America´s Regional iGEM Jamboree Indiannapolis, Indiana

America´s Regional iGEM Jamboree
Indiannapolis, Indiana
Usually the problems are ahead the solutions
Doing Synthetic Biology to bring biological solutions to
everyday problems!
Biosurfactator = rhamnolipids
Rhamnolipid is composed of rhamnose
sugar and b-hydroxyalkanoic acid.
This marvelous molecule:
• Reduce the surface tension of water.
• Have emulsifying properties
• Is a well known Biosurfactant
We found a marvelous gene the
Rhamnosyltransferase complex 1
(RhlAB)
rhlAB: Genetic engineering facts!
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There are reports of several attempts to produce Pseudomona´s Rhamnolipids in
heterologous hosts, like: Pseudomonas fluorescens, Pseudomonas oleovorans,
Pseudomonas putida, and E. coli
Yet, none produces Rhamnolipids in comparable levels as the best P. aeruginosa strains
Breif history: expression of rhlAB gene into E.coli
Ochsner et al. (1995)
Cabrera-Valladares et al. (2006)
Wang et al. (2007)
iGEM Panama Team (2011) intended the RL production in E. coli through synthetic
biology based on BioBrick parts integration. For first time ever!
Our objective is to deliver rhlAB BioBrick part!
• Degradate DNA from 2010
• Panama 2010 BioBrick sample in the registry was invalid.
• Re-designing,
• re-constructing
• and re-assembling the rhlAB gene
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HIND III
2314 bp
2322 bp
6557 bp
4361 bp
pGEM-T + rh1AB
3000 bp + 2314bp
pGEM-T
3000 bp
The rhamnosyltransferase gene complex do not fit with the assembly standard
rh1B gene sequence shows two
illegal restriction sites by PstI
rh1A gene sequence shows
one illegal restriction sites
by PstI
Pst1 Digestion
pGEM-T plasmid
pGEM-T + partially mutated rh1AB
rh1AB with no mutations
• Succesfuly mutated the rhlAB gene
• Re-designed and re-constructed the mutated rhlAB.
• Re-assembled the mutated rhlAB into a BioBrick
part.
Assembly of expression platform for the rhlAB BioBrick
GFP reporter plus Double Terminator BBa
IPTG Inducible Expression Platform
E.Coli based-factory for rhamnolipid production
Acknowledgment
Advisors
Sponsors