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25 Cards in this Set

  • Front
  • Back
Transformation (Review)
* acquisition of naked DNA by bacteria

* DNAse sensitive: treating with DNAse results in loss of genetic exchange

* transformation with chromosomal markers is recA-dependent
Conjugation (Review)
* transfer of plasmid DNA (typical)

* contact-dependent: pili attach to other bacterium, membranes fuse, pieces of DNA transfer over


* DNAse insensitive
Transduction (Review)
* transfer of DNA by phage

* unlike transformation, is filterable: phage can go through filter, but not bacteria

* recA-dependent because deals with double cross-overs
Function(s) of Transposons
* discrete elements of DNA that can jump/hop/excise/insert from one replicon to another

* generate polar mutations - but cannot afford to hurt their host

* encode transpoase

* often confer antibiotic resistance b/c mobile genetic elements
* plasmid

* bacteriophage

* chromosome
General Properties of Transposons
* recA-independent

* ubiquitous in nature: even have them in our normal flora

* frequency of hopping regulated by transpoase
- 10^(-3) to 10^(-7)

* have inverted terminal ends that are cleaved b/c recognizable

* minimal target sequence specificity

* encode txnal terminators at both ends to prevent host cell txn coming in from either end
* a recombinase that regulates the frequency of transposition

* contains its own promoter and repressor

* regulated
Insertion Sequences
* discrete DNA that exists in the chromosome

* lacks any known selectable marker
Simplest Way to Form Transposons
* flank an antibiotic resistance gene between two insertion sequences
Conservative Transposition
* copy number does not increase upon transposition

* no origin of rep'n: can't exist autonomously

Ex. A transposon from one chrm cuts and pastes into another copy of chrm. One chrm has two copies of the transposon and the other has none.
Replicative Transposon
* transposon is replicated after transposition = two copies of transposon produced
- transposon contains origin of rep'n: can exist autonomously

* always results in increase in copy number
Conjugative Transposon
* transposon encodes Tra-like functions (w/o replicons)

* insert into chrm, excises, encodes for a pilus, transfers to another bacterium, and re-inserts
Detecting Pathogenicity Islands
* set of genes not common amongst all bacteria

* presence of known/suspected virulence genes
- virulence genes genetically linked

* different G+C content than rest of genome

* islands usually absent in non-pathogenic bacteria

* inserted into tRNA or duplicated genes

* assciated with mobile DNA elements: IS sequences, transposase, phage integrase
Transposon Mutagenesis
* method of using wide host range plasmids to introduce transposons in wide variety of bacteria

* once introduced into haploid genome of bacteria, the transposon inserts somewhat randomly:
- insertion & null muations, KOs

* the transposon contains a molecular tag used to isolate and characterize the mutated gene and surrounding genes

* generates polar mutations: if inserted into an operon, will affect expression of downstream genes
Transposon Libraries
* collection of bacteria that contains multiple individuals, each with a single transposon insertion

* can consist of a population of many mutants or individual clones in microtiter wells
Making a Transposon Library via Suicide Vectors
* Donor has Tn9 (chloramphenicol resistance) and plasmid that encodes ampicillin resistance

* Recipient has plasmid that encodes nalidixic acid resistance

* want the chrm of the recipient to include Tn9 = plasmid must suicide

1) raise the temperature
2) select for chloramphenicol
3) score for ampicillin sensitivity

* Result: Cm resistant, Nal resistant, amp sensitive bacterium with Tn9 insertion

* plate to produce multiple mutants with Tn9 insertion
Other Ways to Make Suicide Vectors
* use a bacteriophage that can't replicate, can only deliver

* transfer plasmid lacking essential rep'n protein from donor cell:

- delete a plasmid's rep'n fxn and place rep'n genes in chrm of donor bacteria
- plasmid can only replicate in donor (rep'n fxn only acts in trans)
* easily measurable marker of protein (and gene) expression

* usually an enzyme

Ex. lacZ (encodes β-gal), GFP
Transcriptional Fusion
* the reporter relies on the promoter of the target gene to become expressed

* contain their own ribosome binding site and star codon

* no fusion protein produced

Ex. LacZ reporter expresses β-gal under the transcriptional control of another gene
Translational Fusion
* the reporter gene is fused in frame with another protein

* promoter, ribosome binding site, start codon, and some or all of the coding sequece are in-frame
* consists of Tn5 and PhoA (a gene that encodes alkaline phosphatase - no signal sequence)

* generate translational fusions that allow the investigator to identify genes that encode secreted proteins

- many pathogenicity genes are secreted

- fusion ocurs when TnPhoA inserts into a gene in the appropriate orientation and correct reading frame
Activation of PhoA
* alkaline phosphatase is inactivated unless exported outside the cytoplasmic membrane to the periplasm

* cytosol is heavily reducing: cannot form disulfide bonds

* periplasm contains enzymes that catalyze disulfide bond formations (oxidizing environment)
Monitoring Presence of Secreted Fusion
* include a substrate for alkaline phosphatase in the agar

* upon hydrolysis, colony will be blue

- inactive PhoA = white colony
- active PhoA = blue colony
Blue Colonies
bacteria that contain a functional fusion of TnPhoA with a protein that is expressed, transcribed, and transported
What percentage of random TnPhoA should be blue?
(chance of getting an in-frame insertion in the right orientation) * (% of genes secreted)

Ex. (1/6) * 10% = (1/6) * 0.1