TU Mobile genetic elements in bacteria 14/10

Question 1

Intergration of horizontal DNA into chromosome

Answer 1

1. RecA-mediated recombination: Involves the RecA protein, which facilitates the pairing of homologous sequences, allowing the foreign DNA to integrate into the host genome. (they bind to the DNA and put it in the genome)
2. RecA-independent recombination: Includes processes like illegitimate recombination and non-homologous end joining, where DNA is integrated without requiring sequence homology, relying instead on microhomologies.
3. Site-specific recombination: Utilizes enzymes such as integrases, recombinases, or transposases to insert DNA at specific genomic locations, enhancing integration efficiency.

Question 2

Plasmid replication

Answer 2

Theta replication formation of replication bubble + two replication forks moving in opposite directions. -> unwind and replicate the DNA into a multimere (two plasmids together, then they let loose of eachother, seperation), ultimately producing two identical circular DNA molecules.

Rolling circle replication begins with a single-strand break in plasmid. The free 3’ end of the broken strand is extended, displacing the other strand as it rolls out -> synthesis of multiple copies of the DNA, producing long concatamers that can later be processed into individual circular molecules. -> conjugation process uses this

Question 3

integrative & conjugative elements (ICEs)

Answer 3

are mobile genetic elements that can transfer between bacterial cells and integrate into the host’s chromosome. ICEs play a key role in horizontal gene transfer, often carrying genes for antibiotic resistance

Important it has two proteins, xis Excisonase is for excision so the mobile element can get out the chromosome and int for integrase -> integration

Question 4

Transposable elements

Answer 4

Different type of mobile element,

Transposons, also known as “jumping genes,” are mobile genetic elements that can move from one part of the genome to another.

1. Structure:
   • A transposon has two insertion sequences (IS) on either side. These sequences contain the genes for enzyme transposase
   • Between the insertion sequences is usually a gene cluster, which can contain any type of gene (often related to antibiotic resistance, for example).
2. How it Works:
   • Transposase, an enzyme encoded by the transposon, recognizes the inverted repeats at the ends of the insertion sequences.
   • This enzyme helps cut the transposon out of the DNA and insert it into a new location in the genome.

They are classified into two types:
* Simple (Non-composite) Transposons: These consist of a transposase gene flanked by inverted repeat sequences, enabling them to cut and paste themselves within the DNA.
* Composite Transposons: These contain one or more additional genes (often antibiotic resistance genes) between two insertion sequences. They also use transposase for mobility but can integrate more complex functions into the genome

What can it do:
- inactivate a gene product or gene expression
- Activation of gene expression
It can also modulate a hoste gene by providing an alternative promoter to the target host gene

Question 5

How do transposons move?

Answer 5

Replicative Transposition: In this method, the transposon is copied, and the copy inserts itself into a new location in the genome while the original remains intact. This results in an increase in the number of transposons.

Cut-and-Paste Transposition: Here, the transposon is excised from its original location and inserted into a new site. This method does not increase the overall number of transposons in the genome.

Question 6

How to stury MGEs?

Answer 6

• metagenomic assesment (dry lab), is to find what are the mobile elements here that bring antibiotic resistance etc.

Plasmids TRACA: Shared DNA removed using DNase -> Insertion of transposon with origin of replication -> Transform into host strain with medium selective for transposon -> Next-generation sequencing

Transposable elements (PCR amplification): Amplified with PCR and purified -> Ligated into plasmid vector -> Transform into host strain with medium selective for transposon -> Next-generation sequencing

• Reporter construct (wet lab) to observe HGT (horizontal gene transfer), is when, howoften, how does the mobile element transfer from one cell to the other.
  Using fluorescence, conjugation rate of plasmid

don’t think you need to know this, probably just difference wet and dry lab

Question 7

Engineering biology with mobile genetic elements

Answer 7

Introducing gene of interest
Transformation: A method where a gene is introduced into a cell, typically using plasmids or other vectors, allowing the organism to express the new gene.

Transposition: The process by which transposable elements move within the genome, facilitating the integration of a gene of interest into various locations in the DNA.

Recombination: A genetic process where DNA segments exchange or rearrange, allowing the integration of the gene of interest into a specific genomic site through homologous recombination.

Question 8

Cells defend themselves against transposons by

Answer 8

silencing of transposons