Intro to molecular biotechnology

Question 1

What is an expression vector?

Answer 1

a type of plasmid / viral vector designed to enable the expression of a gene of interest in a host cell
DNA of interest is INSERTED into this expression vector.

Question 2

Central dogma

Answer 2

information flows from DNA to RNA to protein

Question 3

DNA and RNA are polymers of

Answer 3

Nucleotides

Question 4

Nucleotides are made of

Answer 4

PhosphaTe group
5C sugar (ribose, or deoxyribose)
Nitrogen-containing base (G, C, A, T, U)

Question 5

define gene (molecular definition)

Answer 5

a sequence of nucleotides that is TRANSCRIBED to produce functional RNA
two types: protein-coding and non-coding genes

Question 6

define bacteria

Answer 6

single-celled organisms with one circular chromosome

Question 7

how are genes regulated (switched on and off)?

Answer 7

via promotor sequences
RNA polymerase binds here

Question 8

transcription vs translation

Answer 8

transcribe DNA to RNA
RNA is translated to proteins

transLATion occurs LATer

Question 9

define molecular cloning

Answer 9

introducing a fragment of DNA of interest (often a gene) into a plasmid (vector), making a recombinant DNA molecule. provides a means of producing identical copies.

Question 10

define recombinant DNA molecule

Answer 10

one produced by laboratory methods of genetic recombination bringing together material from multiple sources

Question 11

four essential questions of molecular cloning

Answer 11

1. where to get DNA from
2. how to cut DNA
3. how to join DNA fragments
4. how to propagate more copies

Question 12

how is the DNA for molecular cloning isolated? overall method

Answer 12

break bacteria cells
centrifuge
organic-aq extraction
treatment (remove RNA) and precipitation

gel electrophoresis

Question 13

how are bacterial cells broken to release DNA?

Answer 13

lysozyme to break down peptidoglycan

detergent (SDS) bursts cell membrane by disrupting lipid bilayer

Question 14

purpose of gel electrophoresis
how does it work?

Answer 14

separate DNA fragments by size
using electric current
negatively charged DNA backbone, migrates through agarose gel

Question 15

how is DNA cut into fragments?

Answer 15

using restriction enzymes / endonucleases

bind to specific sites and cut the backbone

Question 16

what do “sticky ends” refer to for a strand of cut DNA? give an example of a restriction enzyme that does this.

Answer 16

restriction enzyme cuts the backbone leaving single stranded overhangs, as opposed to a “blunt end”

eg. EcoRI

Question 17

how do we join DNA fragments together?

Answer 17

using DNA ligase
seals backbone by forming covalent 3’-5’ phosphodiester bond

requires ATP and Mg2+ ions

Question 18

what are plasmids?

Answer 18

naturally occurring circular DNA molecules found in bacteria

Question 19

what are cloning vectors?

Answer 19

specialised molecules (often but not necessarily plasmids) that will hold any piece of foreign DNA for replication in host cell

Question 20

Name three essential features of cloning vectors

Answer 20

1. origin of replication
2. selection cassette / selectable marker
3. Multiple Cloning Sites (MCS)

Question 21

explain the importance of origin of replication for cloning vectors

Answer 21

origin of replication - a sequence that signals where DNA replication begins, allows vector to be copied in host

Question 22

explain the importance of the selection cassette / selectable marker in cloning vectors

Answer 22

usually antibiotic resistance. only cells that take up the vector survive on a selective medium (identify successful clones)

Question 23

explain the importance of MCS in cloning vectors

Answer 23

Multiple Cloning Sites (MCS) - a short segment of DNA containing multiple unique restriction enzyme recognition sites, provides flexibility for inserting DNA

Question 24

how does blue/white screening work?

Answer 24

lacZ gene codes for an enzyme that can break a compound down into a blue product. lacZ w MCS, vector inserted and disrupts this gene.
blue colonies = bacteria w non-recombinant plasmids.
white colonies = recombinant

Question 25

define constitutive promoter.

Answer 25

a DNA sequence that drives continuous and constant expression of a gene, ie. the gene is always "on" eg. lacZ

Question 26

How do we add DNA fragments to plasmids?

Answer 26

Restriction digest cloning. digest plasmid with restriction enzymes to linearise vector. digest DNA of interest with enzymes to produce compatible ends mix the two and ligate. transform e.coli w ligation mixture. select on antibiotic plates.

Question 27

how is e.coli transformed with the ligation mixture?

Answer 27

transformation of e.coli by ELECTROPORATION cells + DNA from ligation mixture. high voltage pulse induces transient pores, some cells acquire exogenous DNA

Question 28

what is a genomic library?

Answer 28

a collection of DNA fragments that represents the entire genome of the organism

Question 29

how is a genomic library created?

Answer 29

each DNA fragment of an organism is inserted into a cloning vector and propagated, so that it can be studied.

Question 30

why are most cloned fragments in a genomic library non-functional or truncated?

Answer 30

the directional nature of DNA! 3'-5', 50% of inserts in wrong direction. DNA fragment has 6 potential reading frames (3 forward and back), only 1 correct. restriction enzyme is unlikely to excise a perfect open reading frame

Question 31

what is an expression vector used for?

Answer 31

for gene EXPRESSION, ie to perform transcription to produce a protein from the inserted gene

Question 32

key features of expression vectors

Answer 32

Ori, selection cassette, MCS, AND ALSO: inducible promotor (eg. LacUV) +ribosome binding site, transcription terminator etc (more regulatory elements)

Question 33

define ORF

Answer 33

Open Reading Frame a segment of DNA / RNA that when translated can produce a protein. bounded by a start and stop codon.

Question 34

what is directional cloning?

Answer 34

using two different enzyme restriction sites at each end of the DNA fragment to be cloned, such that it inserts directionally into the plasmid. the enzymes must be ZERO CUTTERS within the Open Reading Frame (ORF)!

Question 35

what is an operon? describe structure and purpose

Answer 35

in PROKARYOTES, a cluster of genes that are transcribed together. either expressed together or not at all. structurally: a consecutive sequence of multiple (structural) genes with ONE promotor followed by ONE operator.

Question 36

what does the promotor and the operator of an operon bind to?

Answer 36

promotor binds to RNA polymerase, transcription occurs. operator bings to repressor protein, inhibits transcription

Question 37

define inducible and repressible (operons)

Answer 37

inducible: gene expression is activated by a specific molecule repressive: turned off by a specific molecule

Question 38

how is gene expression regulated in prokaryotes?

Answer 38

3 types of regulatory molecules: repressors activators inducers

Question 39

function of repressors

Answer 39

bind to operators to prevent RNA transcription

Question 40

function of activators

Answer 40

can increase the rate of transcription by helping RNA polymerase bind to promotor / stabilising their interaction

Question 41

function of inducers

Answer 41

can bind to either repressors or activators to alter their activity and control gene expression

Question 42

inducer and repressor for the lac operon - explain mechanism

Answer 42

allolactose (when lactose is present) binds to lac repressor (LacI), preventing it from binding to the operator and allowing transcription to occur

Question 43

define global and specific control of gene regulation

Answer 43

specific: regulates only one operon (eg. LacI for the Lac operon) global: regulates many different operons

Question 44

how does the Lac operator exhibit both specific and global control?

Answer 44

specific control: LacI repressor controls only Lac operon global: CAP protein (also called Crp, the global activator) is a cAMP receptor protein that promotes transcription of the lac operon when glucose levels are low. Overall, cell preferentially uses glucose as an energy source, resorting to lactose metabolism when glucose is scarce.

Question 45

what conditions of glucose/lactose must there be for the Lac operon to be transcribed?

Answer 45

NO glucose (hence high cAMP binds CAP protein) and YES lactose (so that allolactose binds LacI to disengage the repressor)

Question 46

What is the pET vector expression system used for? describe its key features.

Answer 46

pET = plasmid for expression by T7 RNA polymerase. very common bacterial expression system for producing recombinant proteins in high yield. key features: T7 promotor (NOT recognised by e.coli RNA polymerase, only T7 " ") lać operator - extra layer of regulation Ribosome binding site his-tags for protein purification, antibiotic resistance for plasmid selection

Question 47

how does a pET system work? why is it so effective?

Answer 47

T7 RNA Polymerase gene inserted in e.coli under the control of LacUV5 promotor without an inducer, LacI blocks T7 RNA polymerase from transcribing. add IPTG, induce that binds to LacI so T7 RNA polymerase is produced. this then binds to T7 promotor on pET vector --> rapid strong transcription of target gene, ONLY induced by IPTG.

Question 48

Cloning vector vs expression vector

Answer 48

Cloning: for replicating and amplifying DNA fragments Expression: for expressing genes and producing proteins