Kumar Lesson 3

Conjugation

A temporary union of two organisms for the purpose of DNA transfer.

What is a primary difference between polymerase chain reaction (PCR) and traditional cloning procedures such as those used to clone the human growth hormone gene? A. PCR eliminates the need for complicated cloning steps and identification of clone containing desired gene. B. PCR and traditional cloning make use of different types of bacteria. C. PCR is more time-consuming, but the purity of the obtained DNA clone is much higher than in traditional cloning. D. PCR and traditional cloning make use of different types of vectors. E. PCR offer no advantage.

A. PCR eliminates the need for complicated cloning steps and identification of clone containing desired gene. PCR allows researchers to quickly amplify the desired gene (specifically) that can be cloned in a vector by single step of ligation.

How do we describe transformation in bacteria? A. assimilation of external DNA into a cell B. semiconservative replication of DNA C. the creation of a strand of RNA from a DNA molecule D. the creation of a strand of DNA from an RNA molecule E. the infection of cells by a phage DNA molecule

A. assimilation of external DNA into a cell Transformation is defined as the genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous genetic material (DNA) from its surroundings through the cell membrane(s).

What is an expression vector?

An expression vector, otherwise known as an expression construct, is usually a plasmid or virus designed for gene expression in cells. The vector is used to introduce a specific gene into a target cell, and can commandeer the cell's mechanism for protein synthesis to produce the protein encoded by the gene. Expression vectors are the basic tools in biotechnology for the production of proteins.

Expression plasmids often contain the T7 promoter. The intention is to? A. Block the expression of all genes except for the inserted gene B. Increase the expression of only the N-terminal tag. C. Increase the expression of inserted gene D. Assess the amount of control plasmid

C. Increase the expression of inserted gene T7 promoter is stronger compared to bacterial promoters like Lac or Tet. Thus allowing to achieve higher expression of recombinant protein.

How many potential open reading frames are present in a DNA sequence? A. One. B. Three. C. Six. D. More than six. E. None of these.

C. Six. Total six, 3 possible ORF for each strand of DNA.

Site-directed mutagenesis: A. Is a technique to produce specific mutants. B. Can be used to alter gene function in specific ways.C. Can create mutant genes to be studied in living organisms. D. All of these. E. None of these.

D. All of these.

You discover a novel eukaryotic organism that glows in the dark. You believe this trait is due to a single gene, and you wish to clone the gene. Which of the following strategies is most likely to be successful? A. Isolate the genomic DNA from the organism, digest with a restriction endonuclease, insert into a plasmid vector and transform into bacteria. Screen colonies for the ability to glow in the dark. B. Isolate the genomic DNA from the organism, digest with a restriction endonuclease, insert into a plasmid vector and transform into eukaryotic cells such as yeast. Screen colonies for the ability to glow in the dark. C. Isolate mRNA from the organism, reverse transcribe and generate cDNA, insert into a plasmid vector and transform into bacteria. Screen colonies for the ability to glow in the dark. D. Isolate mRNA from the organism, reverse transcribe and generate cDNA, insert into a plasmid vector and transform into eukaryotic cells such as yeast. Screen colonies for the ability to glow in the dark.

D. Isolate mRNA from the organism, reverse transcribe and generate cDNA, insert into a plasmid vector and transform into eukaryotic cells such as yeast. Screen colonies for the ability to glow in the dark. Option D is the best answer. The gene is from a eukaryotic organism that will require removal of introns, so mRNA and reverse transcription steps are needed. Also the functional protein may requires some other chaperon proteins to correctly fold, which can only be achieved in eukaryotic host cells.

Expression of a cloned eukaryotic gene in a bacterial cell involves many challenges. The use of mRNA and reverse transcriptase is part of a strategy to solve the problem of _____. A. nucleic acid hybridization B. post-translational processing C. electroporation D. post-transcriptional processing E. restriction fragment ligation

D. post-transcriptional processing In eukaryotes the introns are removed after transcription (mRNA processing). Bacterial host will not have the ability to process the mRNA and the protein will not expressed properly.

The lacZ gene is sometimes included in a cloning vector. What is its purpose? A. allow resistant transformants to grow in selective medium B. distinguish introns from exons C. allow viral replication D. screen for vectors with inserts E. allow plasmid replication

D. screen for vectors with inserts The lacZ gene in vector is disrupted upon successful insertion of gene (insert) and can be used to screen colonies on x-gal (blue white selection).

Transduction in bacteria

DNA is transferred from a donor cell to a recipient via a bacteriophage

PCR components

DNA sample, primers, dNTPs, Taq polymerase, mix buffer, PCR tube

How PCR works

DNA template, polymerase enzyme, primers, and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius. 94(Denature and anneal primers @3' end) 54 degrees(the annealing (binding) of the primers to the single-stranded DNA templates @ 3' end) Elongation: In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complementary dNTPs to the 3' ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest.

How PCR works

DNA template, polymerase enzyme, primers, and dNTPs. The ability to heat the mixture without denaturing the enzyme allows for denaturing of the double helix of DNA sample at temperatures in the range of 94 degrees Celsius. 94(Denature and anneal primers @3' end) 54 degrees(the annealing (binding) of the primers to the single-stranded DNA templates @ 3' end) In the third step of the cycle, the sample is reheated to 72 degrees, the ideal temperature for Taq DNA Polymerase, for elongation. During elongation, DNA polymerase uses the original single strand of DNA as a template to add complementary dNTPs to the 3' ends of each primer and generate a section of double-stranded DNA in the region of the gene of interest.

Which of the following sequences in double-stranded DNA is most likely to be recognized as a cutting site for a restriction enzyme (endonuclease)? A. AGTC TCAG B. AAAA TTTT C. ACCA TGGT D. AAGG TTCC E. GGCC CCGG

E. GGCCCCGG The restriction site are Palindromic in sequence, i.e. the sequence is same on both DNA strand read 5' to 3'.

What is inclusion body?

Inclusion bodies are foreign proteins that produce insoluble fragments(especially in Eukaryotes). They form when the highly produced protein favor intermolecular interactions between the hydrophobic parts of the incompleted folded polypetide chains leading to aggregation and misfolding of the protein

What are sticky ends and blunt ends generated by restriction enzymes?

Many restriction enzymes make staggered cuts, producing ends with single-stranded DNA overhangs. However, some produce blunt ends. •DNA ligase is a DNA-joining enzyme. If two pieces of DNA have matching ends, ligase can link them to form a single, unbroken molecule of DNA. In DNA cloning, restriction enzymes and DNA ligase are used to insert genes and other pieces of DNA into plasmids.

How are expression vectors different from cloning vector?

The main difference between cloning vector and expression vector is that cloning vector is used to carry foreign DNA segments into a host cell, whereas expression vector is a type of a cloning vector, which contains necessary elements for a host cell to transcribe and translate the gene

The pUC18 vectors used for cloning purpose is an effective tool in screening for clones. What is the basis for blue white selection and why colonies are white on X-gal plate if an DNA fragment is inserted in pUC18 vector?

The pUC18 plasmid contains alpha peptide of lacZ gene (b-galactosidase) and the host E. coli is modified where alpha peptide is inactivated, but contains omega peptide. When combined together they form a functional b-galactosidase which will degrade (metabolize) the chemical X-gal in to galactose and indole, turning colonies blue. The insertion of DNA fragment at multiple cloning site in pUC18 vector disrupt the expression of alpha- peptide of lacZ downstream resulting in loss of b-galacosidase activity and colony remains white (whitish yellow).

How to prevent self-ligation of plasmids during gene cloning?

Use 2 digest enzymes

How the clones are selected and screened?

Using AB (ampr) codes for an enzyme (b-lactamase) that is secreted into the periplasmic space of the bacterium where it catalyzes hydrolysis of the b-lactam ring of the ampicillin. Thus, the gene product of the ampr gene destroys the antibiotic.and blue-white screening (lac Z)

replica plating technique

allows identification of mutants*****

What is a cloning vector?

an agent, such as a plasmid, used to transfer DNA from an in vitro solution into a living cell -can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes.

What is a restriction endonuclease and how it is useful in cloning?

an enzyme produced chiefly by certain bacteria, having the property of cleaving DNA molecules at or near a specific sequence of bases.

Identify the role of antibiotic gene and lacZ in selection and screening of the clones.

o lacZ- that gets cut will not be blue hydolrsis by the protein made by lacZ (B-galactosidase) produced indoxyl which, when oxidixied turns blue if coloby turns white gene was inserted at lac Z

Transformation

process in which one strain of bacteria is changed by a gene or genes from another strain of bacteria

how they can affect the protein purification?

to purify protein usually have to break cells and when centrifuged they become sediment, if the protein becomes an inclusion we can bypass theses steps and just add protein dentuarant to make the inclusion bodies soluble