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BIOL SCI 215 Study Guide - Midterm Guide: Apoptosis, Body Plan, Kras

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School
Northwestern University
Department
Biological Sciences
Course
BIOL SCI 215
Professor
aaronpeterson

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Recombinant DNA technologies
● Molecular cloning
Recombinant DNA technology
● Isolate, study, manipulate genes from any organism
○ Manipulation is genetic engineering
● Key to modern molecular genetics, medicine, and biotechnology
Uses
● Isolate genes to better study the proteins they encode, understand their function in the
cell
● Isolate and study genes that are causing human diseases - screen drugs, design rational
therapies
● Manipulate genes to improve crops, produce useful proteins (e.g. insulin), create useful
bacteria for bioremediation
NOT the same as recombinant chromosomes or cloning (e.g., Dolly)
Methods to find gene that expresses fluorescence in jellyfish
1. Genetic screening - mutagenize jellyfish and breed to find recessive mutations causing
lack of fluorescence and then map that gene to find the gene required for fluorescence
2. Isolate all genes, insert in bacteria and see which bacteria glow up
Two different ways to amplify an interesting gene
1. Cloning
2. PCR
Cloning
- Cut up jellyfish genome
- Place one fragment at random each into one plasmid
- Place one plasmid in each bacteria
- Let bacteria multiply
- BUT cannot do “eukaryotic genes → plasmids → bacteria” because bacteria can’t splice
out introns
- SO use cDNA
Reverse transcriptase (from retroviruses)
● Can create a DNA molecule from an RNA template
● Synthesizes 5’ → 3’
● Design a primer called oligo-dT (poly-T’s) that anneals to polyA tail on mRNA’s 3’ end
● Then DNA polymerase comes and elongates, the new strand, creating cDNA or
complementary DNA
: a copy of DNA from RNA
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Make cDNA from jellyfish RNA, which will lack introns
● Transcription splicing in live jellyfish
● Extract jellyfish RNA
● Make cDNA “in vitro” (in a test tube)
● Use trick to make second DNA strand
Use a plasmid as a vector for delivery. Plasmids can have a high copy number (hundreds per
cell) and are inherited from one generation to another
● Each have one ori (Origin of replication)
Transformation: delivering a plasmid to bacteria
Anatomy of a plasmid
● MCS (multiple cloning site)
○ Useful region for cutting and inserting new DNA
● Ori
● ampR
○ Antibiotic resistance to kill bacteria that do not take up plasmid
Recombinant DNA overview
1. Cut DNA using restriction enzymes
2. Paste DNA using DNA ligase
3. Transform DNA into bacteria
4. Screen DNA to find desired new molecule
Cut DNA with restriction enzyme
● Restriction enzymes are a class of enzymes produced by different species of bacteria
● Each restriction enzyme cuts at a specific DNA sequence
○ Useful as a defense mechanism by bacteria
○ Useful for biotech for us
● E.g. EcoRI (comes from E. coli): cleaves palindromic DNA to get sticky ends
Restriction enzymes cut DNA at specific sequences to leave either “blunt” or “sticky” ends
● Sticky ends = overhanging ends
Why doesn’t the cell’s restriction enzymes destroy its own genome?
● DNA methylation protects bacterial genomic DNA from being cut
● Acts as an immune system
Ligase joins fragments together
● Requires compatible sticky ends
Transformation: Bacteria can be treated to allow uptake of foreign DNA
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● Shock bacteria using salts or electricity so they can take up plasmids
● Only 1 in ~100,000 bacteria cells takes up a plasmid
● Each bacterial colony grew from one cell so are genetically identical
The use of antibiotics and resistance genes allows selection of bacterial cells that contain the
plasmid
● Transformations are inefficient, which requires the use of a selectable marker such as
AmpR
Screen for the desired product
● Often particular cloning strategy requires testing the plasmids growing individual
bacterial colonies for presence of the insert or orientation
● One way to check for insertion is with PCR
● Gel electrophoresis
● Could sequence
To produce jellyfish GFP by expression in E. coli, you should use a vector with a bacterial
promoter because it needs to be recognizable by the bacteria’s RNA polymerase
Plasmid library: each bacterium expresses a different jellyfish protein
We have now isolated a cDNA sequence encoding GFP protein
5/21
Transgenic
Methods
- Electrophoresis - measure lengths of nucleic acids
- Dideoxy sequencing and PCR - you require a primer
Why would you create transgenic animals?
- Give animals/cells a new protein
- Interested in a particular protein and you want to understand the functionality
- Overexpression
- Determine consequence of having excess protein for that gene
- E.g. Gal80
- Misexpression
- Express myoD everywhere in mouse not just in making muscle
- Expressing a gene in new time and place
- Make a labeled version of that protein/tissues to examine using GFP so
that u can track the growth such as during cell divisions
- Create a specific mutation
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Document Summary

Isolate, study, manipulate genes from any organism. Key to modern molecular genetics, medicine, and biotechnology. Isolate genes to better study the proteins they encode, understand their function in the cell. Isolate and study genes that are causing human diseases - screen drugs, design rational therapies. Manipulate genes to improve crops, produce useful proteins (e. g. insulin), create useful bacteria for bioremediation. Not the same as recombinant chromosomes or cloning (e. g. , dolly) Methods to find gene that expresses fluorescence in jellyfish: genetic screening - mutagenize jellyfish and breed to find recessive mutations causing lack of fluorescence and then map that gene to find the gene required for fluorescence. Isolate all genes, insert in bacteria and see which bacteria glow up. Two different ways to amplify an interesting gene: cloning, pcr. Place one fragment at random each into one plasmid. But cannot do eukaryotic genes plasmids bacteria because bacteria can"t splice. Can create a dna molecule from an rna template.

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Related Questions

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.