Jullianes Flashcards
What enzyme is required with no origin of replication?
RecA in bacteria or homologue
RadA in archea
Rad51 eukaryotes
Where does the rekaxosome nick?
the OriT
What tra gene encodes for the relaxase?
traI
Who discovered the transposons?
1940s: Barbra McClintock: discovered mobile genetic elements
What are the foure main affects that are benifical ffrom transposons?
Transposable Elements produce a variety of important effects:
One
They can insert within a gene to cause a mutation or stimulate DNA rearrangement, leading to deletions of genetic material.
Two
Because some transposons carry stop codons or termination sequences, when transposed into genes they may block translation or transcription, respectively.
Three
Other transposons carry promoters and can activate genes near the point of insertion.
Thus, some transposons can alter gene function and turn genes on or off.
Four
They contribute to the evolution of an organism’s chromosome, plasmids and other mobile genetic elements.
What are the five steps of a suicide vector?
One: Load vector with antibiotic resistance
Two: Put vector in new bug
Three: Placed in antibiotic selection: forces a homologues recombination event
Four: Bug take up vector into DNA or Does not
Five: Living bugs PCR check if worked
Where do you get the transposons?
Commercially available:
EZ-Tn5™: can go into plasmids, cosmids, and fosmid.
Custom made:
could have fluorescent tags
Designed primers
Contain thousand of transposons to ensure saturation of the genome
Talk to me about transposon phage mu.
Transposon phage mu: using bacteria phage to use the transposons
Based on the transposition machinery of the bacteriophage Mu
During the lytic phase of its life cycle, bacteriophage Mu replicates its genome by transposing repeatedly inside the host genome
The Mu transposition reaction has been modified into an in vitro reaction catalyzed by a single enzyme - MuA Transposase.
In this system, one in vitro reaction is capable of generating more than a million transposon insertion clones
What has Kiljunen used transposon Mu for?
Shuttle Transposon: uses hosts genes to incorporate genetic elements
Insertion Mutant Library: gene discovery
Isolated pigmentation-defective and auxotrophic mutants, and the respective insertions pinpointed a number of genes previously known to be involved in carotenoid and amino acid biosynthesis pathways.
Further studied by other individual found: motility and adhesion mutants, and the molecular factors involved in hypochlorite tolerance.
What TIS Transposon insertion sequences are there?
Tn-Seq
Tra-Dis
Inseq
HITS
What is TraDis and what did Yasir et al. 2020 use them for?
Transposon-directed insertion sequencing (TraDIS)
Involves inserting transposons into the genome to generate large numbers of mutants
Combination of transposon mutagenesis and high-throughput sequencing
Does not require picking thousands of colonies individually
Yasir et al. (2020): TraDIS-express
Incorporate a promoter in the transposon
If this is located upstream of a gene, then it can promote essential genes that are typically unable to be studies with transposons, the gene is up regulated
If it incorporates down stream, it can transcribe the anti-sense RNA and it will down regulate translation
What are the stregths of transposons?
General Strengths:
Near random:
Salama and Manoil (2006) 30 bacterial species: >170 different nutrient conditions: 300-600 genes per bacteria discovered and essential for viability
Single
Knock-out: if multiple you know nothing
Knockout are mostly stable: rarely can not take
Genetically encoded: easy to do sequencing
I
mprovement from chemical mutagenesis
TIS:
High Through put: pooled transposon libraries many samples (TraDIS)
Sensitive: detects smol changes in fitness
Precise: do promoter regions and all the SMOL things
What are the limitations of the transposons?
Low Frequency of transposition in living systems:
MuA does not insert transposons with uniform efficiency across different DNA sequences
Inaccuracy of transposition systems:
Transposon can create mutations that then created secondary unexpected unknown mutations which are not related to the original transposition
Preferential Insertion: TE although random, do have preferential insertion sites which can create bias
Piggy Bacs: have a preference to the transcription start site.
Partial loss of function:
P element: found in eukaryotes, routinely used as genetic tools to mutate genes, insert in the regulatory regions, typically within 200 nucleotides of the transcription start site.
Easily testable phenotype: required to now the stuff
Gene interruption: can not study essential gene, caviet tradis make beet good time oooh
What is the first stage of the lambda red?
Make a vector gene with the target gene of choice that contained antibiotic resistance
50nt of Gne X
20nt of antibiotic resistance
What are the four was to introduce the lambda machinery?
already in E. coli genome
mini lambda non replicating phage DNA
plasmid
functional phage