BIOL1130 Lecture Notes - Lecture 22: Phosphodiester Bond, Gene Expression, Pipette

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tangerbil581

28 May 2018

School

UWA

Department

Biochemistry and Molecular Biology/Biology

Course

BIOL1130

Professor

Guijun Yan

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9.1 Gene Cloning

1. Differences between animal cloning and gene cloning

2. Experimental process of gene cloning and bacterial transformation (DNA

recombination)

3. Key steps of gene cloning, terminologies

Gene cloning

• take the genes out of organisms to have a look at structures and functions.

• Cure human/animal diseases, better quality, higher production, increase breed

efficiency

Requirements for Gel electrophoresis

• Agarose- sugar, fruit jelly, polymer

• DNA sample

• Pipette

• Well

• Electric field

• When purple dye gets close to bottom, power is turned off, gel is stained with

ethidium bromide, UV light fluoresce as orange bands

How to clone a gene

Requirements

1. A competent bacteria that is capable of taking up DNA, has holes on membrane to

accept foreign DNA

2. Sample DNA that’s to e studied

3. Vector that is cultured in the organism, bacteria gene, E coli, a vector is a plasmid

that can replicate/culture in a host cell. E coli is added to the plasmid and E coli will

culture the plasmids

Steps to form a recombinant DNA

1. Restriction enzyme – restriction endonuclease cuts the foreign DNA at restriction

site

2. R. Endonucelase cuts the plasmid at restriction site

3. DNA ligase joins foreign DNA and plasmid together at sticky ends (phosphodisester

backbone )—hybrid plasmid

Fragments are cut with r.e and ligated into a vector and then introduced into host cells to be

cultured

Vector has antibacterial, therefore foreign bacteria wont grow there, only the bacteria that

needs to be cultured is grown, therefore plasmid has selection markers.

Only the vector ( the plasmid ) that has been taken up by the bacteria E coli will contain

antibacterial + nutrients

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Document Summary

9. 1 gene cloning: differences between animal cloning and gene cloning, experimental process of gene cloning and bacterial transformation (dna recombination, key steps of gene cloning, terminologies. Gene cloning take the genes out of organisms to have a look at structures and functions: cure human/animal diseases, better quality, higher production, increase breed efficiency. Requirements for gel electrophoresis: agarose- sugar, fruit jelly, polymer, dna sample, pipette, well, electric field, when purple dye gets close to bottom, power is turned off, gel is stained with ethidium bromide, uv light fluoresce as orange bands. E coli is added to the plasmid and e coli will culture the plasmids. Fragments are cut with r. e and ligated into a vector and then introduced into host cells to be cultured. Vector has antibacterial, therefore foreign bacteria wont grow there, only the bacteria that needs to be cultured is grown, therefore plasmid has selection markers.

Related Questions

1- if each E. Coli cell contained fifty copies of the plasmids, how many copies of the plasmid were in the microcentrifuge tube after losing the cells?

2- describe how Jeff could use agarose gel electrophoresis to confirm that the plasmid is recombinant?

3- the stripe rust resistance gene is part of a recombinant DNA molecule consisting of cloning vector linked to wheat DNA. What is a cloning vector? Give two examples of specific DNA molecules routinely used as a cloning vectors?

carminemosquito882

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 Î²-lactamase (bla), which provides resistance to the antibiotic ampicillin. The Î²-lactamase protein is produced and secreted by bacteria that contain the plasmid. Î²-Lactamase inactivates the ampicillin present in the LB nutrient agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express Î²-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 cellsâ 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 âcompetentâ (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 ârecoveryâ phase in the absence of ampicillin. During this time the cells begin to produce the Î²-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 42Â°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.

BIOL1130 Lecture Notes - Lecture 22: Phosphodiester Bond, Gene Expression, Pipette

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