BIO 357- Exam 2

How does the automated Sanger sequencing work?

* terminator is labelled by different fluorescence* • Fluorescently tagged ddNTPs - 4 different "colors" (BigDye terminators) • Primer binds template DNA in presence of 4 dNTPs and 4ddNTPs • Newly synthesized DNA fragments terminate with one of 4 fluorescent ddNTPs • Denature DNA • Four separate reactions are pooled into one "lane" • Fluorescence is detected to generate the electropherogram

Which amino acids can be methylated, acetylated, and ubiquitinated?

- Lysine and arginine can be methylated - Lysine can be acetylated - Lysine can be ubiquitinated

Which amino acid can be phosphorylated?

- Serine - Thyrosine - Threonine

How does immuneprecipitation work?

- Use an antibody specific to the target protein OR to an epitope tag fused to the target protein - Express the tagged target protein in a cell - Immunoprecipitate the tagged protein using the antibody It works by binding antibodies to Protein A, Protein G, or a lab-created mix of the two called Protein A/G. Proteins A and G are bacterial proteins that bind very well to antibodies.

What is screen?

A Strategy to use different criteria to screen through transformants to FIND what you want Use Human cDNA library To screen for cDNA clones that can rescue fission yeast cdc2 mutant.

What is a protein domain?

A protein domain is any identifiable longer contiguous subsequence of a protein that can fold, function and exist independently of the rest of the protein chain or structure.

What is selection?

A strategy to set a condition to select away things that you DONT want or for the things that you DO want Auxotroph = mutant that cannot grow on minimal medium, requires certain supplement(s). Prototroph= wild type, it will grow in minimal medium or medium lacking the supplement.

How does RT-PCR (reverse-transcriptase PCR) work?

A technique in which RNA is first converted to cDNA by the use of the enzyme reverse transcriptase, then the cDNA is amplified by the polymerase chain reaction.

This is the Figure 1D from your Writing Assignment #2 (Cong et al., 2013). Lane numbers are marked for your convenience.

ADD IMAGE

Question 4 Part3: In this experiment, Cong et al., concluded that SpRNaseIII is NOT required for editing genome in mammalian cells. Which lane(s) provide(s) results that support this claim? Be explicit and describe the results that support your conclusion.

ADD IMAGE From the lane 4 we can say that, mammalian cell does not require SpRNaselll for editing. Reason: Because, if we compare lane 4 and 5, we can see that, if all the enzymes are present in the smaple including SpRNaselll we can get double strand break. But if we see the lane 4, we can see that, if we give all the enzymes except SpRNaselll, then also we can get double strand break. Conclusion: Thus, we can conclude that lane 4 justifies the question. or Lane 4 is showing separate bands, even in the absence of spRNAasel lll the bindings are formed at 367 and 317bp proving that spRNAase is not required for editing of the genome in a mammalian cell.

You are using yeast two-hybrid analysis (BD vector, AD vector, and the Saccharomyces cerevisiae strain AH109) to understand how two newly discovered proteins, a kinase TUK1 and a potential interacting protein, TAP2, might physically interact. In your initial investigation you fused a portion of TAP2 protein, TAP226-207 (amino acids 26-207 of the TAP2 protein), to BD and revealed that this portion of the TAP2 protein (TAP226-207 ) is interacting with TUK1. You are interested in finding out whether the whole TUK1 protein is required to physically interact with this portion of TAP2 (TAP226-207 ) or maybe just a portion of the of TUK1 is required to physically interact with TAP226-207. You have made several Activation Domain (AD) constructs fused to different portions of TUK1 (Panel A) and tested their interactions withTAP226-207. The results of your yeast two-hybrid experiment are shown in Panel B. (1). Which test serves as a negative control for this whole yeast two-hybrid experiment? (2). Using these yeast two hybrid results, identify the minimal fragment of TUK1 that is responsible for the interaction with TAP226-207. Be explicit and describe the results that support your conclusion. (3). Using these yeast two hybrid results, identify the fragment of TUK1 that DOES NOT interact with TAP226-207. Be explicit and describe the results that support your conclusion.

ADD IMAGE In the yeast two-hybrid experiments the BD is the DNA-binding domain and the AD is the transcription activation domain. For activation of transcription (identified by expression of protein) both AD and BD is needed in the yeast. In this experiment, we want to find out which portion of the TUK1 protein (amino acid numbers) interacts with the TAP226-207 region. Q1) Test 1 acts as the Negative control for the experiment because in this case neither AD TUK1 protein is present nor BD TAP226-207 is present. The response seen in the negative control is the response that will be observed when AD TUK1 protein and BD TAP226-207 does not interact. Q2)The Tests which showed a positive result i.e. interaction between AD TUK1 protein and BD TAP226-207 was seen are - Tests 2, 3, 5 and 6. Now Test 2 - has AD TUK11-725 protein and BD TAP226-207 Test 3 - has AD TUK11-447 protein and BD TAP226-207 Test 5 - has AD TUK1231-541 protein and BD TAP226-207 Test 6 - has AD TUK1231-412 protein and BD TAP226-207 If we compare all the sequences then we can see that the common sequence is AD TUK1231-412 protein this means that the minimal fragment that is responsible for interaction between AD TUK1 protein and BD TAP226-207 is the AD TUK1231-412 sequence. Thus the minimal fragment of AD TUK1 that is responsible for interaction with BD TAP226-207 is AD TUK1231-412 protein sequence. Q3) The Tests which showed a negative result i.e. no interaction between AD TUK1 protein and BD TAP226-207 was seen - Tests 1 and 3 Now Test 1 - Negative control Test 3 - has AD TUK1413-725 protein and BD TAP226-207 In the above question we saw that minimum sequence needed for expression is AD TUK1231-412 protein and test 3 construct does not have the TUK1231-412 protein sequence hence there is no expression which means that AD TUK1413-725 protein sequence is not necessary for the interaction between AD TUK1 protein and BD TAP226-207. Thus the fragment that does not interact with BD TAP226-207 is AD TUK1413-725 protein sequence.

Question 4 Part1: As shown in Figure 1D above, four components are tested in mammalian cells: SpCas9, SpRNaseIII, tracrRNA, DR-EMX1(1)-DR. Which one of these four components functions as the A and B (crRNA) part in the following diagram?

ADD IMAGE Q1)From the gel it is clear that the insertion and deletions called indel is observed in lane number 4 and 5 with two extra bands seen other than the 684 bp band as observed in lane 1, 2 and 3 too. The two extra bands of size 367 and 317p is observed in lane 4 and 5 which is due to the indel (4.7 and 5%), thus here the mismatch region created. The CRISPR will provide the way to produce loss-of function mutations in gene. Thus, in this case, the component acting as A and B of rRNA are tracrRNA, DR-EMX1(1)-DR, because without them the two peaks or bands not produce due to indel. (just compare the lanes where these two components are not present with respect to where they are present.

If you would like to use CRISPR/Cas9 to edit the GOI gene, which region of the following RNA constructs as illustrated in the following diagram should contain the sequences from your GOI gene? a.) A b.) C c.) B d.) D

ADD IMAGE b.) C or a) a

Question 4 Part 3: What technique/assay was used to generate the two fragments (367 bp and 317 bp, indicated by arrows)? State the name of the technique.

ADD IMAGE Here technique used is polymerase chain reaction. Polymerase chain technique is used to amplify single copy of segment dna or few more copies of segment dna into million copies of a particular dna sequence used in clinical and research laboratories for various applications. It is developed in 1983 by kary mullis and he was awarded nobelprize for PCR. Three steps in per done on automated cycle for 20-40 cycles which heated and cooled ona short period of time. First step Denaturation (94°C) Reaction mixture is heated at 94°c for one minute this separate two strands of dsDna. now each strand synthesize complementary strand by acting as template strand. Second step Annealing (45-60°C) This step makes binding of primer to strand of template and these will annealed (hybridized). Third step extension (72°C) Ideal Temperature for acting of tag polymerase ad this will adds nucleotide complimentary to template which has 3-oh primer region which makes to extend the new strand. Final step Final hold(4-15°C) Before steps are repeated for 30 times to produce billions of copies of target dna and for storage

In the yeast two-hybrid system as illustrated below, which of the following statements accurately complete this sentence: A reporter gene... Select one: a. is fused to the activation domain of a transcription factor. b. is fused to the DNA binding domain of a transcription factor. c. is expressed only if the tested protein interaction between X and Y occurs. d. requires the presence of histidine in the growth medium for its expression

ADD IMAGE FROM ipad C) is expressed only if the tested protein interaction between X and Y occurs.

THE GOI gene is known to be 1.5 kb and the pBR322 vector is known to be 4.4 kb. In trying to clone GOI into the pBR322 vector at the BamH1 and the Sal1 site and identify the pBR322-GOl plasmid (see diagram below), plasmid DNA was isolated from various colonies and digested by either one restriction enzyme or two restriction enzymes. The digested DNA was analyzed by gel electrophoresis and the results are shown on the right panel. Lane "M" is a DNA marker (ladder) with known DNA sizes. Isolated plasmids were digested with either BamH1 only (Lane 1 and Lane 2) or Sal1 only (lane 3 and Lane 4), or both BamH1 and Sal1 (Lane 5). From these restriction digestion results shown in the above right panel, the lane(s) that contained pBR322-GOl is (are):

ADD IMAGR FROM IPAD: Lanes 1, 3, anf 5

How are GAL4-AD and GAL4-BD used in Y2H?

AH109 is auxotrophic yeast strain commonly used for Y2T (Leu-, Trp-, His-) BD Gal4 DNA Binding Domain vector (plasmid) supply TRP AD Gal4 Activation Domain Vector (plasmid) supply LEU

What screen strategies can you use to identify your desired recombinant plasmid? (antibiotic-resistance, plasmid prep and restriction digestion, PCR screening)

Antibiotic resistance: it allows a scientist to easily detect plasmid-containing bacteria when the cells are grown on selective media and provides those bacteria with pressure to keep your plasmid. Plasmid Prep: isolate small plasmid DNA from bacteria while limiting contaminating proteins and genomic DNA. The plasmid quality is acceptable for restriction analysis, sequencing, cloning, or other purposes, but should not be used without additional cleanup for embryonic injections. Restriction digestion: Cells containing recombinant plasmids can often be identified as containing recombinant plasmids by screening for the insertional inactivation of a second genetic marker on the plasmid. PCR screening: The PCR technique produces multiple copies of a particular DNA sequence in vitro through repeated cycles of PCR reactions. Recombinant DNA is produced in order to locate the gene. Recombinant DNA is not produced. - 1) Pick a colony, 2) replica plate, 3) Add PCR master mix. 4) Perfrom PCR - Run PCR samples on DNA gel

How can GFP used to study protein function?

Biologists use GFP as a marker protein. GFP can attach to and mark another protein with fluorescence, enabling scientists to see the presence of the particular protein in an organic structure. Gfp refers to the gene that produces green fluorescent protein

Be able to explain a strategy to identify a bacterial colony carrying a recombinant plasmid DNA if you are inserting your gene of interest into the ampicillin-resistance gene on the plasmid pBR322.

Cells containing recombinant plasmids can often be identified as containing recombinant plasmids by screening for the insertional inactivation of a second genetic marker on the plasmid.

Explain how colony-PCR works. Interpret data (gel data) of colony PCR.

Colony PCR is a method for rapidly screening colonies of yeast or bacteria that have grown up on selective media following a transformation step, to verify that the desired genetic construct is present, or to amplify a portion of the construct. PCR strategy: PCR reaction determines to insert size - Colony PCR - conduct PCR on cells from each colony - either grow culture or keep track of colonies picked - Run PCR product on gel to detect with ethidium bromide Now that your PCR is complete, it's time to run the products on an agarose gel to determine their size. Make sure to use an appropriate molecular weight standard for reference and to add a loading dye with glycerol to your samples before pipetting them onto the gel. The figure above summarizes generalized expected results for the three primers previously described. When using insert-specific primers (1), positive clones (+) will give a band, while a negative clone (-) will not. Backbone-specific primers (2) give larger sized products for positive clones (+) compared to negative clones. Finally, orientation-specific primers (3) give the same band (+) or no band (-) result as insert-specific primers but also tell you whether the insert has the correct directionality.

what is so special about dideoxynucleotide or ddNTP?

If a ddNTP is added to a growing a DNA strand, the chain is not extended any further because the free 3′ OH group needed to add another nucleotide is not available. By using a predetermined ratio of deoxyribonucleotides to dideoxynucleotides, it is possible to generate DNA fragments of different sizes.

How can immunoprecipitation be used to identify potential interacting proteins of your interests?

Immunoprecipitation is one of the most widely used methods for isolation of proteins and other biomolecules from cell or tissue lysates for the purpose of subsequent detection by western blotting and other assay techniques.

Explain how Sanger sequencing work. What is so special about dideoxynucleotide or ddNTP?

In automated Sanger sequencing, a computer reads each band of the capillary gel, in order, using fluorescence to call the identity of each terminal ddNTP. In short, a laser excites the fluorescent tags in each band, and a computer detects the resulting light emitted. lecture: Sanger developed a DNA sequencing technique - DNA polymerase synthesizes template-dependent DNA strands in 4 equivalent reactions - Template, primer, dNTP mix, buffer, DNA polymerase. - Each dNTP mix is poisoned with one deoxnucleotide "ddNTP" You use gel electrophoresis to see the fragments. Read the gel from bottom to top for the sequence.

Which amino acid is responsible for disulfide bridge formation?

In proteins, the amino acid cysteine contains a thiol group and readily forms disulfides so linking two cysteine units together.

What determines alpha helices and beta sheets?

Intramolecular hydrogen bonding forms within the polypeptide chain to create a spiral structure. Beta sheets are formed by linking two or more beta strands by intermolecular hydrogen bonds. 3.6 amino acids residues are winded to form an alpha-helix polypeptide.

How can a protein tag be used to purify a protein? Describe an example (e.g. FLAG).

It involves cloning a gene of interest in-frame with a protein tag that will be expressed as a fusion with the resulting protein—and that allows the protein to be captured or detected. AG-tagged recombinant protein can be affinity purified directly from a cell culture lysate or supernatant. The FLAG-tagged protein binds to the FLAG-tag specific monoclonal antibody conjugated on an agarose gel.

Describe how PCR works. Describe the required components in a PCR reaction. Explain the steps in the PCR cycle - melting (denaturing), annealing (renaturing), extension (synthesis).

PCR: laboratory technique for rapidly producing (amplifying) millions to billions of copies of a specific segment of DNA, which can then be studied in greater detail. The key ingredients of a PCR reaction are Taq polymerase, primers, template DNA, and nucleotides (DNA building blocks). The ingredients are assembled in a tube, along with cofactors needed by the enzyme, and are put through repeated cycles of heating and cooling that allow DNA to be synthesized. Denaturation- It is the melting of double-stranded DNA, in this step the DNA will open into two single strands. Annealing- At the moderate temperature, primers are joined with the template of the DNA. the polymerase attaches itself and begins amplifying the templates. Extension- In this step, the DNA blocks that are complementary to the template are coupled with primer and form double-stranded DNA.

How does a R (variable) group in an amino acid residue affect protein structure?

Primarily, the interactions among R groups creates the complex three-dimensional tertiary structure of a protein. The nature of the R groups found in the amino acids involved can counteract the formation of the hydrogen bonds described for standard secondary structures such as the alpha helix.

What's the major difference between RT-PCR and traditional (regular) PCR?

RT-PCR, you can measure the amount of DNA copied by adding a fluorescent dye. The fluorescence increases as the desired sequence are amplified. This makes it easier for tracking the amplification in real time. Whereas in regular PCR, the end products have to be analyzed by techniques like agarose gel electrophoresis in order to ensure that the amplification has taken place.

real time PCR vs PCR

Real-time PCR is quantitative and traditional PCR is not. In other words, trafitional PCR measures accumulated PCR products at the end of all cycles, and real-time PCR measures amplification as it occurs. Real-time PCR uses the enzyme Taq polymerase. Traditional PCR is commonly used for sequencing and real-time PCR is commonly used for pathogen detection.

What's real time PCR?

Real-time PCR is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. This is achieved using a variety of different fluorescent chemistries that correlate PCR product concentration to fluorescence intensity

What and how restriction enzymes are used in recombinant DNA technology?

Restriction Enzymes: Enzymes that cut DNA at a specific sequence of nucleotides. The first step in the development of recombinant DNA technology was the characterization of restriction endonucleases—enzymes that cleave DNA at specific sequences. These enzymes were identified in bacteria, where they apparently provide a defense against the entry of foreign DNA (e.g., from a virus) into the cell.

For the following statement, please indicate whether the statement is TRUE or FALSE. Your research project is to determine the cellular location of a protein but unfortunately your protein is not particularly antigenic (i.e., it does not develop a strong antibody response, so you cannot purchase an antibody specific for the protein). STATEMENT: "You can express the protein fused to the GST tag (Glutathione-S-Transferase Tag) in the cells, and then visualize the fusion protein by adding GFP (green fluorescence protein)."

TRUE GST tag can be used to purify the protein and fused with GFP will produce the fluorescence when observed under a fluorescence microscope. As the GFP is fused with protein of interest fluoresce within the cell will help to determine the cellular location protein.

How does yeast 2 hybrid (Y2H) work? Describe your experimental design if you need to test interactions between two know proteins, or to find proteins that interact with the protein of your interest.

The Y2H technique allows detection of interacting proteins in living yeast cells [17]. As described in full detail in chapter 3, interaction between two proteins, called bait and prey, activates reporter genes that enable growth on specific media or a color reaction. Example experiment: Pull-down assay is an in vitro method used to determine a physical interaction between two or more proteins. It can be used for confirmation of existing protein-protein interactions discovered by other techniques or initial screening to identify novel protein-protein interactions.

Understand and describe levels of protein structures.

The complete structure of a protein can be described at four different levels of complexity: primary, secondary, tertiary, and quaternary structure. • Primary structure = sequence of amino acids • Secondary structure = hydrogen bonding in the backbone chain (alpha helix, beta sheets) • Tertiary structure = folded polypeptide chain • Quaternary structure = assembled protein of multiple subunits

How do you read the sequences from Sanger sequencing gel?

The sequence in the 5'-to-3' direction is read from the bottom to the top of the autoradiogram. strand can be read directly from the autoradiogram. One starts at the bottom and looks across the four lanes to

Describe how you would clone a specific DNA of interest into a plasmid DNA vector

The steps in the DNA cloning process are to isolate the gene of interest and cut it and a plasmid vector with restriction enzymes. The gene of interest and the plasmid are then ligated together with DNA ligase. Then, the recombinant DNA can be inserted into a cell for replication.

What is a yeast auxotroph and how auxotrophs can be in selections in yeast two-hybrid assay?

Yeast auxotroph: a yeast strain that requires a particular addition nutrient which the normal strain does not. Genes can supply nutrient: HIS- , LEU-, TRY-, HIS3, LEU2, TRY1 The yeast 2-hybrid (Y2H) assay is a well-established technique to detect protein-protein interactions. This is an extremely powerful tool for researchers and is often used alongside one or two other methods to examine the multitude of interactions that take place in cells.

Example Question 1: A gene named GOI (Gene-of-Interests) contains two restriction sites, BamH1 and Sal1, as illustrated (see diagram below, Panel A). This GOI gene is cloned into the pBR322 vector (diagram below, Panel B ) using both the BamH1 and the Sal1 site in the pBR322 vector, resulting a pBR322-GOI plasmid (diagram below, Panel C). Cells carrying the pBR322-GOI plasmid will be Select the best answer and explain: a. Sensitive to both Tetracycline and Ampicillin b. Resistant to Tetracycline c. Resistant to Ampicillin d. Resistant to both Tetracycline and Ampicillin

add image: c) resistnat to ampicillin

For one type of screen, how can one find something (cells) that will die on Ampicillin?

pBR322 (Plasmid 1) is 4.3 kb long. - Bacterial cells carrying this plasmid can grow on the media containing both ampicillin and tetracycline. Digest with Pst1 (cuts) - Pst1 cut will interrupt the Ampr gene (Ampr encodes a protein that can degrade degrade ampicillin) Bacterial cells carrying this plasmid can grow on media containing tetracycline, not ampcillin!

How does a protein tag work?

protein tags are peptide sequences that are attached to proteins to facilitate easy detection and purification of expressed proteins. In addition, they can also be used to identify potential binding partners for your protein of interest.

two-hybrid assay

works by requiring an interaction between two proteins, where one has a DNA-binding domain and the other has a transcription-activation domain

Compare and describe protein expression systems.

• Bacteria: Protein expression in bacteria is quite simple; DNA coding for your protein of interest is inserted into a plasmid expression vector that is then transformed into a bacterial cell. Transformed cells propagate, are induced to produce your protein of interest, and then lysed. • Yeast: Yeast is an excellent system for the expression of recombinant eukaryotic proteins. Both endogenous and heterologous proteins can be overexpressed in yeast (Phan et al., 2001; Ton and Rao, 2004). Because yeast is easy to manipulate genetically, a strain can be optimized for the expression of a specific protein. • Insect Cells: Insect cells offer high levels of protein expression with posttranslational modification approaching that of mammalian cells, ease of scale-up, and simplified cell growth that can be readily adapted to high-density suspension culture for large-scale expression. • Mammalian Cell: Protein expression in mammalian cells can also be achieved using viral-mediated transduction by such techniques as the BacMam system. This technology utilizes recombinant baculoviruses for simple transduction of mammalian cells, allowing for production of milligram quantities of protein for structural studies. • Whole animal:

How can protein functions be studied?

• Expression and purification of proteins • Mutation • Localization • Protein-protein interaction (yeast two-hybrid analysis)