Lecture 10: Plasmids & Transformation Flashcards
Plasmid Definition
Small extra-chromosomal dsDNA elements that encode genes of nonfunctional protein
Basic features of plasmids
-Original of replication that is independent of the chromosome replication mechanism
-Usually encodes for nonessential genes
Sinorhizobium meliloti
Soil bacterium that form nodules on legume roots for nitrogen fixation to give plants ammonia.
-Has a plasmid called pSymB
pSymB
-Megaplasmid made of over 1.6 million nucleotides and encodes for 1570 proteins.
-Also encodes for some essential functions
-Contains the only Arg-tRNA and encodes MinCDE for cell division
-Contains genes for exopolysaccharide synthesis, nutrient acquisition, and catabolism of alternate substrates.
Why can’t pSymB be referred too as a second chromosome
It uses a plasmid origin of replication
Chromosome II of vibrio cholera
Has a plasmid P-type replication mechanism. Contains genes for environmental adaptation and stress response meaning it was a plasmid but developed to a chromosome.
R plasmids
Encode for antibiotic resistance genes
ColE1 plasmid
Encodes for colicin E1 bacteriocin that kills bacteria lacking the plasmid
Tol plasmid
Encodes for catabolism of toluene
Replication and Partitioning for Plasmid maintanence
If failure to partition occurs, plasmid can not replicate in both daughter cells as one is at its plasmid limit while the other has no plasmids to replicate
Steps of replication (Theta Replication)
- Rep protein open up oriV
- 3’OH available
- DNA polymerase binds to 3’OH
- DNA polymerase replicates DNA
- Concatenated plasmids resolve
- 2 plasmids are partitioned to opposite ends of the cell
Plasmid Copy
Number of plasmids in a given bacterial cell
Relaxed plasmids
Plasmids that maintain a high copy number
Stringent plasmid
Plasmids that maintain a low copy number
What relaxed plasmids are regulated by
-Antisense RNA that affects a replication step
-Antisense RNA that inhibits an essential RNA or protein
-A protein that binds to certain DNA sequences
Plasmid Compatability
Idea that plasmids might benefit the host bacterium, but they guard against additional, new plasmids that might “Try” to take their place.
Can multiple plasmids coexist in a host bacterium? Can they be compatible
Yes, if they both can replicate and partition to the two daughter cells. Meaning, they are in different compatibility groups.
Plasmids in the same incompatability group
2 different plasmids can’t have the same replication control mechanism, therefore, they will be unable to live in the same cell.
How is replication of plasmid R1 regulated
Rep protein, it recognizes sequences in oriV and helps separate/unwind dsDNA. Different plasmids have their own Rep protein
RepA gene
Gene for rep protein common in low-copy-number plasmids. Can be transcribed from promoters PcopB and PrepA
CopB
Protein that represses PrepA promoter, therefore, repA protein numbers lower in order to lower plasmid replication. In high plasmid concentrations, more CopB is made to keep a negative feedback loop
CopA-RNA
Antisense RNA made by copA gene that binds to the copB-repA transcript to destabilize it in order to regulate RepA production.
RepZ
Rep protein in plasmid Col1b-P9.
-Controls the oriV
-RepZ is translationally coupled to a leader uORF (RepY), but when RepY is translated a pseudoknot forms to keep the ribosome binding site open for RepZ
Antisense ribosome accumulation impact on RepZ
-Antisense RNA zippers up the Inc region, preventing the pseudoknot.
-This keeps RepZ site blocked.
-This is an antisense RNA-sensing riboswitch to control translation
