Outcome 1 Flashcards
What are competent cells
Cells which can uptake exogenous DNA from its enviroment
How is competency induced
By either chemical transformation or electroporation
How does electroporation work
Exposing the cell to electric shocks cuases temporary pores to be created in the cell membrane
Process of chemical tranformation
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.
Describe antibiotic resistance genes
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.
Why are antibiotic resistance genes added to vectors
As a means of screening after transformation to establish which bacteria where sucessfully transformed with a plasmid.
What is the formula for calculating transformation effieciency
= #CFU x Total volume of transformation(ul) / volume of spread(ul) x amount of plasmid used (V of plasmid x C of plasmid)
What are restriction endonucleases
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.
Conditions for restriction endonucleases to work
DNA must be double stranded and unmethylated, recognition site must be within the DNA strand and not at the ends.
Use of restriction enzymes in cloning
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.
What are recognition sites
Palindromic nucleotide sequences usually between 4-8 bases long which are recongnised and cleaved by restriction enzymes.
What is the averae fragment size of a DNA with an Nbp long recogntion site
1/4^N
What are sticky ends
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
What are blunt ends
The result of restriction enzymes cutting both strands at directly opposite points from one another meaning there is no nucleotide overhang
What are klenow fragments
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.
Name Klenow fragment co factors
dNTPS and NAD+ or ATP
What is DNA ligase
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
What is self-ligation
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.
Methods to prevent self-ligation
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
What are genomic libraries
Gene libraries which contain cloned DNA fragments representing the entire genome of a cell/organism of interest.
What is the clark and carbon equation
N = (1-P)/(1-F) where F is insert fragment size/genome size
How are genomic libraries screened
By DNA hybridisation
When can DNA hybridisation be performed
When a specifc nucleotide sequence of the fragment of interest is known.
Describe DNA hybridisation process
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.
What are cloning vectors
Vectors used for the amplification of DNA of interest but not expression
What are the essential components of a cloning vector
An origin of replication, antibiotic resistance genes, multiple cloning sites and selectable markers
5 types of cloning vector
PLasmid vector Bacteriophage ^ vector Cosmid vector BAC YAC
What are expression vectors
Vectors used for the expression of genes to produce proteins
What are the extra structural features of expression vectors
Promoter and regulatory sequences. Extension of cloned gene sequence for tag/fusion partners
What is a promoter
An RNA polymerase binding site upstream from the coding gene that promotes transcription
What are the features of a good promoter
Inducible to control transcription. Low basal expression before induction, high expression rates after induction. Easily induced
Describe the Lac operon
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
What is used instead of lactose for induction and why
IPTG because it cannot be hydrolysed and therefore its concentration level remains contstant.
WHat are tags
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
What are fusion partners
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.
What are the benefits of tag/fusion partners
Higher levels of expression
Detectability
Improved Solubility
Improved purification
Name 2 common fusion partners
GFP and MBP
Name three commonly used tags
His tags (for affinity seperartion, purification) Polycystiene tags (purification) Polyarginine tags (for solubility)
