Horizontal Gene Transfer in Prokaryotes and Recombinant DNA Technology Flashcards
What are the 3 types of gene transfer in bacteria?
Transformation. Conjugation. Transduction.
What is transformation?
Uptake of DNA from the environment and incorporation into the bacterial genome
When was transformation discovered and in wat?
- In Streptococcus pneumoniae.
Describe transformation in Streptococcus pneumoniae
- Two strains S and R - S is lethal to mice and R is not
- Heat killed S strain did not kill mice
- Heat killed S mixed with R did kill the mice
- R had characteristics of S
What were other experiments involving transformation in Streptococcus pneumoniae?
Destroyed different components of the S strain
Only when DNA destroyed was the generation of S strains prevented
What is meant when saying some bacteria are naturally competent? (transformation)
Can actively take up DNA from the environmen
How does transformation work?
Have special pores and complexes located in their cell wall. During DNA entry one is degraded and the other is incorporated into the genome
What bacteria needs to be made competent in order to allow for artificial transformation?
E.coli
What is conjugation?
Direct transfer of either plasmid or genomic DNA from one bacterium to another
Describe the experiment with E.coli to show conjugation.
Strain A = met-,bio-,thr+,leu+,thi+
Strain B=met+,bio+,thr-,leu-,thi-
Neither strain could grown on minimal medium but when mixed and incubated formed colonies
How was cross feeding ruled out during the E.col experiment to show conjugation?
Grew strains separated by a filter. Substances could pass but bacteria could not. Didn’t grow need cell to cell contact
What does unidirectional mean?
One donor and on recipient
Describe unidirectional in relation to conjugation?
Some strains could only receive but not donate but would acquire the ability to donate
How was the ability to donate acquire?
Imposed by ‘fertility factor’ (F)
What is F positive?
Carries F and can donate
What is F negative?
Lacks F and can’t donate
What is the purpose of F in conjugation?
Directs the synthesis of pili that connects to another cell
What is transduction?
Transfer of genetic material between bacterial cells by viruses called bacteriophages
Describe how transduction was discovered
- Used salmonella typhimurium
- U tube, direct contact between 2 autotrophic strains prevented by a membrane
- gained prototrophic colonies on one side of the tube
- genetic exchange was due to the infection of the bacteria by a phage called P22
When does transduction occur?
During lutic phases
What is the mechanism of transduction?
- Phage infects bacteria
- Genetic material of phage replicated
- Phage proteins produced
- Host DNA degraded
- Phage carries some of the host DNA
- Transferred to another bacteria when it is infected
What is generalized transduction?
Any part of the genome can be transferred
What is specialised transduction?
Only transfer specific genes
How is DNA cut?
With restriction enzymes
Why are restriction enzymes produced?
By bacteria as a defense mechanism against bacteriophage
What sequences do restriction enzymes recognise?
Specific palindromic DNA sequences
What are cohesive ends?
Sticky ends. Single stranded overhangs (can get 5’ or 3’ overhangs)
What are blunt ends?
Cut DNA in the same place
Describe hybridization of DNA
- Cohesive ends
- Cut with the same restriction enzymes = same single strand overhangs
- Complemenatary base pairing
How are nicks joined?
- DNA ligase
- Catalyses the formation of a phosphodiester bond between nucleotides
What are cloning vectors?
DNA molecules that can hold pieces of DNA of interest for replication in an norganism
What are the most common cloning vectors?
Modified bacterial plasmids - not part of the bacterial chromosome
How can cloning vectors be amplified?
Using E.coli
Used pUC18 as an example of clonning vectors.
- Bacterial origin of replication
- Selectable marker usually eg antibiotic resistance gene
- Multiple cloning site → referred to as a polylinker → lots of restriction sites/recognition sites
- Multiple cloning site is inside a lacZ gene
What does the multiple cloning site allow us to do?
Cut the plasmid and insert the gene of interest
How does the plasmid get into the E.coli?
Artificial transformation (only a few plasmid will be taken up)
What are the different possibilities that the cells could contain?
No plasmid. Plasmid that has re-litigated with itself. Plasmid with gene of interest.
How do you find out which cells contain a plasmid?
Plate on agar with antibiotic. Cells without the plasmid will be killed as they don’t have the antibiotic resistance gene.
How does blue/white selection?
- Without inserted gene lacZ is functional
- Codes for enzyme B galactosdiase
- Add X gal
- No inserted gene = ill produce a blue substance (oxidation_
- Inserted gene = the reaction won’t take place
What is PCR?
Polymerase Chain Reaction - amplifies DNA
What are the 3 processes of PCR?
Denaturation, annealing, extension.
Describe denaturation.
Heated to 95 degrees - interferes with binding of complmentary pairs. Separated inot single strands
Describe Annealing
- Primers bind to target sequence
2. Binding of primares requires cooler temperatures (55 - 65 degrees)
What are primers?
Small pieces of DNA that are complementary to parts of the target sequence
Describe Extension
DNA polymerase uses the primers as a starting point to synthesise a new strand of DNA - occurs at 72 degrees
What happens when you repeat these steps?
Doubles each time
What DNA polymerase do we use?
Heat stable from the bacterium Thermus aquaticus - lives in hot springs.
What is added at the beginning of PCR?
DNA template. dNTPs. DNA primers. Taq polymerase. Buffer solution.
What is a thermocycler?
Can quickly heat up and cool down. Cycle between different temperatures.
How do we facilitate the insertion of PCR fragments into bacterial plasmid?
Use PCR primers which don’t only bind but also contain restriction site in their 5’ end. Produce PCR products with cohesive ends.
What is reverse transcription PCR?
- If you want eukaryotic protein need to remove introns
- Produce cDNA
- Use reverse transcriptase
- Needs a primer
What is cDNA?
Complementary to mRNA and only contains the sequence of exons not introns
What is reverse transcriptase?
Viral enzyme. Uses RNA as a template to synthesise DNA
What is DNA sequencing?
Determination of the nucleotide sequence of a piece of DNA.
What is Sanger Sequencing?
- Single stranded DNA you want to sequence
- Mixed with DNA primer which binds upstream, DNA polymerase and dNTPS
- Complementary strand is synthesised
- Small amounts of fluorescently labelled ddNTP are added → they don’t carry 3’ OH group and terminate DNA synthesis when they get incorporated
What is the result of Sanger sequencing?
Lots of DNA fragments of different lengths with fluorescent ends to tell you which was the last nucleotide incorporated
How can you separate the DNA fragments?
Gel electrophoresis
