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Transposon Mutagenesis: Part 1 (Plasmids (origin of replication/ori/replic…
Transposon Mutagenesis: Part 1
Lab Objectives
understand DNA as the genetic material through transformation
test/make competent cells for transformation
learn about plasmid vectors, operons, and transposons
understand how transposon mutagenesis can affect gene expression
Transformation
began with Avery, MacLeod, and McCarty with Pneumococcus bacteria who initially found type R bacteria could be changed to type S bacteria if genetic material from S was placed in the culture with R
cannot just occur whenever; E. coli cells must be "competent"
can be made competent by soaking them in a very cold CaCl2 solution before heat shocking them at 90 seconds in 37 degrees Celsius
seems that the positive Calcium ions shield the negative charge of the cell (as DNA is also negative due to its phosphate backbone) and allows the DNA to enter through tiny pores called adhesion zones; additionally, the creation of a heat gradient from the ice cold solution and the heat shock leads to the DNA flowing down the heat gradient into the cell
a wide variety of prokaryote and eukaryotes can undergo transformation but the method of making them competent isn't the same, suggesting that different kinds of cells have different methods of exogenous DNA uptake but use of Calcium ions is a common one
even after they are made competent, transformation will not occur for all of the cells
transformation efficiency is a measure of how many of the cells actually took up the exogenous DNA (total number of cells that successfully took up the DNA/amount of DNA) ---> cells/microgram of DNA
Plasmids
double stranded, small, supercoiled DNA that may exist freely in the host cell (as ours does) or integrate into the host's chromosome
we used pGLO plasmid containing an araC, bla, and gfp gene
GFP gene is a gene for a glowing protein isolated from a jellyfish now controlled by the araC operator which typically controls 3 structural genes for the metabolism arabinose and is therefore controlled by a feedback loop for arabinose
replaces the 3 structural genes with the GFP gene
wild type: so GFP will only be transcribed/translated in the presence of arabinose
arabinose operator has araC structural gene that makes the araC protein which binds to operator at 2 different places and when no arabinose is present it makes the DNA form a loop by bringing the initiator and operator regions close to each other
when arabinose is present it can no longer bind to the araI region but can bind to the araO operator which allows the RNA polymerase to transcribe the mRNA and gets rid of the looping of the DNA (need a proper araC to bind to the araO region in order for effective transcription to occur)
origin of replication/ori/replicon allows the plasmid to replicate using the cell's machinery for replication (so the replicon for this one is consistent with the E. coli's replication machinery so that when the cell divides the plasmid is also replicated and ends up in both of the daughter cells)
stringent plasmids exist only as a few copies in the cell
relaxed plasmids replicate frequently
control of the amount of replication is in the replicon and different compatibility groups of different replicon types exist
the plasmids are allocated randomly
selective marker gene: often a resistance to a specific antibiotic that make it easy to make sure you only have the bacteria the gets transformed
we used a plasmid with a resistance to ampicillin with the bla gene that produces beta lactamase which binds and hydrolyzes ampicillin preventing it from binding to enzymes important for cell wall synthesis (so ampicillin doens't kill bacteria but does prevent their replication)
restriction enzyme recognition sites are places where restriction enzymes (which cut the plasmid) can recognize and cut
can allow researches to insert things into the plasmid or to digest it into a known pattern (so as to run a gel, for example)
Transposons
small piece of DNA that can insert itself randomly into the genome with the help of an enzyme (tranposase) and if it does this into a structural or regulatory gene it can affect gene expression
mix the transposon DNA and the tranposase forms a transposome
when inserts can disrupt the activity of the genes there but also confers resistance to the antibiotic kanamycin (here we are using it to mutate the plasmid DNA not the genomic DNA)
allow for selective growth of mutated plasmids
usually very species specific
characterized by 19bp mosaic end on each side which are around a gene for kanamycin resistance
KanR gene codes for an enzyme that phosphorylates kanamycin or neomycin which prevents the antibiotic from inhibiting protein synthesis
also has many restriction enzyme sites
experimental procedure
control plates: show that before transformation and mutation that the cells are not already resistant to the antibiotics (to show that you are actually screening for transformed/mutated ones by adding these antibiotics)
need to add the 2 antibiotics to show they both transformed/mutated for the mutated plasmid one
kanamycin ensures that the bacteria won't have a mutation in the bla gene so they either have a mutation in araC, GFP, or some in between region (so they shouldn't glow)