BCH210H1 Lecture Notes - Lecture 7: Multiple Cloning Site, Recombinant Dna, Restriction Enzyme

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ochreminnow2

2 Oct 2018

School

UTSG

Department

Biochemical Engineering and Applied Chemistry

Course

BCH210H1

Professor

Wright

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BCH210H1 Full Course Notes

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Proteins and their mutants can be obtained in quantities sufficient for research purposes. Clone the gene of interest from genome or cdna from mrna. Insert the gene in an expression vector . Insert the plasmid into a bacterial host. Grow the bacteria bacteria express protein. Clone - collection of molecules or cells that"s all the same to the original molecule or cell. Can be an exact copy of a gene. Can be an altered version of gene. Can be cleaved at specific sites by restriction enzymes. Dna can be inserted into new site. Recombinant dna molecules can be replicated by many fold in bacterial host. An origin of replication (binds dna polymerase) Multiple cloning site for insertion of foreign dna (restriction enzyme cut sites) Promoter for protein expression (binds rna polymerase) Nuclease cleavage at a restriction site linearizes the circular plasmid to that a foreign. Recombinant plasmids are hybrid dna molecules consisting of plasmid dna sequences, plus inserted dna elements.

Related Questions

1. We can construct our gene library by cloning the PstI fragments into either plasmid pBR322 or phage lambda. Which one should we choose and why?

2. Suppose we want to insert the PstI fragments into the Bam HI site within the gene for tetracycline resistance (tet^r) in plasmid pBR322. If both of these restriction enzymes produce cohesive ends, how can this be done?

3. After transforming E.coli with the plasmids, how can we identify the cells that contain a chimeric plasmid (containing DNA insert)?

4. If a million tetracycline-sensitive, ampicillin-resistance clones are grown on nutrients agar plates, how are we going to detect the rare clone or clones that carry the gene for protein P?

5. After selecting a clone carrying the gene for protein P, its ells are propagated to high density in nutrient broth. The chimeric plasmid (with an insert of the gene for protein P) is then extracted and purified from the rest of the cellular DNA. How can we now isolate the gene from the plasmid?

6. How can we demonstrate that the gene we have isolate is indeed the one for protein P?

lemonrat870

1) Proteins can interact with double-stranded DNA via non-covalent interactions. Among the different types of interactions listed below, which one(s) is/are most likely used by DNA-binding proteins to recognize DNA? Select all that apply.

Hydrogen bonds,Hydrophobic interactions,Electrostatic interactions,Disulfide bonds

2) What reagents are needed for a typical PCR reaction?Select all that apply

Two primers,Human DNA polymerase,Taq polymerase,DNA ligase,Four dNTPs,Buffer with Mg^2+,Template DNA,Four NTPs

3) A new investigator would like to express and purify a newly discovered protein. She decides to clone the gene into a plasmid that contains the sequence for a hexahistidine tag. Plasmids that contain a sequence for a hexahistidine tag will produce a recombinant protein with six extra histidine residues at the N- or C-terminus of the protein of interest.

Order the following steps for construction of the expression plasmid, expression and purification of the protein. The gene of interest will be introduced between the NotI and HindIII restriction sites.(1-5)

Purify the protein using a column of immobilized Ni²⁺.

Introduce NotI and HindIII rrestriction sites at the ends of the gene of interest via PCR.

Digest the plasmid and DNA encoding the genes with NotI and HindIII restriction endonucleases

Transform the plasmid into E. coli for protein expression.

Ligate the cut plasmid and DNA insert.

Once a genetic disorder in an adult is treated successfully through gene therapy, it is highly unlikely that the person's children will inherit the gene(s) for the disease.Answer TrueFalse Vaccines produced by genetically engineered bacteria contain the antigen(s) of the disease-causing organism, not the organism itself. Answer TrueFalse .1 points Question 11 Restriction enzymes are used in genetic engineering to cut DNA between specific base pairs in a DNA strand, resulting in DNA segments that are then used for further study and analysis. Answer TrueFalse .1 points Question 12 Plasmids are useful to scientists involved in recombinant DNA technology because foreign genes can be inserted into the plasmids and then the plasmids can be inserted back into bacteria. Answer TrueFalse

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BCH210H1 Lecture Notes - Lecture 7: Multiple Cloning Site, Recombinant Dna, Restriction Enzyme

BCH210H1 Full Course Notes

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