Parts

Summary

  The Pollumunch project utilizes 10 Basic Parts and 2 Composite Parts for the remediation of polycyclic aromatic hydrocarbon (PAH) pollution in soil and aquatic environments. All components comply with the RFC[10] or RFC[1000] standards and have undergone functional testing, making them available for community use and further development. Access is provided through a registration system.

Basic Part

Number Short Description Long Description
1 BBa_25CTZG9Q phnR A gene that can express the presumed regulatory protein which can recognize PHE and form compounds to drive the phnS promoter to express the downstream genes.
2 BBa_258EKT2X phnS A promoter derived from Pseudomonas putida and others, regulating the expression of the downstream phn gene cluster. In our design, it forms the PHE sensing element together with phnR and a short upstream regulatory sequence.
3 BBa_259NHHZB nahC The coding sequence for 1,2-dihydroxynaphthalene dioxygenase, an enzyme that catalyzes the oxidation of 1,2-dihydroxynaphthalene to 2-hydroxy-2H-chromene-2-carboxylic acid—a rate-limiting step in the nah pathway metabolism of naphthalene.
4 BBa_25S0JKP8 nahABFEDGC A sequence encoding enzyme systems from PAH-degrading bacteria capable of metabolizing naphthalene, including coding sequences for naphthalene dioxygenase, cis-dihydrodiol dehydrogenase, 1,2-dihydroxynaphthalene dioxygenase, and a series of other enzymes, which can convert naphthalene to salicylic acid through a series of oxidation metabolic steps.
5 BBa_2529H49P phnC A gene derived from the phn gene cluster of an obligate phenanthrene-degrading bacterium, encoding 1,2-dihydroxyphenanthrene dioxygenase. In our project, we attempted to introduce this gene into the primary naphthalene-degrading nah gene cluster to bypass the rate-limiting step in phenanthrene degradation, thereby enabling the nah gene cluster to simultaneously degrade phenanthrene.
6 BBa_259AOPHA nahABFEDG-phnC A segment responsible for converting naphthalene to salicylic acid (nahABFEDGC) was extracted from the naphthalene-degrading gene cluster (nah gene cluster). In our project, nahC was replaced with phnC from the phn gene cluster. This engineered genetic part was designed to enable phenanthrene to be initially degraded into a biphenyl-ring intermediate via the phenanthrene metabolic pathway, followed by further degradation to salicylic acid through the naphthalene metabolic pathway.
7 BBa_K5363011 EGFP EGFP is a widely used reporter protein that emits bright green light for monitoring cellular processes.We used it as a reporter element downstream of phnR-P-phnS.
8 BBa_25QO7C82 phnR-P-phnS This part consists of the regulatory protein-coding gene phnR, the PhnR binding sequence, and the promoter phnS. It can sense the presence of phenanthrene and output green fluorescence, and was used in our project to validate the function of phnS-P-phnR.
9 BBa_K5291038 T7 The T7 promoter is a strong, sequence-specific bacteriophage promoter commonly used to drive high-level gene expression.The constructs we developed, pET28a(+)-rhlAB and pET30a(+)-nahABFEDG-phnC, utilize this promoter to facilitate IPTG-induced expression of downstream genes after transformation into the expression host BL21(DE3).
10 BBa_25W0IRQ2 rhlAB The key genes for rhamnolipid biosynthesis in Pseudomonas aeruginosa are rhlA, which encodes an acyltransferase, and rhlB, which encodes a dTDP-glucuronic acid decarboxylase.

Composite Part

Number Short Description Long Description
1 BBa_25ULB7WQ pET30a(+)-phnR-P-phnS-EGFP We used this plasmid to validate the function of phnR-P-phnS by applying the inducer salicylic acid and detecting the green fluorescence from the reporter gene EGFP.
2 BBa_25KJBYVH pET30a(+)-nahABFEDG-phnC We replaced nahC in the nahABFEDGC cluster with phnC, enabling the nah gene cluster to degrade phenanthrene (PHE). During the experimental phase, we employed the T7 promoter to induce gene expression with IPTG.
3 BBa_25Y79J0P phnR-P-phnS-EGFP This part is designed to detect PHE concentration. The two regulatory proteins encoded by phnR and phnS act together on the operator region upstream of phnS. The phnR gene operates constitutively, and upon detecting SA (a metabolic intermediate of PHE), it activates the expression of phnS and its downstream genes. This part can even quantitatively measure PHE concentration and produce graded output signals, although this particular feature was not utilized in our experiments.