Jullianes Flashcards

1
Q

What enzyme is required with no origin of replication?

A

RecA in bacteria or homologue
RadA in archea
Rad51 eukaryotes

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

Where does the rekaxosome nick?

A

the OriT

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What tra gene encodes for the relaxase?

A

traI

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Who discovered the transposons?

A

1940s: Barbra McClintock: discovered mobile genetic elements

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

What are the foure main affects that are benifical ffrom transposons?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

What are the five steps of a suicide vector?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Where do you get the transposons?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

Talk to me about transposon phage mu.

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

What has Kiljunen used transposon Mu for?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

What TIS Transposon insertion sequences are there?

A

Tn-Seq
Tra-Dis
Inseq
HITS

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

What is TraDis and what did Yasir et al. 2020 use them for?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

What are the stregths of transposons?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

What are the limitations of the transposons?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

What is the first stage of the lambda red?

A

Make a vector gene with the target gene of choice that contained antibiotic resistance

50nt of Gne X
20nt of antibiotic resistance

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

What are the four was to introduce the lambda machinery?

A

already in E. coli genome

mini lambda non replicating phage DNA

plasmid

functional phage

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

How do you get the lambda and gene of choice into the bug

A

electroportaiton

17
Q

What are the benifits of culturomics?

A

Biotechnologies

Species interaction

Viable Cells

New Antibiotics

Co-culturing

Natural environment (Enhanced Culturing)

Diffusion Chambers

Anaerobic chambers

50% of enzymes produced by bugs: 99% of the industrial citric acid is produced by aspergillus

Measurable phenotypes

100 species persample

Detects minority samples

18
Q

What are the draw backs of culturomics?

A

Need to know all the requirements for growth

Needs to be a species at a time (time consuming and expensive)

Lack of funding do to the cost

Co-cultures need to be purified

19
Q

What are the benificts of Metagenomics?

A

Biotechnologies

High throughput: Large amount of samples in a short space of time

Improved knowledge of microbial ecosystems

Integrative workflows

Determination of different taxa

Functional analysis

200 species per sample

20
Q

What are the negatives of Metagenomics?

A

Only if you can express the gene in a bug

Difficult to get species specific information

Might not be viable (Dead)

Extraction bias: based on the extraction protocol

Primer bias

Bioinformatic bias

Depth bias: smaller communities tend to be ignored

21
Q

What are the 6 steps of culturmomics?

A

Step 1

Divide the samples diversity the samples into different culture media

Step 2

Multiplication and optimisation of culture conditions

Step 3

Maldi-tof Mass Spec

Identification

Step 4

16S & 18S Sequencing

90.65% Similarity

Step 5

New Species Announcement

Step 6

Increase of knowledge

22
Q

What is the great plate anomoly?

A

•Initially detected with a microscope•Thought only 3% of bugs can be cultivated•Advent of metagenomics known there are far less cultivated then originally thought, closer to 0.1%.•Microbial Dark Matter: now called viable but not culturable

23
Q

What is cultureomics?

A

•Multiple culture conditions combined with the rapid identification of the microorganisms•Maldi-tof: cost affective and Time affective16S & 18S: Time and Cost high

24
Q

What is an environmental culture?

A

•Mimic natural environment•Candidatus pelagibacter ubique 2002•Cultured in a sea water derivativeMore then ¼ of the reads of metagenomics

25
Q

What is a co-culture?

A

•Growth promoting factors from one bug for the other bug•Kill co-bug not required/used concentrated media co-bug was inProchlorococcus requires a co-bug: the co-bug reduces oxidative stress

26
Q

What is an ichip?

A

Two membranes allows the diffusion of nutrients but keep the microorganisms pure and isolated.•Increase of cultured cells increased up to ~300fold. (Study dependant)•Axenic culture.•Serial dilution of samples in molten agar, load chip, and put in the desired environment.•2-4 hours typically•384 (1mm) diffusion chambers•Modified I-chip purpose built for use (smol or to be used in tandem with a Maldi-tof)•Domestication: Microorganisms unable to grow in the lab that grow in an ichip, can then go onto be grown in a lab, some take multiple round in an ichip for growth and others still can not grow in a lab.

27
Q

What is the RNA called in CRISPR-CAS9

A

guid RNA

28
Q

The car complex is called?

A

Unwinds array DNA, creating a tracr RNA and spacer DNA Interference complex