Outstanding Composite Part “RBS+BIND-bearPETase(★BBa_K5436124)”

The part was designed to create Whole-cell Biocatalysts, called "BIND-bearPETase," for breaking down PET plastics. We developed a system that fuses bearPETase with CsgA to present PETase on the E. coli surface, improving stability and achieving the reuse of enzymes and no need for purification.

Additionally, this part contributes to expanding the use of the BIND-System, potentially lowering costs and enhancing enzyme stability and reusability for future iGEM projects.

Fig. 6.1. BIND-PETase Module graphical abstract

For detailed part characterization, see the Registry Page(★BBa_K5436124).

Basic Parts for BIND-PETase

In addition to the composite parts, which was optimal, we devised several other PETases and registered them as Basic Parts. Some of them worked well when implemented in the BIND-System, and some did not.

Parts No.	Parts Name	Description
BBa_K5436005	Optimized RBS for BIND-System	Optimized RBS for BIND-System.
BBa_K5436006	CsgA-taa	CsgA without stop codon, fusing enzymes into Curli Fiber.
BBa_K5436030	duraPETase	PETase variants designed by directed evolution. This is designed by Y. Cui et al.(2021)^[1] which work as whole-cell biocatalysts.
BBa_K5436007	ESM_PETase	In silico Evolution PETase(ID1), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436008	ESM_PETase_affinity_screening	In silico Evolution PETase(ID2), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436009	ESM_PETase_affinity_REU_screening	In silico Evolution PETase(ID3). Althouth it has less homology around 60% with PETase(WT), it has hydrolase activity a little bit.
BBa_K5436010	DuraPETase_EvoProtGrad_ESM2_evocouplings_affinity_screening	In silico Evolution PETase(ID4), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436011	DepoPETase_EvoProtGrad_ESM2_evocouplings_affinity_screening	In silico Evolution PETase(ID5), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436012	LCC_mut_IsPETase_M1M3	In silico Evolution PETase(ID6), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436014	DepoPETase-M4_DuraPETase-M10	Rational Design PETase(ID22), combining mutations from exisiting PETase variants, depoPETase and duraPETase. Working well as whole-cell biocatalysts.
BBa_K5436016	DepoPETase-M2_DuraPETase-M10	Rational Design PETase(ID23), combining mutations from exisiting PETase variants, depoPETase and duraPETase. Working well as whole-cell biocatalysts.
★BBa_K5436015	BearPETase, DepoPETase-M2_DuraPETase-M10	Rational Design PETase(ID24), combining mutations from exisiting PETase variants, depoPETase and duraPETase. Working best as whole-cell biocatalysts. For more detail, please refer to the page of its composite parts*BBa_K5436124
BBa_K5436017	DepoPETase-M3_DuraPETase-M10	Rational Design PETase(ID25), combining mutations from exisiting PETase variants, depoPETase and duraPETase. Working as whole-cell biocatalysts.
BBa_K5436018	DepoPETase-M2-2_DuraPETase-M10	Rational Design PETase(ID26), combining mutations from exisiting PETase variants, depoPETase and duraPETase. Working as whole-cell biocatalysts.
BBa_K5436019	DepoPETase-M2-1_DuraPETase-M10	Rational Design PETase(ID27), combining mutations from exisiting PETase variants, depoPETase and duraPETase. Working as whole-cell biocatalysts.
BBa_K5436020	BamHI Linker	Linker between CsgA and PETase, encoding Gly-Ser.
BBa_K5436021	6xHisTag	6x Histidine Tag

Composite Parts for BIND-PETase

We also registered all the BIND-PETase we functionally measured as composite parts.

Parts No.	Parts Name	Description
BBa_K5436100	BIND-System Module	Optimized RBS for BIND-System(BBa_K5436005)+CsgA-taa(BBa_K5436006)+BamHI Linker(BBa_K5436020) By combining it with the two parts below, any enzyme can be incorporated into the BIND-System, expanding its applicability. This part will allow future iGEMers to use it for broader purposes. For more detailed experimental results, refer to our wiki Engineering Success tab and results tab.
★BBa_K5436124	RBS+BIND-bearPETase	Optimized RBS for BIND-System(BBa_K5436005)+CsgA-taa(BBa_K5436006)+BamHI Linker(BBa_K5436020)+bearPETase(★BBa_K5436015, PETase(ID24)) Most outstanding composite parts, inducing whole-cell biocatalysts “BIND-bearPETase” with high efficiency and high industrial value. For more detail, please refer to Registry Page(★BBa_K5436124)
BBa_K5436130	RBS+BIND-PETase(WT)	Optimized RBS for BIND-System(BBa_K5436005)+CsgA-taa(BBa_K5436006)+BamHI Linker(BBa_K5436020)+PETase(WT)(BBa_J428065) BIND-PETase equipped with PETase(WT), which works as whole-cell biocatalysts.
BBa_K5436131	RBS+BIND-PETase(E. coli optimized)	Optimized RBS for BIND-System(BBa_K5436005)+CsgA-taa(BBa_K5436006)+BamHI Linker(BBa_K5436020)+PETase(2 mut.)(BBa_K2910000) The BBa_K2910000 is incorrect. This sequence has no mutations and is just the optimized sequence for E. coli expression. This works as whole-cell biocatalysts.
BBa_K5436132	RBS+BIND-PETase(5 mut.)	Optimized RBS for　BIND-System(BBa_K5436005)+CsgA-taa(BBa_K5436006)+BamHI Linker(BBa_K5436020)+PETase(5 mut.)(BBa_K2910003) BIND-PETase equipped with PETase(WT), which doesn’t work at all as whole-cell biocatalysts. This doesn’t work at all as whole-cell biocatalysts.
BBa_K5436133	RBS+BIND-duraPETase	Optimized RBS for BIND-System(BBa_K5436005)+CsgA-taa(BBa_K5436006)+BamHI Linker(BBa_K5436020)+duraPETase(BBa_K5436030) BIND-PETase equipped with duraPETase, which works as whole-cell biocatalysts.
BBa_K5436101	RBS+BIND-PETase(ID1)	BIND-System Module(BBa_K5436100)+PETase(ID1)(BBa_K5436007) BIND-PETase equipped with In silico Evolution PETase(ID1), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436102	RBS+BIND-PETase(ID2)	BIND-System Module(BBa_K5436100)+PETase(ID2)(BBa_K5436008) BIND-PETase equipped with In silico Evolution PETase(ID2), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436103	RBS+BIND-PETase(ID3)	BIND-System Module(BBa_K5436100)+PETase(ID3)(BBa_K5436009) BIND-PETase equipped with In silico Evolution PETase(ID3). Inducing whole-cell biocatalysts. Designed through in silico evolution pipeline. Althouth it has less homology around 60% with PETase(WT), it has hydrolase activity a little bit.
BBa_K5436104	RBS+BIND-PETase(ID4)	BIND-System Module(BBa_K5436100)+PETase(ID4)(BBa_K5436010) BIND-PETase equipped with In silico Evolution PETase(ID4), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436105	RBS+BIND-PETase(ID5)	BIND-System Module(BBa_K5436100)+PETase(ID5)(BBa_K5436011) BIND-PETase equipped with In silico Evolution PETase(ID5), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436106	RBS+BIND-PETase(ID6)	BIND-System Module(BBa_K5436100)+PETase(ID6)(BBa_K5436012) BIND-PETase equipped with In silico Evolution PETase(ID6), which doesn’t work at all as whole-cell biocatalysts.
BBa_K5436122	RBS+BIND-PETase(ID22)	BIND-System Module(BBa_K5436100)+PETase(ID22)(BBa_K5436014) Inducing whole-cell biocatalysts “BIND-PETase”. It worked well as whole-cell biocatalysts and was a candidate for BIND-bearPETase.
BBa_K5436123	RBS+BIND-PETase(ID23)	BIND-System Module(BBa_K5436100)+PETase(ID23)(BBa_K5436016) Inducing whole-cell biocatalysts “BIND-PETase”. It worked well as whole-cell biocatalysts and was a candidate for BIND-bearPETase.
BBa_K5436125	RBS+BIND-PETase(ID25)	BIND-System Module(BBa_K5436100)+PETase(ID25)(BBa_K5436016) Inducing whole-cell biocatalysts “BIND-PETase”. It worked well as whole-cell biocatalysts and was a candidate for BIND-bearPETase.
BBa_K5436126	RBS+BIND-PETase(ID26)	BIND-System Module(BBa_K5436100)+PETase(ID26)(BBa_K5436017) Inducing whole-cell biocatalysts “BIND-PETase”. It worked well as whole-cell biocatalysts and was a candidate for BIND-bearPETase.
BBa_K5436127	RBS+BIND-PETase(ID27)	BIND-System Module(BBa_K5436100)+PETase(ID27)(BBa_K5436018) Inducing whole-cell biocatalysts “BIND-PETase”. It worked well as whole-cell biocatalysts and was a candidate for BIND-bearPETase.

Basic & Composite Parts for Electrical Gene Manipulation

We registered several parts to test the expression of Cre Recombinase through electrical stimuli. Although we successfully induced Cre expression using IPTG, the attempt to express Cre Recombinase through electrical stimuli was unsuccessful. However, it is likely that further investigation can be conducted to achieve success.

Parts No.	Parts Name	Description
BBa_K5436200	PlacIq+LacI	A unit for efficient production of LacI protein.
BBa_K5436201	Lac Promoter	IPTG inducible promoter, suppressed by LacI.
BBa_K5436202	loxP-double terminator-loxP	A terminator sandwiched by the loxP sites. The region between loxP sites are cut off by Cre Recombinase.
BBa_K5436210	PlacIq+LacI+T7 Terminator+Lac Promoter+Cre	A unit for IPTG-inducible expression of Cre Recombinase in E.coli.
BBa_K5436211	Pcon+SoxR+T7 terminator+pSoxS+Cre	A unit for electrically-inducible expression of Cre Recombinase in E.coli. Constitutively expressing the SoxR required for pSoxS promoter (BBa_K4216006) and expressing Cre Recombinase by Electrical Stimuli.
BBa_K5436212	Pcon+loxP-double terminator-loxP+mRFP1	Sequence of BBa_K5436202 and mRFP1 (BBa_J428036) combined with a constitutive promoter (BBa_J23100). When Cre Recombinase is present, mRFP1 is likely to be expressed in E.coli.
BBa_K5436213	pSoxS+mRFP1	pSoxS promoter (BBa_K4216006) and mRFP1 (BBa_J428036) sequence. It may be possible to express mRFP1 in Electrical Stimuli in E.coli.

References

Y Cui et al, Computational Redesign of a PETase for Plastic Biodegradation under Ambient Condition by the GRAPE Strategy. ACS Catal. 2021, 11, 3, 1340–1350. doi: 10.1021/acscatal.0c05126 ↩︎