Transposons

Question 1

Genetic libraries

Answer 1

Using trnasposase gene, fuse to strong promoter, we can increase transformation frequency.

Question 2

Mini transposon

Answer 2

They are stabilized once the transposase is excised from the genetic strand.

Question 3

Synthesis of mini transposon

Answer 3

insert origin or replication, use restriction enzymes to cut before the IR. Recover plasmid and use sequencing technique.

Question 4

Promoterless reporter gene

Answer 4

Gene is transcribed to produce mRNA which is translated by ribosome to produce polypeptide sequence.
The promoterless reporter gene disrupts and fuses witht he gene strand. since there is no promoter on the reporter gene, the gene controls the transcription.

Question 5

use of reporter genes to construct transcriptional fusions

Answer 5

Reporter gene mini transposons are ideal for identifying a set of genes belonging to a common regulon.

Construct library of mutants > damage DNA > isolation of mutant expressing reporter gene > clone and sequence

Question 6

Regulons

Answer 6

The way bacteria organise gene expression, in which by operon or dispersed genes which have their own promoter.

SOS regulon:

• Damaged DNA (UV)
• RecA protein can recognise damaged DNA, can form complex with single stranded DNA (damaged)
• Activates RecA protein, which cleaves a specific protein called LexA
• This results in various genes getting turned on.

There are 3 responses:
1)Excision repair (via uvrA uvrB)
Excise oligo nucleotide containing damage

2)Recombinantial repair (recA)
repair gap after damage

3)Error-prone repair (umuC umuD)
complex with DNA Pol3, which forces replication during damage