6. Genetic Multiplication of Bacteria Flashcards
E.Coli lives where normally in the human body?
gut
Why do we know more about E.Coli than other bacteria?
- grows rapidly
- simple nutritional requirements
- well established genetics
- genome sequence is known
The E.Coli K12 genome is a … chromosome with … base pairs and encoding for … proteins
- circular
- 4.6 million
- around 4,400
Of the 4.6 million base pairs, how many are of unknown function?
40%
The average protein contains how many genes in E.Coli?
around 300
What are operons?
many gene encoding enzymes of a single biochemical pathway are clustered into operons
Define ‘plasmids’
- small circular DNA present in varying copy number per cell
- often carry genes which are useful but not essential e.g genes for antibiotic resistance
Plasmids are released by … and absorbed by …
What does this allow?
- dead bacteria
- those still living
- genetic info is exchanged
Horizontal gene transfer has potential to change … and …
genotype and phenotype
3 methods of horizontal gene transfer
- transformation
- transduction
- conjugation
How are genes transferred between bacteria in the mouth? Why?
- dental plaque
- to increase fitness of bacteria e.g strep mutans
- can make dental plaque a resevoir for antimicrobial resistance
How does transformation occur?
- dead bacteria breaks open
- transfer of free DNA into recipient
How does conjugation work?
- plasmid is simply transferred to another bacteria
- both bacteria living
How does transduction work?
- viral delivery of the genes
- virus attaches to recipient and carries gene
Which of the 3 horizontal gene transmission methods do these relate to…
- DNA uptake?
- phage-mediated DNA transfer?
- mating?
- transformation
- transduction
- pilus-mediated transfer; conjugation
DNA is incorporated into the chromosome of recipient cell by …
homologous recombination
How could you select rare genetic recombinants? Use tryptophan for example
- Trp- cells in test tube added to growth medium without means no growth
- if you add DNA from TRP+ cells to TRP- cells, then to an agar medium without means recombinants form colonies
Goals of genetic manipulation in biology
- to understand gene function
- knock-out genes
- change individual amino acids in proteins
- clone and over-express proteins
- construct fusions with reporter genes e.g encoding fluorescent proteins (track expression and location)
Goals of genetic manipulation in biotechnology
- production of recombinant proteins e.g insulin
- develop vaccines e.g hep B
- generate improved probiotic bacteria
What is recombinant DNA technology?
- creation of recom DNA (making new combos of unrelated genes in a test tube)
- cloning recom DNA (amplifying it by introducing it to living cells and produces identical copies)
- using recombinant DNA (expressing the cloned gene to make protein)
4 things needed for recombinant DNA
- DNA/RNA (raw material purified from tissue/cell culture)
- enzymes to manipulate DNA/RNA
- vectors (act as vehicle to carry recom DNA into host cell)
- cells (to amplify and maybe express recom DNA)
4 enzymes used in recom DNA technology
- restriction
- DNA ligase
- taq polymerase
- reverse transcriptase
Role of restriction enzymes
- cleave DNA at specific sequences creating complementary sticky ends
- restriction endonucleases act as molecular scissors
Most restriction enzymes recognise … base pair … sequences
- 4-8
- palindromic
Restriction enzymes first used in a lab in …
1970s
How are restriction enzymes produced?
naturally produced by bacteria as a self defence mechanism
- cleaves foreign bacteriophage DNA
Role of DNA ligase
sticks fragments of DNA together
Role of Taq polymerase
- PCR
- creates multiple copies of DNA fragment
Role of reverse transcriptase
copies RNA into DNA
Properties of vectors
- unique restriction sites for insertion of new DNA
- introduced to host cell relatively easily
- efficient origin of replication
- contain gene to allow selection of cells which contain plasmid e.g antibiotic resistance
- contain regulatory sequences to allow expression of inserted gene
How big are plasmids?
around 2-200 kbp
Why are plasmids commonly used vectors?
- replicate independently of bacterial chromosome
- have many functions like self defence, antibiotic resistance
- have been modified for use in genetic engineering
If plasmids aren’t used as vectors, what 2 things might be?
- bacteriophage
- cosmids/phagemids
Define ‘bacteriophage’
- viruses which infect bacteria
Define ‘cosmids/phagemids’
genetically engineered hybrids which replicate as a plasmid but can be packaged as a bacteriophage
Bacteriophage and cosmid vectors are useful for what?
cloning larger fragments of DNA
How would you clone a human gene in E.Coli?
- creation of recombinant DNA (obtain the DNA of interest and insert DNA into vector like a plasmid)
- cloning recom DNA (plasmid inserted to E.Coli host, selectively culture thr plasmid containing cells and purify or express the recom plasmid)
- transformation (plasmid inserted into bacterial cell)
How would you insert a plasmid into a bacterial cell?
- with CaCl2 on ice or heat shock (420oc, 2 min)
- or electroporation
Bacteria containing pUC18 plasmids can be selected by …
growing on agar and ampicillin
Each bacterial colony arises from a … … E.Coli cell
Each cell in this colony contains …
The cells in this colony are therefore …
- single transformed
- copies of the same plasmid
- clones
Why can you not use animals as a hormone source?
- animal protein not identical to humans so side effects
- difficult to purify
- possible contamination with pathogens e.g BSE
What was the first recombinant drug licensed for human use?
insulin
How does recombinant insulin work?
- insulin made in beta cells of Islets of langerhans in pancreas
- vital to controlling blood glucose
- insulin deficiency results in diabetes mellitus and death if untreated
- regular injections of animal insulin from pigs/cows can alleviate symptoms
How to create recombinant insulin?
- gene for proinsulin contains 2 introns
- mRNA is reverse transcribed to produce pro-insulin cDNA with no introns
How does recombinant DNA link to dentistry?
- can be used to manipulate genes and produce GM organisms
- create genetically altered strain of strep mutans called SMaRT which doesn’t produce lactic acid
- protects against tooth decay
