Outcome 1

Question 1

What are competent cells

Answer 1

Cells which can uptake exogenous DNA from its enviroment

Question 2

How is competency induced

Answer 2

By either chemical transformation or electroporation

Question 3

How does electroporation work

Answer 3

Exposing the cell to electric shocks cuases temporary pores to be created in the cell membrane

Question 4

Process of chemical tranformation

Answer 4

Actively growing cells are centrifuged into cell pellet. Cell pellet is introduced into an ice cold CaCl2 buffer and allowed to incubate for ten minutes. Exogenous DNA of interest is introduced and gently mixed. Mixture is placed in a water bath at 40-42C for 45 seconds to heat shock cells and then returned to ice bath. Pre-warmed recoverey broth is added to mixture. Mixture is incubated at 37C for 30 minutes and plated onto agar with screening agent.

Question 5

Describe antibiotic resistance genes

Answer 5

Sequences of dsDNA which confer a resistance to a specific antibiotic when expressed by coding for either enzymes which inactivate or degrade the antibiotic or coding for a transmembrane pump to remove the antibiotic.

Question 6

Why are antibiotic resistance genes added to vectors

Answer 6

As a means of screening after transformation to establish which bacteria where sucessfully transformed with a plasmid.

Question 7

What is the formula for calculating transformation effieciency

Answer 7

= #CFU x Total volume of transformation(ul) / volume of spread(ul) x amount of plasmid used (V of plasmid x C of plasmid)

Question 8

What are restriction endonucleases

Answer 8

Enzymes which cleave dsDNA at specific recognitino sites by catalysing the hydrolysis of the phosphodiester bond between the 5’phosphate group of one nucleotide and the 3’OH group of the next nucleotide.

Question 9

Conditions for restriction endonucleases to work

Answer 9

DNA must be double stranded and unmethylated, recognition site must be within the DNA strand and not at the ends.

Question 10

Use of restriction enzymes in cloning

Answer 10

Used to isolate DNA fragment of interest by cleaving its phosphodiester bonds to a larger DNA molecule. Used to insert DNA into vectors by cleaving vectors at recognition sites and allowing insertion of DNA fragments by complementary base pairing.

Question 11

What are recognition sites

Answer 11

Palindromic nucleotide sequences usually between 4-8 bases long which are recongnised and cleaved by restriction enzymes.

Question 12

What is the averae fragment size of a DNA with an Nbp long recogntion site

Answer 12

1/4^N

Question 13

What are sticky ends

Answer 13

The result of cleaved DNA in which the restriction enzymes broke the phosphodiester bond of the nucleotides at points of the two DNA strands which are not directly opposite from one another. Resulting in nucleotide overhang on one strand

Question 14

What are blunt ends

Answer 14

The result of restriction enzymes cutting both strands at directly opposite points from one another meaning there is no nucleotide overhang

Question 15

What are klenow fragments

Answer 15

Components of DNA Polymerase 1 that catalyse the polymerisation of nucleotides in the 5’ to 3’ direction. They attach dNTPS to the template strand by catalysing phophodiester bonds.

Question 16

Name Klenow fragment co factors

Answer 16

dNTPS and NAD+ or ATP

Question 17

What is DNA ligase

Answer 17

An enzyme which catalyses the formation of phosphodiester bonds between the 5’phosphate group of one nucleotide and the 3’OH group of another. Can repair gaps in the sugar phosphate backbone such as with Okazaki fragments

Question 18

What is self-ligation

Answer 18

Intra-molecular ligation where a plasmid may rejoin it’s cut ends and repair the backbone without the insert DNA. OR insert DNA might circularise its self.

Question 19

Methods to prevent self-ligation

Answer 19

Alkaline phosphatase - an enzyme which dephosphorylates, ligation requires a free 5’phoshphate group.
Cutting both the insert DNA and vector with 2 different restriction enzymes.
Maininting a 3:1 ratio of insert DNA : plasmid concentration

Question 20

What are genomic libraries

Answer 20

Gene libraries which contain cloned DNA fragments representing the entire genome of a cell/organism of interest.

Question 21

What is the clark and carbon equation

Answer 21

N = (1-P)/(1-F) where F is insert fragment size/genome size

Question 22

How are genomic libraries screened

Answer 22

By DNA hybridisation

Question 23

When can DNA hybridisation be performed

Answer 23

When a specifc nucleotide sequence of the fragment of interest is known.

Question 24

Describe DNA hybridisation process

Answer 24

1- Master plate has nitrocellulose layer placed on it, some of each colony will be transferred to nitrocellulose layer.
2 -A DNA probe is synthesised that is complementary to one of the strands of the fragment of interest. The DNA probe contains 32P atoms to make it radioactive.
3- The cells are lysed and the dsDNA is denatured into single strands.
4- Radioactive DNA probe is inserted and binds to the complementary ssDNA. Excess probe is washed away.
5- Fragments of interest identified under photographic film, master plate colonies of interest can be identified.
6- Colonies of interest can be cultured for amplification and transformed into cloning or expression vectors.

Question 25

What are cloning vectors

Answer 25

Vectors used for the amplification of DNA of interest but not expression

Question 26

What are the essential components of a cloning vector

Answer 26

An origin of replication, antibiotic resistance genes, multiple cloning sites and selectable markers

Question 27

5 types of cloning vector

Answer 27

PLasmid vector
Bacteriophage ^ vector
Cosmid vector
BAC
YAC

Question 28

What are expression vectors

Answer 28

Vectors used for the expression of genes to produce proteins

Question 29

What are the extra structural features of expression vectors

Answer 29

Promoter and regulatory sequences. Extension of cloned gene sequence for tag/fusion partners

Question 30

What is a promoter

Answer 30

An RNA polymerase binding site upstream from the coding gene that promotes transcription

Question 31

What are the features of a good promoter

Answer 31

Inducible to control transcription. Low basal expression before induction, high expression rates after induction. Easily induced

Question 32

Describe the Lac operon

Answer 32

An operon is a set of adjacent genes, regulatory sequences and signals that control their expression.
The Lac operon contains an operator region which a repressor protein binds to preventing RNA polymerase from binding to the promoter. Lactose binds to the repressor, releasing it from the operator and allowing RNA polymerase to bind to the promoter

Question 33

What is used instead of lactose for induction and why

Answer 33

IPTG because it cannot be hydrolysed and therefore its concentration level remains contstant.

Question 34

WHat are tags

Answer 34

a tag sequence is a short sequence of amino acids that is engineered onto the N- or C- terminus of the cloned protein to aid with purifica intion or confer detectability to the protein

Question 35

What are fusion partners

Answer 35

a fusion protein gene is one that codes for a ‘fusion partner’ protein such as maltose binding protein or GFP. This gene is added to the same open reading frame as the clone/insert/gene of interest so that they are transcribed as a single unit i.e. a fusion protein. The fusion partner protein confers solubility or fluorescence (detectability) or facile purification to the fusion protein.

Question 36

What are the benefits of tag/fusion partners

Answer 36

Higher levels of expression
Detectability
Improved Solubility
Improved purification

Question 37

Name 2 common fusion partners

Answer 37

GFP and MBP

Question 38

Name three commonly used tags

Answer 38

His tags (for affinity seperartion, purification)
Polycystiene tags (purification)
Polyarginine tags (for solubility)