CBG.21- Bacterial transformation

Why do bacteria naturally transform?

- Diversity: increase genetic diversity by horizontal gene transfer - Nutrition: source of nucleotides - Repair: uptake of homologous DNA would allow to repair damage via recombination

Describe process of cotransformation to map genes?

- Donor cell that has wild type alleles in fragments of 20kB (only the genes close enough in these 20kB fragments will be transformed) - Recipient cell is e.g deficient in his and trp genes - Transformants are plated onto a medium containing his and trp respectively. - Growth of transformed cells can then be checked for. (plating on minimal medium containing no his or trp) - 40% cotransformants suggest that genes are close Genes that are closely linked on bacterial chromosome can cotransform.

1928, Frederick Griffith- Can n extract of dead bacteria genetically transform living cells?

-> Streptococcus pneumoniae -> Two strains: rough (small colonies) and smooth (large colonies) -> Observed that, when injected into a mouse, the smooth was very virulent, whereas rough was not. [smooth is covered with a polysaccharide capsule, which protects it from the immune system and makes it virulent] -> When he autopsied the dead mice, he could go grow the very same virulent strain that he injected -> No bacteria could be recovered from the living mice that had been injected n non-virulent strain -> However, when injected with the non-virulent strain and heat-shocked virulent strain, mouse died and virulent recovered. Conclusion: The virulent strain contained something ('transforming principle') that could transform the non-violent bacteria

1944 Avery, MacLeod, McCarty: What is the transforming principle?

-> Used a filtrate of the dead virulent strain and treated it with different type of enzymes: RNase, protease (e.g trypsin) and DNase -> Then mixed these with the rough, non-virulent bacteria---> RNase filtrate and protease filtrate were transformed (hence, transforming principle was not RNA or protein) -> DNase sample did not become virulent when incubated. Conclusion: DNA is essential in transformation

A strain of Escherichia coli is arg- trp+ leu- lac+. On what media would this bacterium grow?

1) Rich medium 2) Mediums containing arginine and leucine

Summarise the processes of horizontal gene transfer?

1) Transformation 2) Conjugation 3) Transduction

Transformation process?

1. Double stranded DNA initially binds but bacteria take up single stranded DNA from environment (if competent) 2. This is incorporated into the bacterial chromosome via recombination (2x crossovers) 3. After replication and division, one of the resulting cells is transformed

Blue-white screening?

A method of selecting the desired recombinant bacteria (out of millions of cells) that contain an inserted gene from foreign DNA. Based on alpha complementation: on plasmid you will have a gene that encodes a fragment of β-galactosidase [alpha fragment] that corresponds to N-terminus of β-galactosidase. Rest of β-galactosidase gene will be present in bacteria that you are using as a recipient. Therefore, when recipient bacteria receives alpha fragment, it can reconstitute full β-galactosidase activity. By monitoring β-galactosidase activity, you can observe whether plasmid has been taken up. Monitoring β-galactosidase: -> Ability of enzymes to cleave X-Gal, which releases X chromophore giving you a blue colour to the colony. White colonies do not recover any β-galactosidase activity; the lac-Z gene has been interrupted showing that the gene of interest has been inserted.

To summarise, what do antibiotic resistance and blue-white screening allow you to select for?

Antibiotic resistance: select for presence of plasmid Blue-white screening: select for presence of plasmids that carry gene of interest

What does stable transformation require?

Chromosomal integration of incoming DNA via a double crossover

Plamsid?

Extra-chromosomal piece of DNA that can self-replicate

What does the dsDNA bind to in transformation?

Extracellular appendages called pseudeopilus (+) or pillus (-) One strand will then be degraded *Gram neg requires PiQ, which allows protrusion of pillus through OM

What is the rate of cotransformation equal to?

It is inversely proportional to the distance between the two genes being cotrasnformed.

What can cotransformation be used to do?

Map genes: e.g p,q and o are genes. You can monitor strains that have received the genes. DNA can be extracted from donor bacteria; it will breakdown into fragments p and o are on a different chromosome so unlikely they can be cotransformed. Hence, closer genes are on chromosome, the higher the transformation frequency

Describe the experiment that supports the molecular mechanisms of transformation?

Mix Arg+ DNA and Arg- recipient cells: 1. Bacteria that is auxotroph for Arg is with extracellular DNA that provides capacity to synthesise Arg. [only those that can transform the Arg will grow on minimal medium] 2. At different time intervals, add DNase. Hence, any DNA that is in extracellular DNA will be degraded. 3. Extract DNA from cells in step 2 and transform new cells Results: Time interval 1- all extracellular DNA degraded; none is incorporated into cells---> NO arg+ cells Time interval 2- Still too early for incorporation---> single stranded fragment of Arg+ but NO Arg+ cells [supports that for DNA to bind onto cell, it has to be double stranded] Time interval 3- dsDNA can be bound and internalised; DNA transformed---> Arg cells! *stage at time interval 2 is known as eclipse phase: strains of DNA can be taken up but cannot be expressed as not chromosomally incorporated.

Is artificial transformation effective?

No it is relatively inefficient compared to natural transformation

Artificial transformation?

Not every bacterial strain are naturally competent hence must be artificially transformed: -> Chemical transformation - CaCl2 treatment of bacterial cells at 4 degrees - Causes changes in membrane permeability to allow entry of plasmid DNA (+ charge on calcium neutralises charge on bacterial cell wall, dissipating electrostatic repulsion and weakening the cell wall) -> Electroporation - Electric field changes the permeability of bacterial membrane (allows entry of plasmid ) - Membrane repairs and restores natural permeability once electric field is removed

Phototrophs and auxotrophs?

Phototrophs: synthesise all essential nutrients Auxotrophs: require supplement (e.g aa) Auxotrophs are used by bacterial geneticists

Competence?

State that bacteria enter to take up naked DNA from their environment Leads to assembly of a DNA uptake molecular machinery ie. pilus, PiQ

Does the transformation process need direct contact?

The transformation process DOES NOT need direct contact between donor and recipient strains; DNA taken up is environmental

What is transformation used for?

Transfer genes between bacteria

Define transformation?

Uptake of extracellular DNA

Structure of a cloning vector?

[PLASMID] -> Origin of replication: essential for propagation in daughter cells -> Polylinker: unique areas for inserting foreign DNA. Unique restriction sites are found here. -> Antibiotic resistance: essential for selection of cells that have taken up plasmid e.g ampR (transformed cells will be resistant to beta lactams like ampicillin)

Plasmids are engineered to have specific restriction enzymes target sites in specific places. In the pBR family of plasmids, the only NheI site is contained within a functional tetR gene from E. coli. NheI is used to cut the plasmid open, a gene of interest is ligated into the gap, and the plasmid is transformed into bacteria. The cells are then plated out onto agar containing ampicillin and another agar containing tetracyclin. In which plate will i) the untransformed cells grow ii) The transformed cells iii) the transformed cells containing a transformed plasmid

i) Neither ii) Both iii) Just the ampicillin agar (Transformed plasmids have a broken tetR gene so would be found only on the ampicillin plates)