Team:BGU ISRAEL/Basic Part
Basic Part
Protocatechuate Degradation Pathway
Pseudomonas putida is a bacterium capable of utilizing protocatechuate, a toxic molecule for most bacteria. The protocatechuate degradation pathway converts protocatechuate into 3-oxoadipate, which is further metabolized by the bacteria and enters the TCA cycle.
We chose to submit the genes encoding the enzymes in this pathway.
We also characterized the utilization of protocatechuate by bacterium using M9 minimal medium with protocatechuate as a sole carbon source.
We have not been able to submit the pcaB gene, encoding the 3-Carboxymuconate Cycloisomerase, since the sequence contained 4 PstI restriction cut sites, which we did not have time to alter using point mutations.
| Part Name | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K2091000 | Coding | Protocatechuate 3,4-dioxygenase Alpha Subunit | B. Vaknin | 606 |
| BBa_K2091001 | Coding | Protocatechuate 3,4-dioxygenase Beta Subunit | B. Vaknin | 720 |
| BBa_K2091002 | Coding | 4-Carboxymuconolactone Decarboxylase | B. Vaknin | 393 |
| BBa_K2091003 | Coding | 3-Oxoadipate enol-lactonase | B. Vaknin | 792 |
LC-Cutinase Protein Variants
LC-cutinase is an enzyme found to have the ability to degrade PET. We have chosen to improve this enzyme using rational mutagenesis. 4 mutant variants of the LC-cutinase protein were produced using the PROSS algorithm and one Codon-optimized version of the protein was designed using the Genome Compiler software.
We have tested and characterized these proteins using kinetic trials, bacterial growth assays and scanning electron microscopy imaging.
We have cloned and submitted all 5 of our LC-cutinase variants:
| Part Name | Type | Description | Designer | Length |
|---|---|---|---|---|
| BBa_K2091004 | Coding | LC-Cutinase Codon Optimized | I. Bariah, E. Zajfman, I. Segal | 948 |
| BBa_K2091005 | Coding | LC-Cutinase Variant F4 | I. Bariah, E. Zajfman, I. Segal | 948 |
| BBa_K2091006 | Coding | LC-Cutinase Variant F7 | I. Bariah, E. Zajfman, I. Segal | 948 |
| BBa_K2091007 | Coding | LC-Cutinase Variant R4 | I. Bariah, E. Zajfman, I. Segal | 948 |
| BBa_K2091008 | Coding | LC-Cutinase Variant R7 | I. Bariah, E. Zajfman, I. Segal | 948 |
