Practical Stuff Flashcards
What do expression plasmids need extra from cling vectors?
Both need an ori and selectable marker eg AmpR gene
Expression need MCS and an expression marker eg. LacZ gene- beta-galactosidase metabolises X-gal producing a blue product- it will be interrupted if the gene has been successfully introduced- no blue
What are the three most important signals for expressing genes in bacteria?
Promoter- determines the rate that mRNA is synthesised. Can use inducible or repressible promoters.
Terminator
Ribosome binding site- the Shine-Delgarno sequence and Kozak sequence
How could you introduce DNA into the bacteria?
Micro injection
Viral transfection
Heat shock
Electroporation
What are he limitations of using E. coli ?
Limited post translational modification eg. No glycosylation
Improper folding
Degradation
Lack of ability to perform intron-exon splicing
Codon bias- difficulties in host tRNA translating the mRNA
Foreign genes may contain bacterial termination signals
What are the pros and cons of using yeast in cloning?
Is eukaryotic so will be more homologous than E. coli- post translational modification and protein degradation
High yeilds
But still codon bias and proteins are often hyper glycosylated
What are the pros and cons of using mammalian cells?
Difficult and costly and cells usually require a surface to grow on rather than a suspension
If gene is over expressed it can have deleterious effect on the cell so it will be down regulated
But the protein can be accurately post translationally modified and spliced etc.
Describe vectors
A vehicle used to transfer genetic material to a target cell
It needs to be capable of independent replication within a host and of carrying a fragment of foreign DNA
Eg. Plasmids- 10kb, bacteriophages lambda phage-23kb, cosmids 30-44kb, YACS 0.2-2Mb, BACS 300kb
What is gene editing?
Enables manipulation of virtually any gene in a diverse range cell types and organisms
Based in the use of engineered nucleases
Outline the methods of genome editing
Zinc-finger nucleases (ZFNs)- zinc fingers recognise a codon- attached to Fok1 which cleaves in pairs- two different series of zinc-fingers are required on either side of the cleavage site
Transcription Activator-Like Effector Nucleases (TALENS)- also uses Fok1 but uses repeat variable diresidues (RVD) to recognise individual nucleotides- one either side of the cleavage site
Clustered Regularly Interspaced Pandendromic Repeat (CRISPR)- CRISPR Associated Protein 9 (Cas9)- Cas9 is the nuclease, requires tracrRNA and crRNA- cleaves at the end of the non-complementary strand to the guide sequence on the crRNA
Give an overview of using ZFNs in gene editing
Sequence specificity- long target site
Modular customisable DNA binding domains
Limitations- poor targeting density
Target site limitations- G-rich and methylation sensitive
Not all newly synthesised ZFNs are able to cleave chromosomal DNA
Low success rate
Has a high amount of off target effects
Variable cytotoxicity
Costly and difficult to produce and deliver
Give an overview of using TALENS for gene editing
Can be used to recognise small sequences High success rate Low off target effects Less costly to produce Limitations- large constructs Highly homologous- self-recombination Target site limitations- 5' thymine and methylation sensitive More difficult to deliver
Give an overview of using CRISPR-Cas9 for gene editing
Has a high success rate Capable of multiplexing Low cytotoxicity Affordable Easy to modify Easy to deliver Limitations- restriction site limitations- end with an NGG or NAG Has variable off target effects Big contruct
How could you deliver reprogrammable nucleases?
Via plasmid DNA In vitro transcribed mRNA Non-integrating viral vectors Purified protein ➡️ Electroporation Liposome transfection Micro injection
What are the applications of genome editing?
Pre-clinical
-Targeting non-coding regions
-Creation of genetic variation and study their effects
-Generation of knock-out and knock-in models by direct injection into embryos
-Create an isogenic cell line to model human disease
Biotechnology
-GM crops and livestock
-Production of therapeutic proteins in cultured cell lines
Therapeutics
-Treatment of HIV in humans
-Treatment of haemophilia B in mice
-Gene correction and addition in patient-derived pluripotent stem cells for patients with genetic diseases
What cell activity does genome editing rely on?
Double strand break repair
NHEJ repair can lead to deletions or inserting that can knock out a gene
Insertion of donor DNA, single nucleotides or tags
