BIO 113 Gene Cloning II (Vectors)

Order of insert size (large to small)

Bacterial artificial chromosome (BAC) > Cosmid > Replacement vector > Insertional vector = Plasmid vector

________ is a virus that infects bacteria?

Bacteriophages - have a head (contains DNA/RNA) and a tail

What is the most commonly used phage?

Lamda Phage

In Phage genome, related genes with the same function are clustered together. True or False

True

Function of the Sense RNA

Used to make proteins and study regulations - It was made by using the antisense DNA strand as a template

What is a catenane?

a series of linear phage genomes joined together

What is an Expression Vector?

- a cloning vector designed so that a foreign gene inserted into the vector is expressed in the host organism - the protein encoded by that gene is made in the host

What is a Virus?

- a very small infectious particle made nucleic acid enclosed in a protein coat (capsid) - can be either DNA or RNA, but not both - viruses are obligate intracellular parasites, so they can only multiply within a host

How to make man made viral particles: In vitro packaging?

- add your target gene into a replacement vector - it will replicate and form catanane - add the packaging extract containing heads, tails, and proteins to the test tube - viral particles assemble IN TEST TUBE - more copies are still needed so allow the phages to infect E. coli cells. (Transduction) - each plaque you see represents clones

Function of Antisense RNA

- it's complementary to the sense RNA - can be used as a probe to detect mRNA in the sample

What is a phage insertional vector?

- nonessential region has been deleted and the two arms ligated together. - Insertional vectors contains at least 1 unique restriction site where foreign DNA can be inserted

Features of pBR322?

- small - has an origin of replication - 2 antibiotics resistance genes - unique restriction enzyme recognition sites

Features of pUC18?

- small - one antibiotics resistance gene - has an origin of replication - unique restriction enzyme recognition sites - has a lacZ gene that has poly linker region aka multiple cloning site (MCS)

Briefly explain the lysogenic cycle?

- the phage injects its genome into the chromosome of the host bacteria - it replicates ALONG with the host chromosome - when condition is good the viral DNA is excised and it can then enter the lytic cycle

Briefly explain the lytic cycle?

- the phage injects its genome into the host and starts using host's materials - the phage genome replicates and protein coats are made - phage particles assemble and then the host bacteria is lysed releasing the phages

What additional features are found in an expression vector?

1. A strong promoter: so RNA polymerase can bind - and multiple cloning site will be present down stream from this promoter 2. A ribosome binding site: so that bacteria's ribosome can recognise mRNA made by foreign DNA. *Improves translation 3. A terminator sequence: prevents transcription of other genes

What are the 2 main sources of vectors?

1. Bacterial Plasmids 2. Bacteriophages

What are the two types of phage cloning vectors?

1. Phage insertional vector 2. Phage replacement vector

What are the key features of plasmid cloning vector?

1. Small (<10kb) therefore easy to manipulate and present in many copies 2. Has an origin of replication 3. Has a selectable marker: an antibiotics resistance gene 4. Unique restriction enzyme recognition sites or MCS

Advantages of using 2 restriction enzymes for digestion?

1. Solves wrong orientation problem 2. Reduces requirement for alkaline phosphatase to prevent self-ligation of a vector.

Steps of a cloning experiment using cosmid?

1. cosmid is digested and target DNA is inserted 2. catenanes form 3. in vitro packaging of phage particles 4. phage particles are used to infect E. coli 5. Culture on selective medium 6. Result: colonies containing circular recombinant cosmid grow NOT plaques

What is the function of the cos sites?

1. make the linear dsDNA to be circular when it enters the host - the circular DNA can then replicate by a rolling circle mechanism and form catenane 2. Essential for cleavage of catenane and packaging the DNA into the phage head. Enzymes responsible for cleaving and packaging only recognise the cos site

What is the packaging constraint on phage particles?

1. only DNA between 37 to 52 kb in length can be packed inside the phage head 2. Cos site must be present on both ends

Examples of commonly used plasmid cloning vectors?

1. pBR322 2. pUC18

What is your choice of vector dependent on?

1. size of insert (DNA fragment you want to amplify) 2. which host will you use

Additional features of transcription vector + its uses

1. two promoters: can make both sense and antisense 2. multiple cloning sites between the promoters USES: - can be used to make hybridisation probes - can be used to study RNA processing - can be used to study protein synthesis

What is a plasmid?

A plasmid is a circular DNA that is independent of the bacterial chromosome.

What is a cosmid vector?

A plasmid vector that has cos sites. Insert can be any DNA fragment (even without λ genes) as long as cos sites are 37-52 kb apart - also has origin of replication, restriction site, and selectable marker - the cos site allows them to be packaged in phage particles

What is phage replacement vector?

Foreign DNA replaces the stuffer fragment (non essential) of the vector - has two recognition sites flanking the stuffer fragment - carry larger pieces of DNA than insertional vector

What is a Vector?

a carrier DNA molecule into which a gene is inserted creating a recombinant DNA molecule. - a vehicle that carries your target DNA into the host - vectors must be capable fo replicating in the host organism

What is a transcription vector?

a cloning vector designed to produced only the related RNA molecule coded by the gene - only transcription no translation

Briefly explain the phenomenon of insertional inactivation.

a phenomenon where the insertion of a foreign piece of DNA into a vector inactivates a gene carried by the vector

What is a polylinker? aka Multiple cloning site

a synthetic double stranded oligonucleotide in the vector that carries many unique restriction enzyme recognition sites. - as a result there is flexibility in choice of restriction enzyme used for digestion.

What is a shuttle vector?

a vector that can replicate in the cells of more than one species. e.g. in both E. coli and mammalian cells

Explain the single-strain and two-strain system for producing packaging extract?

a) Single-strain system: all the proteins needed for λ packaging are prepared from 1 bacterial strain infected with defective λ phage carrying a mutation in the cos sites b) Two-strain system: each strain is infected with a defective λ phage. The cell extract mixture will contain all factors need for packaging

What is a restriction enzyme?

an enzyme that cuts DNA at specific recognition site. - the foreign DNA can then enter the vector

What is the most common source of the promotor?

bacteriophage - viral promoters are very efficient. They ensure that the required protein is made in large amounts

How do we ensure that the phage only enters the lytic cycle?

delete the genes responsible for lysogenic cycle. - this deletion does not interfere with the lytic cycle

What structure is the phage genome?

linear dsDNA with cos sites at both the 3' and 5' ends - the cos sites are made of 12 nucleotides and are complementary