Transcript Transposable Mutagenesis for an E. coli Methionine

Transposable Mutagenesis for an E.
coli Methionine or Histidine Synthesis
Gene
Jon Kissel
Mary Maschek
Transposon mutagenesis
Mutating prototrophic bacteria to become
auxotrophic using a transposon
Making plasmid library from bacterial
genome
Sequencing plasmid with interrupted gene
Transposons
Mobile DNA elements
Randomly insert into genome
Can contain resistance genes
Types of transposons
Size
Number of genes
Bacteriophage Mu
Lysogenically inserts into bacterial
genome
Inserts at multiple locations
Can recombine out of genome and form a
plasmid library
Plasmid Libraries
Contain bacterial genome
Individual genes are located on each
plasmid
Can be used to transform wild-type
bacteria
Inserting the Transposon
EZ::TN™ <R6Kγori/KAN-2>Tnp
Transposome™ Kit
Electroporation
EZ::TN™ <R6Kγori/KAN-2>Tnp
Transposon
Epicentre EZ::TN™ <R6Kγori/KAN-2>Tnp
Transposome™ Kit protocol
Finding the Auxotroph
Plate cells on minimal medium + met/his
Replica plate on minimal medium
Select for colonies that don’t grow
Making the Library
DNA purification
Restriction digest
End-It DNA Repair Kit
DNA ligation
Finding Resistance
Mix library with wild-type E. coli
Plate on kanamycin
Select for kanamycin-resistance
Plasmid Purification
Good ole Biology 107
Run on gel just to be sure
Sequencing and BLAST
Send purified plasmid to IUPUI
Primers come with transposome kit that
tell sequencers where to begin
BLAST returned sequence
Epicentre EZ::TN™ <R6Kγori/KAN-2>Tnp Transposome™ Kit
Timeline
September 13-20 – make plates, LB broth
September 20-27 – begin work with transposome and
obtain auxotroph
September 27-October 15 – ligate genomic DNA,
plasmid library ready
October 15-22 – isolate kanamycin-resistant colony
October 22-29 – isolate and purify plasmid
October 29-November 5 – enter sequence into BLAST
November 5 to December 8 – work on manuscript
Budget
EZ::TN™ <R6Kγori/KAN-2>Tnp
Transposome™ Kit
$375?
End-It DNA Repair kit
$70
Sequencing at IUPUI
$17
Grade Agreement
Obtaining auxotroph
15 points
Purifying DNA
5 points
Digesting DNA
5 points
Assembly of plasmid library
10 points
Obtaining kanamycin-resistant colony
10 points
BLAST results give methionine or histidine synthesis
gene
5 points