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Transposon Mutagenesis of Anabaena PCC 7120 to Isolate Nitrogen Fixation Mutants

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Transposon Mutagenesis of Anabaena PCC 7120 to Isolate Nitrogen Fixation Mutants Powered By Docstoc
					Experiment 1

TRANSPOSON MUTAGENESIS OF

ANABAENA PCC 7120 TO ISOLATE NITROGEN
FIXATION MUTANTS

Transposons
•‘Jumping Genes’- hop from one piece of DNA to another. • Inactivate the genes they jump into.

•Types: Replicative and ‘Cutand-paste’ variety.
•We will be using the latter

Some Bacterial Transposons

   

Simplest are called ‘IS elements’ (Insertion Sequence). Contain inverted repeats and encode a ‘transposase’. Composite Transposons- right and left ends flanked by IS elements of the same type and carry genes in-between them. We will be using a Tn5 derivative- nearly random hopping ensures any gene can be mutated.

pRR106- TnRR1
•TnRR1 is carried by plasmid pRR106
•Derivative of Tn5 •Transposase •Resistance to Streptomycin and Spectinomycin •Origin of Transfer (OriT) •Origin of replication in E.coli (OriV)-maintenance

Today’s Lab: Transformation into HB101 (pRL528)

•pRR106 is introduced into HB101-it will be correctly methylated because pRL528 encodes Anabaena methylase.

•Otherwise, Anabaena will recognize the DNA as foreign and degrade it- protection against phages (viruses).

Transposition into the Anabaena genome

Genetic selection for Fix- Mutants using PCC 7120

Isolation of the Transposon from Mutant
MspI MspI MspI
Anabanea DNA Cut (MspI); ligate IPCR product: Run on gel, purify and sequence

Primer

Primer

Overview
      

Transform E.coli with plasmid containing transposon. Introduce pRR106 into Anabaena sp. PCC 7120 by conjugation with transformed E.coli strain. Transposon ‘hops’ into the Anabaena genome. Select for Anabaena ex-conjugants that have received the transposon using antibiotics. Negative selection for Fix- mutants by placing them in a media that lacks fixed nitrogen source. ‘Rescue’ dying colonies. Phenotype, isolate transposon from mutant, and identify site of insertion


				
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