Amgen Lab

Why is mFP placed in binding buffer?

-Binding Buffer(high salt solution) -to change the conformation of the protein, causes hydrophobic regions to now point out (and hydrophillic to now point in) so that it will stick to the hydrophobic resin. -hydrophobic proteins will be released

Why do you think it is necessary to use very small and exact volumes of reagents in biotechnology?

-In this field you would use very small amounts of the reagents and the correct measuremts of the reagent amounts is necessary for procdeured to be successful

What does the term genetic deices mean? What examples of gentic disease do you know about?

-a genetic disease is a disease caused by an absent of defective gene in the DNA -suckle cell anemia: disease resulting from defective red blood cells- treatment focuses on reliveing pain/controlling infection and complciations

What do the restriction enzymes, Bam HI and HindIII, do to the plasmid?

-cut the plasmid (as restriction enzymes do at specific nucleotide sequence(restrcition site0

What is the purpose of the first stop on the micropipette? scond stop?

-first stop: measure correct volume/draw out -second stop: expel liquid (adds air)

Draw and label the pARA-R plasmid. Desribe functions.. -rfp -pBAD -araC -ampr

-rfp: gene that codes for red fluorescent protein(mFP) -pbad: promotor region for rfp; RNA polymerase complex bids to Pbad to allow for transcription of mFP -araC: gene that codes for araC protein(always being transcribed and translated) -ampr: gene that codes for ampr protein(ampicillin resistance) always being transcribed and translated

What was the purpose of the Wash Buffer?

-slightly lower salt solution -filter out proteins that are less hydrophobic than the mFP

How do bacteria reproduce?

-usually binary fission(asexual reproduction) -cell grows and divides=identical cells

What does it mean that the E. coli bacteria is "competent"(aka. it is a compitent cell)?

-was treated with calcium(Ca2+) that neutralized -meanign it is more likely to accept a plasmid

How are the P+ and P- tubes different?

P+ contains a plasmid while P- does not

The para-R plasmid you digested in Laboratory 2A was replicated in a bancterial cell. What configurations -supercoiled,nicked circle, multimer, regular/normal- might the plasmid have before digestion?

The plasmid can have all three configurations.

In order to create a plasmid that can produce the red fluorescent protein in bacteria, what components are needed in the plasmid?

The plasmid needs an origin of replication, a promoter sequence, the gene of interest (rfp), and a gene for antibiotic resistance that allows for identification of bacteria that have taken in the plasmid.

What is a bacterial colony?

a "pile" of identical bacterial cells

Why did you have to add distilled water to the R- tube?

acts as a control for volume- balance to the weight of the R+ tube

What type of resin was used in the chromatography colummn?

hydrophobic

what property of the protein was used to purify out the protein in chromatograohy colummn?

hydrophobicity

How did actual gel results compare to gel predictions?

if the procedure was done correctly, students should see the same number of bands as predicted in each lane.

What role do enzymes have in nature?

protect bacteria from infection by bacteriophage viruses

Why was ampicillin included in the overnight culture?

to allow "transformed" E. coli cells to grow and divide

What is the purpose of "heat shock"

to cause the plasmid to enter the E. coli(host organism)=transformation!

List and explain 2 Examples from reading where a defect in protein production can lead to complicarions for an organism

-hemophellia: make little/no clotting factor protein so blood doesn't clot properly -diabtes: glucose cannot enter the cell due to complications of insulin hormone

During the labs, you were often reminded to avoid contact with the pipette tips- for example, you were asked to put the pipette tip on without using your hands, to avoid setting gown the micropipette, to use the ejector button to remove the tip, and to keep the tip box closed. If you were working with plasmids and bacterial cells, why would these precautions be important?

working with bacterial cells and plasmids, you would want to be very careful to avoid any cross-contaminations from occurring.

Why is a protein's conformation important for carrying out its function?

A specific conformation results in binding sites on the outside of the protein. The binding sites allow the protein to attach to other molecules, which is how a protein can carry out its function. Proteins have one of four functions: catalyzing reactions, transporting molecules, providing a signal, or forming structures.

What is the importance of micropipettes and gel electrophoresis in the gentic engineering process?

Micropipettes are used to transfer very small and exact volumes of reagents and gel electrphoresis is used to seperate and identify plasmids and short linear pieces of DNA

What was the purpose of adding ampicillin (amp) to the agar plate?

To select for bacterial cells that contain the plasmid

What is the formula used to calculate transformation efficiency? (include units)

Transformation efficiency= total # of cells growing on agar plate(cfu)/ amount of DNA spread on agar plate (ug)

How might the column chromatography procedure be adjusted or modified to increase the purity of the red fluorescent protein sample?

Use more wash buffers that have more graduation in the concentration of salt during the elution process. Collect to red eluate in small batches and keep the middle bathes only.

Explain the relationship between Pbad, arak protein, arabinose, and transcription of rfp.

When arabinose if present, it binds to araC protein. RNA polymerase then binds to this complex and can transcribe the rfp gene

Compare the lanes that have linear fragments with the lanes that have plasmids. Is there a difference in the shape of the bands between these two DNA forms?

Yes, the bands for the linear fragments have trailing edges.

How would you identify a restriction site?

same Dna sequence on both directions(same nucleotide sequence just backwards)

What was the purpose of soaking the gel in the stain EtBr(Ethidium Bromide) and then rinsing it in distilled water?

to stain the DNA(enable you to see DNA bands)

How many restriction sites are on the pARA-R plasmid?

two

Which solution contained a single dye: s1,s2,s3. How do you know?

S3; contained a single dye because it only had one band

What substance is "food" for the bacteria? What 2 forms was it found in for the lab?

-LB -2 forms: liquid and in gel(bottom of petri dish)

what would the window of a micropipette ( P-20) look like if you dialed 6.3ul? -P200:63ul? -P1000: 630ul

-P20: 063 -P200:063 -P1000: 063

Adding human DNa to bacteria makes it possible to make human insulin. What do you already know about DNA

-location of DNA in cell: eukaryote -DNa found in chromosomes of nucleus and in mitochondria and chloroplasts; prokayote: DNA found in a chromosome in the nucleoid and in plasmids

What was the purpose of the Elution Buffer?

-low salt concentration -"reflip" mFP back to orgiginal conformatioin(hydrophobic point in and hydrophillic point out) -mFP is now released from resin !!

What products might you expect to see in the R- and the R+ tubes? Create a table that shows all the possible fragments and plasmids by tube. Include the length (bp size) of each possible fragment or plasmid, and arrange them first by size and next by speed through the gel, from fastest to slowest.

1. R-: the plasmid can have all 3 configurations, and the supercoiled configeration should move the fastest 2. R+: (1) pBAD-rfp fragment; (1) ampR-ori-araC fragment

Summarize Lab 4 procedure:

1. check agents: (R-,R+, LD, DL, SB flask) 2. pippette 2.0 ul LD into R- and R+ 3. centrifuge 4. cover gel in GEU with 1x SB 5. Pippette 10.0 ul each of DNA ladder(DL), R-,R+ into designated well 6. run gel 7. check to see if purple loading dye is moving toward the positive(red) electrode

Summary procedures in lab A

1. check reagent (RD) 2. cover gel in pippetting practice plates with SB 3.Centrifuge RD tube 4. Pipette 10.0ul RD into each well in plate 5. Check reagants s1,s2,s3 6. cover gel in Geu with SB 7. Centrifuge s1,s2,s3 8. draw out location of solutions in gels 9. Gplace over GEU 10.run gel 10 min

Summarize Lab 2a procedure

1. check reagents 2. label 2 tubes R+ and R- 3. Pippette 4.0 ul 2.5 B into R+ and R- tubes 4. Pippette 4.0 ul pR into R+ and R- 5. Pippette 2.0 ul RE into R+ tube .6 mix reagents in R+ tube 7. Pippette 2.0 ul of dH20 into R- tube 8. Mix reagents in R- tube 9. Centrifuge R+ and R- 10. place R+ and R- tubes into 37 degree C. water bath and incubate for 60 min 11. Place R+ and R- tubes in freezer at -20 C

The molecular weights for the dyes are 452.38 atomic units (au) for orange G, 669.98 au for bromophenol blue, and 538.62 au for xylene cyanole. How do these weights compare with your original conclusions about the weights of the dyes?

Base on the order of movement of the dyes through the gel (yellow moved the farthest, then purple, then blue), students should have originally predicted that the orange G (yellow dye) was the smallest, then bromophenol blue (purple), then xylene cyanole (blue) was the largest. But based on the molecular weights above, the purple was actually bigger than the blue, yet it moved farther in the gel.

What is genetic engineering?

Branch of Bio tech that uses special procedures and techniques to chnage an organism's DNA

What is the structure and function of Dna?

DNA is a double-stranded molecule, and each strand is made up of nucleotides. There are four different nucleotides, which are distinguished by a subpart called a base. The bases are cytosine, guanine, adenine, and thymine. The two strands of DNA are connected by hydrogen bonds between adjacent bases, which are called base pairs. In the base pairs, cytosine is always paired with guanine, and adenine is always paired with thymine.

Why was DNA loaded into the negative end of the gel electrophoresis box?

Dna has a negative charge as well so you turn on the gel box, the electrical charge will cuase the negative DNA to move towards the positive end of the box and therefor move through the gel. -DNA phosphate groups make DNA negative

What step in Lab 5 actually caused Transformation?

Heat shock

Where would you expect to see the rfp gene and the ampR gene in the gel photograph? Are you able to locate these two genes? Explain your answer.

In the R+ lane, we would expect to see a band between the 1,000 bp and 500 bp DNA ladder fragments, which is the 807 bp fragment that carries the rfp gene. In the R+ lane, we would expect to see a band between the 4,000 bp and 5,000 bp DNA ladder fragments, which is the 4,495 bp fragment that carries the ampR gene. We see both of these bands in these locations.

What was the purpose of "PBAD" on the plasmid

It is the promoter for the rfp gene

Ampicillin is an antibiotic that kills bacteria cells by disrupting the formation of cell walls. However, the pARA-R plasmid has the ampicillin resistance gene, which produced a protein that breaks down ampicillin. What is the purpose of growing bacteria that have been transformed in the presence of ampicillin?

Only cells that have a plasmid that contains the gene for ampicillin resistance will be able to grow in the presence of ampicillin. You can select the cells that have been transformed, which are very few.

What property of the protein was used to purify out the protein in the chromatography columns?

Position of hydrophobic R-groups

Why did E. coli only grow on P+ side of the LB/amp plate?

The plasmid produces ampicillin-resistant protein which interferes with the ampicillin antibiotic in the plate. Any bacteria containing a plasmid producing this protein would be a survive and grow when exposed to ampicillin

What characteristic of red fluorescent protein is used as the basis for separation by column chromatography?

The red fluorescent protein has more hydrophobic amino acids than hydrophilic amino acids and when unfolded sticks to the resin on beads in the column.

What is the sequence of the sticky end that results when DNA is cut with BamHI? with Hindlll?

They over hand end for BamHI is GATC. The overhanging end for HindIII is AGCT

Does the gel photograph show that you are using the correct recombinant plasmid? Describe the evidence you used to make this assessment.

This is the correct recombinant plasmid because the R+ lane has two fragments of the correct sizes.

In what circumstances might it be important to use gel electrophoresis to seperate and identify plasmids and short linear pieces of DNA?

This would be important if you are making s recombinant plasmid an dhave to cverify that you have been successful

What was the purpose of adding lysis buffer to cell culture?

To "bust open" cell; allowing access to proteins

What was the purpose of "araC" on the plasmid in the presence of arabinose

To make a protein (araC) that will be part of a complex which will bind with RNA polymerase

In the R+ lane, do you see evidence of complete digestion? Explain your answer.

Yes, there are only two bands in the lane, showing that the plasmid was completely digested into its two fragments.

Do you think there was a difference in the amount of charge on one of the dye molecules? Explain the reasoning for your response.

Yes, there must have been a difference in charge in the purple dye, or else it wouldn't have moved farther than the smaller blue molecule. -dyes in lab have different ratios of charge to mass-although heavier molecules will move more slowly than smaller molecules if both have the same electric charge,molecules with more negative charge will move faster than molecules with less negative charge if both have the same weight. Xylene cyanole migrates more slowly than bromophenol blue although it is smaller than bromo. blue becasue bromo clue has a greater charge to mass ratio. In addition, longer, branched molecules will be tangled up in gela nd move slower than shorter ones

Why did E. coli grow on both the P- and P+ sides of the LB plate?

since the LB plate was just bacteria food, all bacteria cells could grow

What are 4 confugeratiosn of a plasmid?

supercoiled,nicked circle, multimer, regular/normal

How are the R+ and the R- tubes different?

the R+ tube contains the restriction enzyme while the R- does not

Why is it important to identify and verify a recombinant plasmid?

-error during procedure/other problems may occur -reagents may not be correct -products of genetic engineering are not visible-mistake will go unnoticed. -must ensure recombinant plasmid that has desired gene/important gene sequences

What are 2 purposes of using the loading dye?

1. make DNA more dense-allow sample to sink into wells 2. make movement of DNA visible in gel- doesn't run off gel

gel electrophoresis separates molecules based what 2 properties? Which property was used in lab 4a?

1. size (used in lab 4a) 2. charge

What is the relationship among genes, proteins, and traits (or observable characteristics)?

A gene contains the code for making a protein, and proteins are molecules that are used in making and running the cell, so they are responsible for traits. Often, a trait is the outcome of multiple proteins.

All living organisms contain DNA. In what ways is DNA different organisms the same, and in what way does it vary?

All DNA has the same structure and uses the same code and transcription and translation processes. Among different organisms, the DNA sequences will vary because the organisms make different proteins.

Using your understanding of genes and how they are expressed, explain why it is possible for a bacterial cell to make a human protein from the instructions encoded in a human gene.

Because the DNA in all organisms uses the same code and transcription and translation processes, the bacterial cell can create a human protein from a human gene. -GENE CODE IS UNIVERSAL

Why are the colonies red on the LB/amp/ara plate but not on the LB/amp plate?

Both have "transformed" colonies but in order to make the red fluorescent protein, arabinose needs to be present in order to bind to the araC protein which then allows for the binding of RNA polymerase to Pbad.

Why do DNA restriction fragments an plasmids separate when analyzed by gel electrophoresis?

DNA molecules, including fragments and plasmids, move through the gel during the procedure of gel electrophoresis. Separation occurs during the procedure because lighter and more compact molecules move faster than heavier and less compact molecules.

What is the purpose of the "marker"/DNA Ladder?

DNa fragments of known size(ladder) allow you to estimate the size of DNA fragments/bands you run through a gel

Recombinant plasmids are engineered so that they can replicate in the cell independently of the chromosome replication. Why is it important to have multiple copies of a recombinant plasmid within a cell?

IN genetic engineering the goal is to produce a lot of product, such as insulin. More copies of the plasmid and its gene will result in the production of more product within the cell.

How might the conformation (shape) of a protein be important for its function? Focus on one of the following protein functions: acting as an enzyme (speeding up reaction rates), transporting molecules, signaling, or forming structures

If a problem is a catalyst, its shape can hold reactants in an orientation that speeds up the reaction. If a protein transports other molecules or acts as a signal, its shape can match the shape of another molecule in such a way as to hold on to it. If a protein forms a structure, its shape can fit in with other proteins that also form that structure.

What ae important features of a plasmid vector that are required to clone a gene. Explain purpose of each feature

Important features of a plasmid vector are (1) a sequence for the initiation of DNA replication, ori, which allows the plasmid to replicate in the bacteria; (2) a promoter sequence for initiating transcription of the inserted gene; and (3) a gene encoding a protein for antibiotic resistance, which allows for identification of bacteria that have taken in the plasmid.

A protein is a long linear molecule what it is made, but it immediately folds into a specific three-dimensional conformation. What properties of the amino acids in a protein control the folding process?

Protein folding is the result of the formation of weak noncovalent bonds between amino acids, the tendency of hydrophobic amino acids to become buried inside the protein, and the formation of covalent disulfide bridges between sulfur-containing amino acids.

How can solutions of different salt concentrations, which will unfold proteins to varying degrees, be used to help purify red fluorescent protein using column chromatography?

Proteins are unfolded in the binding buffer, which has a high salt concentration. When added to the column, hydrophobic proteins in the binding buffer stich to the beads in the column, whereas hydrophilic proteins pass through the column. The hydrophobic proteins that stich to the column can be released by adding buffers with lower salt concentrations. These buffers allow the proteins to refold, which releases them from the column. Several different buffers can be added to release proteins with different relative amounts of hydrophobic amino acids.

Why are proteins sometimes called workhouse molecules?

Proteins carry out almost all cell processes and from cell structures

What was happening in the 37 degree Celsius water bath?

Restriction Enzymes were cutting the plasmid(hydrolyzing the bonds at the restriction sites)

In the R- lane, do you see evidence of multiple configurations of plasmids? Explain your answer.

Students should see two or three different bands in the R- lane, which is evidence of multiple plasmid configurations. -no restriction enzyme= different forms of plasmids(not digested)

Bacteria, sea anemones, and humans seem, on the surface, to be very different organisms. Explain how a gene from humans or a sea anemone can be expressed in bacteria to make a product never before made in bacteria.

The DNA code and the processes of transcription and translation are the same in all living organisms. Once a human or sea someone gene is paired with a bacterial promoter on a plasmid and the recombinant plasmid is taken up by the bacteria, the bacteria can read the DNA code and make the protein.

What do bacteria and humans have in common that makes it possible for a human gene to be expressed in bacteria?

The DNA structure and code are the same in bacteria and humans. The cell machinery that carries out transcription and translation is the same in bacteria and humans.

Why is it possible for bacteria to make a human protein, such as insulin, or a sea anemone protein, such as the red fluorescent dye?

The DNA structure and code, and the cell machinery that carries out transcription and translation, are the same across all living organisms.

Due to a mishap in the lab, bacteria carrying a plasmid with an ampicillin-resistant gene and bacteria carrying a plasmid with a gene that provides resistance to another antibiotic (kanamycin) were accidentally mixed together. (Hint: Make sure that you do not kill off one of the kinds of bacteria you are trying to sort out!)

The bacteria need to be divided into two batches; one batch should be treated with kanamycin, and the other batch should be treated with ampicillin.

Why would bacteria retain a gene that gives them resistance to antibiotics? How is the existence of bacteria without antibiotic resistance affect medicien today?

The bacteria with the antibiotic resistance gene will reproduce more because they have a selective advantage over other bacteria that do not carry that gene. This selective advantage is a huge concern in medicine because now there are strains of bacteria that cause disease but cannot be killed by antibiotics.

Given that only 1 in 1,000 cells were transformed in Laboratory 5A, what can you say about the size of bacteria cells?

The cells must be extremely small, as we used 50pL of suspended cells in each group and yet that volume must contain thousands of cells because we obtained several transformed colonies.

Does the eluate containing your red fluorescent protein appear less bright or brighter than it did in the cell lysate following centrifugation? If there is a noticeable difference in the intensity of the red color, what might account for that?

The eluate is brighter that the cell lysate. The eluate contains mostly separated red fluorescent protein, while the cell lysate contained all the cell proteins.

Why did the red colonies only appear on the LB/amp/ara plate and not the LB/amp plate?

The rfp gene cannot be expressed unless the cell is given arabinose, as the arabinose operon will only turn on the rfp gene promoter in the presence of arabinose.

What will happen when bacteria cells that contain the pARA-R plasmid are not given arabinose?

The rfp gene cannot be expressed unless the cell is given arabinose—only in the presence of arabinose will the AraC activator protein turn on the rfp gene promoter.

How is the information encoded in the rfp gene expressed as a trait? Be sure to use what you have preciously learned about gene expression and the relationship between DNA, RNA, protein and traits.

The rfp gene in the plasmid is made up of DNA, and the gene is copied into messenger RNA. This process is called transcription. The messenger RNA is used to build the protein at the ribosome by attaching to transfer RNA molecules that match up codons and amino acids; the amino acids are then joined together to make the protein. This process is called translation. The protein can contribute to a trait, and in this case the red fluorescent protein makes the cells red.

What properties of the amino acids in a protein relate to protein folding?

The sequence of amino acids determines folding. Protein folding is the result of formation of weak noncovalent bonds between amino acids, the tendency of hydrophobic amino acids to become buried inside the protein, and the formation of covalent disulfide bridges between sulfur-containing amino acids.

Bacteria can be killed by an antibiotic unless that carry a plasmid that has the gene for resistance to that antibiotic. Biotechnologists call these genes selectable markers because only bacteria that carry the gene will survive an antibiotic. If the uptake of DNA by bacteria is inefficient (as discussed in the reading), why is a selectable marker critical in cloning a gene in bacteria?

You want to know which bacteria cells have the plasmid and can make the protein you are purifying. The selectable marker will allow you to kill off the bacteria that do not have the plasmid with the gene of interest.