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Team Tango Alpha
mreB Project Description
The goal of our project is to clone the gene, mreB, from Caulobacter crescentus (stalked shaped cell) into Escherchia coli (rod shaped cell) to see if mreB will affect E. coli's cell shape in some way. We expect to see the E. coli cells lyse or change from their normal rod shape.
MreB is a rod-shape determining gene that appears as bands or spirals encircling the cell. It is known to associate with mreC and penicillin-binding proteins to catalyze precursors for the peptidoglycan cell wall and correctly position polar bacterial proteins. It is also homologous of actin in eukaryotes.
Accession # NC_011916 1,044 base pairs No introns
Forward primer: the first 34 bases of our gene sequence. We also added in the Biobrick enzymes EcoR1 and Xba1 in front of the primer.
Reverse primer: the last 20 bases of our gene sequence. We also added in the Biobrick enzymes Pst1 and Spe1 in front of the primer.
This promoter is inducible by L-arabinose. When induced, AraC binds and changes the conformation interacting with Aral1 and Aral2 operator sites, permitting transcription.
Biobrick part containing ampicillin marker gene.
Procedure 1. Grow C. crescentus and E. coli on the respective medias 2. PCR to extract mreB gene + Biobrick (BB) enzymes 3. Isolate and purify mreB + BB enzymes 4. Cut mreB + BB enzymes and ligate with pSB1A3 5. Perform bacterial transformation to import the plasmid into E. coli 6. Grow E. coli bacteria on experimental plates (No ampicillin, ampicillin, and ampicillin + L-arabinose) 7. Verification testing by examining the physical cell shapes under a light microscope and sequence verification by sending a sample of cloned DNA to Iowa State.
Important Results and Milestones