Molecular Biotechnology Flashcards
Molecular cloning
Introducing foreign DNA into a host organism via a vector
Vector
System (plasmid, virus) that facilitates the introduction of the foreign DNA sequence in the host organism - thus the production of the protein
Restriction enzymes
- Aid bacteria to remove foreign DNA by cleaving specific DNA sequences of 4-8 base pair length known as restriction sites
- They cut DNA at the restriction sites if the DNA is not methylated
Palindromic DNA sequences
They’re identical if read from 5’ to 3’ end of the coding strand or from the 5’ to 3’ end of the complementary strand
Blunt ends
The result when DNA strands are cut in the same location on both strands - both strands have the same length
Overhangs
The result when DNA strands are cut at different locations on each strand - they are small single-stranded portions that extend on one strand
Sticky ends
Overhangs have these
What kind of ends does EcoRI generate? What about HindII?
EcoRI generates sticky ends while HindII generates blunt ends
Hybridization
Resulting DNA molecule is hybrid of 2 original DNA molecules
Insert
Gene of interest inserted into a vector that can be removed by restriction enzymes - cloned into the MCS
What if the blunt ends are produced using different restriction enzymes?
The restriction enzyme is usually absent from the recombinant DNA and it’s more difficult to remove insert if needed
Terminal transferase
Used to generate complementary sticky ends on insert and vector - adds oligonucleotides to end of DNA molecule to be recombined
Addition of synthetic linker to a molecule
Another method to recombine DNA molecules that don’t have matching restriction enzyme cleavage sites
Linker
Small synthetic DNA molecule sequence between the insert and the vector sequence
How is genomic DNA obtained
By isolating the genomic DNA of the organism that contains the gene of interest to be cloned
How is complementary DNA obtained?
It’s obtained by isolating mRNA of organism producing protein of interest
Reverse transcriptase
- Generates DNA strand complementary to mRNA sequence
- In presence of primer annealed to RNA, it can synthesize DNA strand by using RNA strand as template
RNase
Hydrolyzes RNA to remove RNA from DNA-RNA hybrid
Upstream processing
Unit operations that occur up to end of fermentation (cell growth + production in bioreactor)
Downstream processing
Series of purification steps that follow fermentation - 1st step is usually cell separation from broth via centrifugation or filtration and 2nd step is usually cell lysis if product is intracellular or in the cell periplasm
Fermentation broth
Mixture of medium + products secreted by cells in bioreactor
Homogenization
Process of cell lysis in bioprocess engineering - causes product loss
Post translational modifications that occur in eukaryotic proteins but not prokaryotic proteins
- Formation of disulphide bonds - introduced during downstream processing
- Protein glycosylation
E. coli as a host organism
- Grow rapidly + produce lots of recombinant protein
- But they’re gram negative + possess outer membrane so the recombinant proteins may be trapped in the periplasm of Gram-negative bacteria
Bacillus subtilis as a host organism
- Doesn’t possess outer membrane so better suited for production of secreted proteins - cheap!
- But isn’t well established yet + genetically unstable which leads to mutations that eliminate/reduce production of protein - leads to selection of cells that don’t produce protein of interest during upstream processing
Yeast as a host organism
- Used as production place for recombinant proteins that require simple post translational modifications - cheap!
- Can be used to produce recombinant proteins using plasmids but production is transient unless plasmid DNA becomes incorporated into yeast genome
Mammalian cells as host cells - why is CHO used?
- Used to produce glycosylated recombinant proteins for therapeutic use - Current bioprocesses use CHO due to high cell specific productivity (product produced per cell per hour) + resistance to stress + elevated growth rates - best for product safety + quality
Insect cells as host cells
- Can apply post translational modifications, grow rapidly + use very safe vectors - produce recombinant proteins used in vaccines
Plants as host organisms
- GMO’s: resist pests or increase nutritional value or render crop sterile
- But potential invasion of habitat by GMO and cost of buying GMO seed that is infertile are concerning
Plasmids
- Used as cloning vectors + exist naturally in bacterial cells
- They allow exchange of genetic info between bacteria by transformation or conjugation
Transformation
Uptake of genetic info from extracellular medium
Conjugation
Transfer of genetic material between cells via direct cell-cell contact
Plasmids under relaxed vs stringent control
Plasmids used in molecular cloning apps are under relaxed control (they replicate independently of cell division) while plasmids not used for cloning are under stringent control (they replicate once/cell division)
Origin of replication
DNA sequence allowing for initiation of replication within plasmid by recruiting transcriptional machinery proteins - some plasmids have >1 ORI sequence to allow their replication in >1 host
Antibiotic resistance gene
Allows for selection of plasmid-containing bacteria
Multiple cloning site
Short DNA segment which contains many restriction sites allowing for easy insertion of DNA - in expression plasmids, MCS is often downstream from promoter
Promoter region
Drives transcription of target gene - vital component for expression vectors: determines which cell types the gene is expressed in + amount of recombinant protein obtained
Selectable marker
Plasmids have these for use in cell types other than bacteria - include proteins that confer antibiotic resistance to host (organisms that haven’t incorporated the plasmid can’t grow in presence of that antibiotic while hosts that incorporated plasmid are resistant + can grow in antibiotic-containing medium)
Primer binding site
Short single-stranded DNA sequence used as initiation point for PCR amplification/sequencing - can be exploited for sequence verification of plasmids
Expression plasmids
On plasmids used to express foreign proteins
Reporter gene
- Easily detected to help monitor vector or effect of gene construct introduced into cells - spans the MCS
- If insert added to MCS the reporter gene is disrupted
Green fluorescent protein
- Cloning plasmid is amplified in E.Coli but bacteria doesn’t express GFP as they don’t have transcription factors required to bind + recruit polymerase to CAG promoter
CAG promoter
Active in all mammalian cells + contains enhancer element from CMV promoter, the beta-actin promoter + splice acceptor of beta globin gene which enhances activity of beta-actin promoter
Tropism
Specificity of a virus for a given target cell - depends on types of receptors present on target cell surface which determines virus binding + uptake by cells
Capsid
Protein structure containing viral DNA or RNA - can limit insert size added to viral genes
Why are replication deficient viral vectors produced?
To reduce risk to human manipulators + to create more space for foreign DNA in viral genome
How are replication deficient viral vectors produced?
Must use production host organism that differs from target host organism
Transduction
Intro of foreign genetic material into animal cells via viral vectors
Transfection
Intro of foreign genetic material into animal cells via non-viral vectors
Competent
Bacteria that are permeable to DNA - electrical or chemical treatments are used to make bacteria competent
How are chemically competent bacteria obtained?
By exposing bacteria to divalent cations in cold conditions followed by heat shock
How are electro-competent bacteria obtained? AKA electroporation?
By placing cells in distilled water + then applying electric field - electric field disrupts interactions between negatively charged phospholipids, creating small spaces in membrane allowing for DNA entry
Antibiotic resistance cassette
If plasmid contains this, only bacteria that were transformed with plasmid will form colonies on substrate
Clones
If colonies arise from single parent cell - but even if clone contains vector of interest, doesn’t mean it also expresses gene of interest
Blue-white screening
- Method of distinguishing clones that contain insert in MCS from clones which contain the vector without an insert - MCS is in middle of DNA sequence coding for lacZ gene
- Beta-galactosidase cleaves substrate to generate blue product + clones that contain intact lacZ gene appear blue
- If lacZ gene disrupted due to insert presence in MCS, cells won’t produce blue product + will appear white
Stable expression
Aim of clonal selection is to achieve this in host for protein of interest - continuous capacity of cells to express gene of interest
Why is it preferable to express gene of interest under control of inducible promoter?
- This allows for rapid cell expansion without diversion of cell resources to protein production - so production of protein of interest is induced once cells have reached good cell concentration in bioreactor
- Reduces negative selective pressure caused by production of foreign protein/product
Basic steps of molecular cloning
Identifying gene of interest, inserting it into cloning vector, transforming cells w/vector, selecting clones that express selectable marker + confirming that clones stably express protein of interest
Directional cloning
Insert can only recombine with vector in 1 direction so vector must have complementary sticky ends available for annealing
Non-directional cloning
- 2 ends of insert generated by restriction enzymes are the same - since both ends are same the insert can recombine w/vector in any direction
- Can also occur if 2 dif restriction enzymes used to cut 2 sides of insert but 2 sides result in same ends
When is non-directional cloning preferable?
When insert + vector don’t have matching restriction sites
Con of non-directional cloning
Reduces probability that gene of interest is inserted in proper direction
PCR general process
Synthetic DNA primers used with DNA polymerase to replicate DNA in test tube + prior to replication, DNA strands are separated by helicase + other proteins unwinding being replaced by DNA melting at high temps
Trick to developing practical PCR method?
Identification of heat-resistant DNA polymerase from organisms that live in high temp environments + thermocycler
Thermocycler
Automated apparatus used for PCR
Steps of PCR
- Reaction mixture w/template DNA, heat-resistant DNA polymerase, dATP, forward + reverse DNA primer + buffer solution prepped
- Template DNA denatures
- Primer anneals
- DNA extension by DNA polymerase
- steps 2-4 repeated for up to 45 cycles
Primers used in PCR
Single-stranded synthetic DNA strands - sequence complementary to that of target DNA to which they anneal (ensures specific amplification of target DNA sequence)
How must primers align with target DNA sequence in PCR?
- 3’ end of each of 2 primers must align w/target DNA sequence - forward primer anneals at 5’ end of DNA + anneals w/non-coding DNA strand so has same sequence as 5’ end of coding strand
- Reverse primer anneals at 3’ end of DNA molecule + its sequence is same as non-coding DNA strand
- This allows extension of both o.g. DNA strands at 3’ end of each primer
Why are non-specific sequences added to 5’ end of primers that will be incorporated into PCR product?
- Introduces small mutations into PCR product + can contain additional nucleotides that represent restriction sites (after PCR amplified DNA molecule contains new restriction sites)
- Can add additional codons - used to add histidine residues so that PCR amplified sequence contains them as they facilitate protein purification
Quantitative PCR
- Used to quantify initial amount of DNA template in sample by measuring relative amount of DNA in PCR mixture as function of cycle # - relative increase in DNA concentration monitored using molecule which detects double stranded DNA
- AKA real time PCR b/c relative concentration of DNA measured in real time during PCR
- After DNA exceeds detection limit, exponential increase in DNA concentration as function of PCR cycle + eventually primers or dATP become limiting rather than template DNA
What if PCR efficiency is 100%?
DNA molecules obtained after each PCR cycle doubles
Why is qPCR used to quantify amount of DNA molecule of interest relative to another?
Because relative increase in fluorescence is easy to quantify
Gel electrophoresis
Used to separate charged samples based on electrophoretic mobility
Agarose gel electrophoresis
Samples deposited in agarose matrix + separated by applying electric field where negatively charged molecules (like DNA) migrate towards anode
What happens if electrophoresis experiment is interrupted before smallest DNA molecules exit the gel completely?
Distance traveled by DNA through gel is greatest for smallest DNA molecules
Agarose gel electrophoresis as analysis of molecular cloning results (AKA restriction analysis)
- Once host cells have amplified cloning vector it can be purified from cells + vector can be digested using restriction enzymes which linearizes DNA - size of DNA fragments measured w/this process
- Dif restriction enzymes lead to dif bands visualized
Genome
Represents all DNA present in cell
Transcriptome
Represents genes transcribed into mRNA by cell
Proteome
Represents all proteins in cell
Metabolome
Represents all metabolites in cell
Genomics + transcriptomics + proteomics + metabolomics
Represent techniques + data analysis methods used to study genome, transcriptome, proteome + metabolome
