Part:BBa_K653000

Re-designing and re-assembling of the rhamnosyltransferase BioBrick part: "The Biosurfactator"

This year`s BioBrick construction effort will be due to a re-designing and re-assembling of our last year surfactant BioBrick project and improving with a new concept in which we degrade hydrocarbons as well as using the surfactant BioBrick, that we will describe next.

Pseudomonas aeruginosa species of bacteria produce the natural rhamnosyltransferase gene complex (RhlAB). This is the key enzyme responsible for transferring the rhamnose moiety to the b-hydroxyalkanoic acid moiety to biosynthesize rhamnolipid, which is a biomolecule with surfactant properties, but the natural RhlAB gene has illegal restriction sites that make it incompatible with the Assembly Standard Protocol 10, specifically the Pstl1 restriction sites.

The design and construction of the rhamnosyltransferase gene into a BioBrick part was the main goal and achievement of the iGEM Panama Team 2010, but since the beginning of this year’s iGEM project we failed in producing the rhamnolipid compound into our E. coli based-factory, because our last year’s BioBrick appeared to have been denaturalized. We then ordered our BioBrick part BBa_K424018 (iGEM Panama team 2010) from The Registry and once it arrived we ran some tests and we noticed that the part was no longer fitted into the plasmid backbone.

As scientist and iGEMers, we have the responsibility to deliver a functional BioBrick, thus according to iGEM’s competition new slogan, "Quality not Quantity", we have embraced the challenge of re-designing and re-assembling the same BioBrick part from last year in order to comply this new standard based on quality. So we bring the Biosurfactator!!!

Re-designing "The Biosurfactator" The first Central American BioBrick

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 103
  Illegal BamHI site found at 663
  Illegal XhoI site found at 839
  Illegal XhoI site found at 2125
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1018
  Illegal NgoMIV site found at 1739
  Illegal NgoMIV site found at 1852
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 328
  Illegal BsaI site found at 1368
  Illegal BsaI.rc site found at 512
  

How to use it as a BioBrick? :

We developed this standarized part to be inserted into E. coli for rhamnolipid production through the mutation of the gene that is responsible to synthesize the key enzyme rhamnosyltranferase (RhlAB) to be compatible with Assembly Standard 10. We have achieved this by performing several silent mutations using the QuikLighting Multi Site-Directed Mutagenesis Kit from Stratagene. This rhamnosyltransferase BioBrick (Rh1AB_BB) is ready to be tested on an expression plasmid device following the Assembly Standard Protocol 10.

Application of The Biosurfactor :

ITB_Indonesia 2015 team used this coding sequence to make BBa_K1685011 part for rhamnolipid production device under T7lac promoter. The supernatant of IPTG-induced E. coli BL21(DE3) transformant showed surfactant activity comparable to Tween 20%.