Transformtion Of E.Coli Flashcards
(20 cards)
What is vertical gene transfer?
New genes are passed onto daughter cells only through cell division
What is horizontal gene transfer?
Bacteria acquire new genes from neighbouring bacteria in their environment
Movement of DNA from one cell to another takes place by 3 mechanisms
- bacterial transformation
- transduction
- conjugation
What is bacterial transformation?
Process by which bacterial cells take up naked DNA molecules from the surrounding medium and thus acquire new genes
What is natural transformation?
Where bacteria take up DNA molecules present in their environment as a result of the death and breakdown of other bacteria
The incoming DNA is often incorporated into the bacterial genome and thus bacteria acquire new genes
Describe artificial transformation
Naked DNA from any source (e.g. plasmid DNA) can be introduced into bacteria in a lab setting —> don’t usually use transformation as a major route for genetic exchange naturally
The bacteria must undergo treatment to enhance their ability to take up DNA —> cells that had this are in a specific physiological state known as competence
Most common way to be treated for induction of competence is by using calcium chloride
How is calcium chloride used to induce competence in bacteria?
- Bacteria are treated in the early log-phase of growth with a solution of ice cold calcium chloride
- Plasmid DNA can be introduced into bacteria by mixing competent bacterial cells with plasmid and quickly raising the temperature to 42 degrees for a short amount of time (heat shock)
- Bacteria can then take up DNA but the exact mechanism of uptake is unknown
- Bacterial cells are allowed to ‘recover’ in liquid medium and begin to express the antibiotic-resistance gene before plating on plates containing antibiotic
What is meant by transformation efficiency?
The measure of how successful a transformation has been
This is dependent on the competency of the recipient cells, the type of plasmid and the protocol followed
What is involved in the pGLO system?
- recombinant plasmid = pGLO
- gene coding for green fluorescent protein = GFP
- gene coding for a regulatory region = araC
- gene coding for beta-lactamase, an enzyme that catalyses the breakdown of the antibiotic ampicillin = bla
- origin of plasmid replication = ori
In the presence of arabinose, araC protein promotes the binding of RNA polymerases to the promoter of GFP
Where did the GFP gene originate?
In bioluminescent jellyfish, Aequorea Victoria
The GFP encoded by the gene allows jellyfish to fluoresce
Significance of GFP in bacteria
If a plasmid is transformed into bacteria and is GFP induced, the bacteria also expresses GFP and glow green under UV light
Plasmid will also carry a gene for resistance to ampicillin (makes bla ineffective) as well as a gene-regulation system that can be used to control the expression of GFP
Control of gene expression in bacteria
The gene for GFP is only switched on (induced) when the sugar arabinose is present in the growth medium
What is bacterial conjugation?
The transfer of DNA from a donor cell to a recipient by the direct contact of the cells
What is required for bacterial conjugation?
The donor must contain a conjugative or mobilisable genetic element, most often a conjugative plasmid
This plasmid can either be free in the cell or can be integrated into the chromosome of the donor cell and usually, only the plasmid will be transferred from the donor but occasionally part of the main chromosome can be transferred as well
How is GFP production controlled?
- Arabinose is a sugar which can be metabolised by bacteria
- Bacterial cells produce enzymes that break down arabinose only if the sugar is present in the environment
- If arabinose is absent, the genes are usually switched off
- In the pGLO, genes responsible for arabinose metabolism have been replaced with genes coding for GFP
- So when arabinose is present, GFP is induced
What can conjugative plasmids also be known as?
Sex or fertility plasmids
Cells containing such an element are called F+ cells and those that do not are called F-
Steps of conjugation
- Establishment of contact by the donor cell to the recipient cell by means of a long protein filament called the conjugation pilus (or sex pilus)
- The pilus draws the cells together enabling the outer membranes of the two cells to fuse, forming a conjugation bridge encoded by genes on the conjugative plasmid itself
- One strand of the plasmid is nicked and transferred through the conjugation bridge into the recipient cell by the action of an attached protein called the ‘pilot’
- During this process, the transferred strand is replaced in the host by rolling circle replication and the complementary strand is made in the recipient cell
- Both cells now contain the double-stranded plasmid and are deemed to be F+. Cells now separate
Bacterial conjugation in the lab
Can occur simply by mixing the donor and recipient cells together and leaving them for an hour at 37 degrees
The mixture of cells is then plated on nutrient agar plates containing the suitable antibiotics that will select for recipient cells that have received the copy of donor DNA
What are conjugative pili?
Can be classified as one or the other of 2 types and the type of conjugative pili a donor bacterium expresses is one determinant of the efficiency (frequency) of plasmid transfer in different environments
What are the types of pili?
Long, flexible pili such as those encoded by the F plasmid, form durable contacts with recipient bacteria in both liquid environments and on solid surfaces
Short, brittle pili such as those encoded by the R (for resistance) plasmid, only form durable contacts with recipient bacteria when the cells are immobilised on a solid surface
Thus, F-type plasmids will transfer between cells a high frequencies in both liquid and solid environments whereas R-type plasmids will only transfer at high frequencies between cells on a solid surface
How is transformation efficiency calculated?
Total number of transformants (multiply by DF)
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Total amount of pGLO DNA used in tranformation
