Team:Stony Brook/Contribution
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Contribution
Our Contributions to the Registry
Our team added four new parts to the registry. While the full engineering design cycle was completed only for the PgsA-MlrA fusion composite part, the other parts represent the culmination of a lot of literature review. Also, we added new documentation to the usage and biology section of the JNU_China PgsA parts page. Specifically, we elaborated on how we applied their part for our anchoring construct.
PgsA-MlrA Fusion Protein
This composite part links MlrA with the gene for PgsA (poly-γ-glutamate synthetase A), a protein natively found in Bacillus subtilis. The aim of this is to anchor MlrA, or microcystinase, to the outer membrane of E. coli. Specifically, it is composed of a codon-optimized subunit of PgsA (poly-γ-glutamic acid synthetase) (BBa_K2963020), an anchoring motif to MlrA with 6x His (BBa_K1907002), with a 2 amino acid Gly-Ser linker (BBa_J18920)
pTac Promoter
The sequence for this promoter was derived from the paper by Browning et al. It is a hybrid of trp and lac UV5 promoters, and is IPTG inducible.
GSH/T25 Fusion Protein
This composite part protein consists of the antioxidant glutathione (GSH) (BBa_K2571001) attached to the T25 subdomain of adenylyl cyclase (BBa_K1638002) with a 54 amino acid Gly-Gly-Ser Linker (BBa_K3128010). The sequence was set under the control of a constitutive promoter (BBa_J23112), has ribosome binding site (Part:BBa_J61102 ), and terminator (BBa_K3128010). This biobrick is one part of a bacterial two-hybrid assay that serves as a novel detection system for microcystin-LR ( MC-LR). It is used in conjunction with a fusion protein combining protein phosphatase 1(PP1) and the T18 subdomain of adenylyl cyclase (BBa_K3802002).
PP1/T18 Fusion Protein
This composite part consists of the catalytic domain of human protein phosphatase 1 (PP1)(BBa_K1012001) attached to the T18 subdomain of adenylyl cyclase(BBa_K1638004) with a 54 amino acid Gly-Gly-Ser Linker (BBa_K3128010).The sequence was set under the control of a constitutive promoter (BBa_J23112), has ribosome binding site (BBa_J61102 ), and terminator (BBa_B0015).This biobrick is one part of a bacterial two-hybrid assay that serves as a novel detection system for microcystin-LR (MC-LR). It is used in conjunction with a fusion protein combining Glutathione (GSH) and the T25 subdomain of adenylyl cyclase (BBa_K3802003)
| Name | Type | Description | Length (bp) |
|---|---|---|---|
| BBa_K3802000 | Composite | PgsA-MlrA Fusion Protein | 2429 |
| BBa_K3802001 | Regulatory | pTac Promoter | 147 |
| BBa_K3802002 | Composite | GSH/T25 Fusion Protein | 1621 |
| BBa_K3802003 | Regulatory | PP1/T18 Fusion Protein | 1900 |
