The aim of the following construct is to synthesize polyhydroxyalkanoates from available carbon sources.
The genes which were considered for the design were the ones that involved PHA production starting at 3-ketoacyl-ACP, which is in Pseudomonas putida KT2440 and in most E.coli strains. The first gene in the construct is fabG that turns 3-ketoacyl-ACP into (S)-3-hydroxyacyl-ACP, and phaG uses it to produce (R)-3-hydroxyacyl-CoA, the precursor for PHA. The following genes are phaC1 and phaC2, which are both synthases for polyhydroxyalkanoates. All the sequences have been optimized for their use in E.coli BL21 and restriction sites have been removed.
T7 Promoter BBa_I712074
To use this promoter we have to transform an E.coli BL21
This promoter is repressed during the exponential phase of the growth curve. It can be activated with IPTG (isopropyl-beta-d-thiogalactopyranoside) a synthetic sugar that works as an inductor.
The main function of this biobricks is to regulate and optimize carbon usage necessary for PHA synthesis.
The ACC gene encodes for Acetyl-CoA Carboxylase, which has been shown to increase the synthesis of fatty acids, and since this pathway will be altered to include PHA synthesis it will also increase PHA production.But an overexpression of ACC leads to a lack of carbon necessary for the Krebs cycle. In order to avoid this problem ACC is controlled by the pBad promoter, allowing for a normal metabolism in the absence of arabinose.The iLOV protein will be used as a reporter for the expression of ACC.