Team:KU Leuven/Parts

Favourite parts

1. BBa_K1060002 contains an open reading frame that codes for a sesquiterpene synthase (GenBank Accession No. AY835398), (E)-b-farnesene synthase, that has been isolated from Artemisia annua (Picaud et al., 2005). The enzyme converts farnesyl diphosphate into E-β-farnesene. E-β-farnesene is an alarm hormone produced by aphids. It is also an important semiochemical in aphid localization. It is released in the cornicle secretions of many aphid species (Franscis et al., 2005) to alert surrounding aphids of the presence of natural enemies (Kunert et al., 2005). Detection of these short range chemical cues leads not to the aphid directly, but only indicates the presence, improving prey detection of the predators and parasitoids.
   Group favourite: it was a milestone in our project work. We succeeded to clone the beta farnesene synthase of Artemisia annua in E. coli. To apply to the iGEM standards, we removed an EcoRI site in the gene (AY835398.1).This gave us one of the basic parts we needed to create our system.


2. BBa_K1060009 is a construct that constitutively expresses beta farnesene synthase. It is BBa_K1060008 behind a medium (constitutive) promoter and medium RBS (BBa_K608006).
   Group favourite: This generator will convert farnesyl pyrophosphate into the aphid alarm hormone, (E)-β-farnesene. This was the final device used for our Aphid experiments.


3. BBa_K1060011 is similar to BBa_K1060009. However, in this biobrick we added a lac operator in front of the beta farnesene synthase. This makes it possible to switch of (E)- β-farnesene production by using biosensors expressing LacI.
   Group favourite: after the design of BBa_K1060009 this is the next step in the design of our glucose model.

Other parts

1. BBa_K1060000 contains the aroG gene, coding for 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and is a part of the Shikimate pathway. It converts phosphoenolpyruvate and erythrose-4P to chorismate. In our project chorismate was one of the precursors needed for the biosynthesis of methyl salicylate (see also BBa_K1060003). As basic chorismate levels inside E. coli are limited. We wanted to upregulate the biosynthesis of chorismate. The basic aroG gene can be repressed by phenylalanine. However, mutatations in the amino acid sequence (Pro150Leu and Leu175Asp) can prevent this repression by phenylalanine.


2. BBa_K1060001 contains EBF synthase from Streptomyces coelicolor. In this organism the enzyme albaflavenone synthase (GenBank: AL939123.1) also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate (Zhao et al., 2009). It results in the production of (E)-beta-farnesene (61%).


3. BBa_K1060003 is a generator for Methyl salicylate production. We requested the BBa_J45700 brick from MIT 2006 and recloned into the pSB1C3 standard plasmid backbone. For the assembly of this part we refer to the original MIT brick BBa_J45700 => "We assembled a complete wintergreen odor biosynthetic system (BBa_J45700) by combining the salicylic acid (BBa_J45320) and wintergreen odor generators (BBa_J45120). BBa_J45700 catalyzes the conversion of the cellular metabolite chorismate to methyl salicylate or wintergreen odor."


4. BBa_K1060004 is a construction intermediate. It contains the BSMT1 enzyme (BBa_J45120) for the production of methyl salicylate followed by a double terminator (BBa_B0015).


5. BBa_K1060005 is a construction intermediate. It contains the pchBA enzyme (BBa_J45320) for the production of BBa_B0015).


6. BBa_K1060006 is an intermediate construct existing of the gene BBa_K082003 which codes for GFP (+degradation LVA tag), followed by the double terminator BBa_B0015. This is a part of our feed-forward-loop.


7. BBa_K1060007 is an intermediate construct existing of the cinI BBa_C0076 gene followed by the double terminator BBa_B0015. cinI codes for an autoinducer synthetase and is derived from Rizobium etli. The quorum sensing molecule produced by this autoinducer synthetase is 3OH,C14:1-HSL. This construct was made to be incorporated in an oscillator, that can be used for the alternating production of two different components. A quorum sensing molecule is necessary to get a colony-wide synchronization of oscillation. More information can be found on the oscillator page.


8. BBa_K1060008 is a construction intermediate in contains EBF synthase (BBa_K1060002) followed by the double terminator BBa_B0015.

Note: BBa_K1060010, BBa_K1060012 and BBa_K1060013 are still under construction.