulation system, which can be used to control expression of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white (wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is included in the nutrient agar medium.

The transformation procedure involves three main steps. These steps are intended to introduce the plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly acquired genes. 1) Cells are first treated with a solution of CaCl2, which is thought to neutralize the repulsive negative charges of the phosphate backbone of DNA and the phospholipids of the cell membrane, allowing the DNA to adhere to the cells. This is referred to making the cells ‘competent’ (i.e. the cells are capable, or competent, to take-in exogenous DNA). 2) Cells are then subjected to a heat shock, which is thought to increase the permeability of the cell membrane, allowing the cells to take-in the plasmid DNA. 3) Cells are allowed to grow in a ‘recovery’ phase in the absence of ampicillin. During this time the cells begin to produce the β-lactamase protein, which will allow them to survive when they are subsequently placed on agar plates containing ampicillin.

For the first week section of lab

To eac

h of two microfuge tubes, add 250ul of transformation buffer. Keep these tubes on ice.

Using a sterile loop, pick one colony of bacteria from the LB plate. Transfer this colony to one of the microfuge tubes.

Gently vortex the tube until the colony is dispersed. Repeat for the second tube. Add 0.08ug of DNA to one of these tubes (+DNA tube).

Incubate both tubes on ice for 10 minutes. Incubate both tubes at 42°C for 50 seconds.

Incubate both tubes on ice for 2 minutes. Add 250ul of LB broth to each tube and incubate at room temperature for 10 minutes. Flick each tube gently. Add 100ul of t

his transformation mix to the appropriate plates.

Results For plates:

+DNA: Has the E. Coli pGlo plasmid and - DNA plates do no have EColi pGLo plasmid .

Amp means ampiclin and Ara means arabinose on agar plates

+ DNA Plate LB/amp/ara--> white 9 colonies, Glow: yes

+DNA LB/amp--> 7 colonies, GLow: no

--DNA LB/AMP- 1 colony so no colony growth and no glovw

-DNA: LB--> many colonies, Glow: no

Please explain significance of result or thand the reason for the results in scientific terms based on the background information provided and your own knowledge to be detailed. Would these results be expected as wll.

can anyone paraphrase the following procedure for me please. I prefer to be typed because the handwriting sometimes is not clear

First experiment: Bacterial Transformation and Affinity Purification of Protein:

Transformation procedure:

Bring 2 test tubes and label them +pGLO and –pGLO,

Transfer 250 ul of transformation solution to each tube and then place them in ice.

Pick a single colony of bacteria using a sterile loop from the bacterial plate and immerse the loop in +PGLO tube and start spinning the loop to mix, repeat the same step for –pGLO and place both tubes in ice again.

Then, immerse a new sterile loop in the pGLO DNA plasmid stock tube and withdraw a loopful of the plasmid and immerse it in the +pGLO tube to load it and place the tube in ice again. No pGLO plasmid will be added to the –pGLO sample as labeled.

Incubate the tubes in ice for 10 minutes and take the following labeled plates from the instructor: LB/amp + pGLO, LB/amp/ara + pGLO, LB/amp – pGLO, and LB – pGLO. There will be a total of four plates.

Heat hock will start by transforming both tubes to 42C water bath for 50 second and then returning them to ice again.

Add 250 ul of LB nutrient broth to both tubes and incubate at room temperature for 10 minutes.

Using a new sterile pipet for each plate, transfer 100 ul of the –pGLO solution to the, LB/amp – pGLO and LB – pGLO agar plates and 100 ul of +pGLO solution to the LB/amp + pGLO, and LB/amp/ara + pGLO plates.

Spread the suspension using sterile loops and place your plates upside down in the 37C incubator overnight.

Bacterial growth will occur and result can be analyzed the day after.

* For data analysis, observe the result in the plates under normal light conditions and then under UV light and right your observations. This will include observing the number of colonies, colonies color and growth. Transformed bacteria will look green in color under UV with the presence of arabinose sugar in the media and will look white in color if arabinose is absent. No growth will be shown if the bacteria are not transformed.

Second experiment: Affinity purification of green fluorescent protein:

Affinity purification procedure:

Re-observe you plates from previous experiment under natural light and UV light. Identify several green colonies on the LB/amp/ara plate and with the use of a loop scoop up some cells and immerse the loop in a new culture tube labeled (+), the same procedure is done for the LB/amp plate in a tube labeled (-). Disperse the colonies in the culture tubes and shake vigorously. And place in an incubator for 24 hours.

Transfer the entire culture to a 2-milliliter microtube labeled +. Centrifuge for 5 minutes and remove the supernatant.

Re suspend the pellet in 250 ul of TE solution and then add 1 drop of lysozyme and mix then place the tube in the freezer to help rupture the cells.

For bacterial lysis, thaw the microtube and centrifuge for 10 minutes at maximum speed. And prepare the chromatography column.

Before performing the chromatography, add 2 milliliters of Equilibration Buffer to the column, and Drain the buffer from the column until it reaches the 1 milliliter.

After centrifugation, transfer 250 μl of the supernatant into the new microtube and transfer 250 μl of Binding Buffer to the microtube and Place in the refrigerator.

Label 3 collection tubes 1, 2 and 3. Load 250 μl of the supernatant in Binding Buffer into the top of the column and Let the entire volume of supernatant flow into tube 1.

Transfer the column to collection tube 2. Add 250 μl of Wash Buffer and let the entire volume flow into tube 2.

Transfer the column to tube 3. Add 750 μl of TE buffer and let the entire volume flow into tube 3.

Examine all collection tubes using the UV lamp and note any differences in color between the tubes.