Introduction
Here we show the collection of parts that we either used
or created during the course of our project.
For every part is provided the name, the Registry accession number,
and a complete brief description.
Our project aims to test the functioning of different enzymes,
on the same molecules. For this purpose we structured our parts
to have access to different sets of parts: we have sets of basic
parts and composite parts that present the same structure but with
different coding sequences.
In this way, we can keep the experimental settings as similar as
possible while testing the functioning of different enzymes.
Registry parts that we used
A promoter derived from the fusion of the lac and trp promoters.
It’s a strong promoter inducible with IPTG. The sequence also contains
the LacO operator sequence for regulation.
Strong RBS based on Ron Weiss thesis.
A plasmid backbone belonging to the JUMP collection. It contains two different
terminators, a Kanamycin selection marker and a medium copy number ORI:
BBa_J428303.
pJUMP29-1A serves as a standard backbone from the JUMP collection, a series
of plasmids for Golden Gate or Biobrick cloning, with Kanamycin resistance.
This part offered a perfect backbone for our work since it encodes for a
terminator right after the suffix sequence:
BBa_J428305
, which is a T0 lambda terminator.
The coding sequence of a native dehalogenase found in the known
Delftia acidovorans genome, codon optimized for E. coli expression.
This is an improvement on part
BBa_K3347010
, which encodes
for the sequence of Dehalogenase type two.
The sequence was modified by adding bases to insert the NdeI restriction site upstream and BamHI restriction site downstream. This structure is developed for an intracellular expression cassette, and the presence of two different restriction sites at the beginning and the end of the sequence, allows to remove and change the coding sequence via directed cloning.
Any sequence of interest would need the same enzymatic sites at the end in order to be exchanged with this part.
The sequence was modified by adding bases to insert the NdeI restriction site upstream and BamHI restriction site downstream. This structure is developed for an intracellular expression cassette, and the presence of two different restriction sites at the beginning and the end of the sequence, allows to remove and change the coding sequence via directed cloning.
Any sequence of interest would need the same enzymatic sites at the end in order to be exchanged with this part.
This 32 bases sequence is a part that only contains two BsaI recognition
sites, and was designed to be used to insert a terminator via golden
gate assembly.
This sequence was initially necessary in order to insert a terminator, since it couldn’t be synthesized inside the expression cassette. We subsequently choose to keep the terminator of the backbone instead.
This sequence was initially necessary in order to insert a terminator, since it couldn’t be synthesized inside the expression cassette. We subsequently choose to keep the terminator of the backbone instead.
This part was a result of searching with PROTEIN BLAST in
Synechocystis PCC 6803 proteome for alignments with the five
Delftia acidovorans dehalogenases.
The sequences used as query were selected from the ones proposed by USAFA iGEM 2020 Team as best candidates for PFAS degradation: the search yielded significant results only for DeHa4. We propose DeHaS as an enzyme with good degrading activity on PFAS since its similarity with DeHa4 and considering the high Synechocystis’ survival capabilities in contaminated areas, and we used this sequence as a part of the enzymatic surface display system: BBa_K5109020.
A 3D model was developed through homology modeling for this enzyme and docking simulations with various PFAS molecules demonstrated positive and encouraging results. The simulations revealed a specific binding pocket within the enzyme, suggesting a potential catalytic site where PFAS could be degraded.
The sequences used as query were selected from the ones proposed by USAFA iGEM 2020 Team as best candidates for PFAS degradation: the search yielded significant results only for DeHa4. We propose DeHaS as an enzyme with good degrading activity on PFAS since its similarity with DeHa4 and considering the high Synechocystis’ survival capabilities in contaminated areas, and we used this sequence as a part of the enzymatic surface display system: BBa_K5109020.
A 3D model was developed through homology modeling for this enzyme and docking simulations with various PFAS molecules demonstrated positive and encouraging results. The simulations revealed a specific binding pocket within the enzyme, suggesting a potential catalytic site where PFAS could be degraded.
Coding sequence of a Laccase from Escherichia coli genome.
The sequence was chosen from bioinformatic research and is an improvement of part BBa_K863006 , with the removal of undesired restriction sites that were removed since they interfered with our cloning experiments.
This enzyme has been extensively used in previous iGEM projects, where it has been well-characterized. Additionally, we confirmed that it contains all three domains characteristic of the laccase family.
This enzyme was chosen as a putative candidate for PFAS degradation, since it has a promising background in bioremediation.
The sequence was chosen from bioinformatic research and is an improvement of part BBa_K863006 , with the removal of undesired restriction sites that were removed since they interfered with our cloning experiments.
This enzyme has been extensively used in previous iGEM projects, where it has been well-characterized. Additionally, we confirmed that it contains all three domains characteristic of the laccase family.
This enzyme was chosen as a putative candidate for PFAS degradation, since it has a promising background in bioremediation.
This part consists of the sequence encoding Synechocystis
PCC 6803's laccase, which is an enzyme selected to be part
of a surface display system:
BBa_K5109022.
We selected this enzyme because it is endogenously expressed in Synechocystis PCC which is well known for its high survival capabilities in PFAS-contaminated environments. Additionally, a BLAST search demonstrated significant similarity to a laccase expressed in Pleurotus ostreatus, which has been shown in previous studies to effectively degrade PFOA and may be applicable for the degradation of other PFAS compounds. A further BLAST analysis revealed its similarity to E. coli's laccase, which we have also selected for our project.
We selected this enzyme because it is endogenously expressed in Synechocystis PCC which is well known for its high survival capabilities in PFAS-contaminated environments. Additionally, a BLAST search demonstrated significant similarity to a laccase expressed in Pleurotus ostreatus, which has been shown in previous studies to effectively degrade PFOA and may be applicable for the degradation of other PFAS compounds. A further BLAST analysis revealed its similarity to E. coli's laccase, which we have also selected for our project.
The coding sequence of a carrier, consisting of Lpp’s signal peptide
(first 20 aminoacids of its entire sequence) and of a truncated
version of OmpT (coding aminoacids 7-158).
This sequence comes from a bibliographic research that indicated Lpp-OmpT as the best and most used candidate for surface display systems. Our main reference was: “Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif”
Hui CY, Guo Y, Liu L, Zheng HQ, Wu HM, Zhang LZ, Zhang W. Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif. Biotechnol Lett. 2019 Jul;41(6-7):763-777. doi: 10.1007/s10529-019-02676-4. Epub 2019 Apr 25. PMID: 31025146.
This sequence comes from a bibliographic research that indicated Lpp-OmpT as the best and most used candidate for surface display systems. Our main reference was: “Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif”
Hui CY, Guo Y, Liu L, Zheng HQ, Wu HM, Zhang LZ, Zhang W. Development of a novel bacterial surface display system using truncated OmpT as an anchoring motif. Biotechnol Lett. 2019 Jul;41(6-7):763-777. doi: 10.1007/s10529-019-02676-4. Epub 2019 Apr 25. PMID: 31025146.
The coding sequence encoding a carrier and a linker, to
use upstream a gene of interest in an expression cassette
to create a display system.
This part improves part BBa_K5109003 : the presence of the linker allows this part to function as a carrier. The linker sequence also encodes for a thrombin cleavage site. By supplying thrombin to the medium in which the cells are cultured, the linker can be cleaved, enabling the isolation of the surface-expressed protein.
Upstream and downstream the coding sequence, there are NdeI restriction sites: those allow the cloning of this part upstream a coding sequence, via restriction with NdeI and following ligation.
This part improves part BBa_K5109003 : the presence of the linker allows this part to function as a carrier. The linker sequence also encodes for a thrombin cleavage site. By supplying thrombin to the medium in which the cells are cultured, the linker can be cleaved, enabling the isolation of the surface-expressed protein.
Upstream and downstream the coding sequence, there are NdeI restriction sites: those allow the cloning of this part upstream a coding sequence, via restriction with NdeI and following ligation.
Coding sequence of Dehalogenase type II from part
BBa_K3347010
with the addition of an His Tag at the end of the sequence.
BsaI restriction site and BamHI restriction site were added respectively upstream and downstream the coding sequence, by adding bases.
Those modifications are identically made on the Multicopper Oxidase, the Alfa/Beta hydrolase and the ECOL laccase.
This procedure provides a set of four different enzymes with different coding sequences but structured in the same way, allowing the interchange of them in the same expression tool.
Together, Deha2, DehaS, LacE and LacS form a set of similar basic parts but with a different coding sequence.
Creating a set of different enzymes with the same design allows it to proceed in parallel with different cloning: this way, a better comparison between the functioning of different enzymes can be analyzed.
BsaI restriction site and BamHI restriction site were added respectively upstream and downstream the coding sequence, by adding bases.
Those modifications are identically made on the Multicopper Oxidase, the Alfa/Beta hydrolase and the ECOL laccase.
This procedure provides a set of four different enzymes with different coding sequences but structured in the same way, allowing the interchange of them in the same expression tool.
Together, Deha2, DehaS, LacE and LacS form a set of similar basic parts but with a different coding sequence.
Creating a set of different enzymes with the same design allows it to proceed in parallel with different cloning: this way, a better comparison between the functioning of different enzymes can be analyzed.
Coding sequence of Synechocystis sp. dehalogenase type
II from part
BBa_K5109010
, with the addition of an His Tag at the end of the sequence.
BsaI restriction site and BamHI restriction site were added respectively
upstream and downstream the coding sequence, by adding bases.
Dehalogenase S is the new name chosen for this part after the improvement.
Dehalogenase S is the new name chosen for this part after the improvement.
Coding sequence of ECOL laccase from E. coli, from part
BBa_K5109011
, with the addition of an His Tag at the end of the sequence.
BsaI restriction site and BamHI restriction site were added
respectively upstream and downstream the coding sequence,
by adding bases.
After improving the sequence, we renamed it Laccase E.
After improving the sequence, we renamed it Laccase E.
Coding sequence of Synechocistis sp Multicopper Oxidase from part
BBa_K5109012
, with the addition of an His Tag at the end of the sequence.
BsaI restriction site and BamHI restriction site were added respectively upstream and downstream the coding sequence, by adding bases.
After improving the sequence, we renamed it Laccase S.
BsaI restriction site and BamHI restriction site were added respectively upstream and downstream the coding sequence, by adding bases.
After improving the sequence, we renamed it Laccase S.
Improvement of part
BBa_J428341
that modifies a single base in the sequence, removing an
undesired NdeI site. Mutation of the site is obtained
via PCR with mutagenic primers.
Mutagenesis was mandatory on pJUMP29-1A because an NdeI site was present in the backbone: all our composite parts present one or more NdeI restriction sites, which are fundamental to follow the project design.
In order to do so, we performed a mutagenic PCR that changed the backbone sequence through mutagenic primers.
Mutagenesis was mandatory on pJUMP29-1A because an NdeI site was present in the backbone: all our composite parts present one or more NdeI restriction sites, which are fundamental to follow the project design.
In order to do so, we performed a mutagenic PCR that changed the backbone sequence through mutagenic primers.
Expression cassette for intracellular expression of the dehalogenase
type II from Delftia acidovorans.
This part describes a construct for intracellular expression of Dehalogenase type II, featuring specific restriction sites at both ends: BBa_K5109009 . Dehalogenase type II was previously characterized by the iGEM20_USAFA team as part BBa_K3347010 , demonstrating its potential as a foundation for subsequent bioremediation research.
The construct includes an inducible promoter, which incorporates the LacO operator sequence for regulation through IPTG. Part BBa_K864400 was selected to establish an inducible rather than constitutive expression system: in the presence of LacI, expression is repressed, while addition of IPTG activates gene expression. A strong ribosome binding site (RBS), part BBa_B0030 , was also employed.
Due to synthesis limitations, a dedicated terminator was not included; instead, the expression cassette utilizes the terminator embedded in the chosen backbone, pJUMP29-1A, specifically BBa_J428341 , which is positioned immediately after the biobrick suffix, allowing it to function as a terminator for the expression construct.
The selection of restriction sites was performed with precision: NdeI and BamHI cutting sites are strategically positioned at the 5' and 3' ends of the gene of interest, respectively. This configuration enables the excision of the gene and its replacement with alternative enzyme expression sequences via standard restriction and ligation protocols. Furthermore, the NdeI site was engineered for the insertion of the Lpp-OmpT anchor, facilitating surface expression applications.
Additionally, the two BsaI sites located at the downstream end are designated as part BBa_K5109002 . This 32-base sequence enables the insertion of supplementary terminators through GoldenGate cloning methods.
This part describes a construct for intracellular expression of Dehalogenase type II, featuring specific restriction sites at both ends: BBa_K5109009 . Dehalogenase type II was previously characterized by the iGEM20_USAFA team as part BBa_K3347010 , demonstrating its potential as a foundation for subsequent bioremediation research.
The construct includes an inducible promoter, which incorporates the LacO operator sequence for regulation through IPTG. Part BBa_K864400 was selected to establish an inducible rather than constitutive expression system: in the presence of LacI, expression is repressed, while addition of IPTG activates gene expression. A strong ribosome binding site (RBS), part BBa_B0030 , was also employed.
Due to synthesis limitations, a dedicated terminator was not included; instead, the expression cassette utilizes the terminator embedded in the chosen backbone, pJUMP29-1A, specifically BBa_J428341 , which is positioned immediately after the biobrick suffix, allowing it to function as a terminator for the expression construct.
The selection of restriction sites was performed with precision: NdeI and BamHI cutting sites are strategically positioned at the 5' and 3' ends of the gene of interest, respectively. This configuration enables the excision of the gene and its replacement with alternative enzyme expression sequences via standard restriction and ligation protocols. Furthermore, the NdeI site was engineered for the insertion of the Lpp-OmpT anchor, facilitating surface expression applications.
Additionally, the two BsaI sites located at the downstream end are designated as part BBa_K5109002 . This 32-base sequence enables the insertion of supplementary terminators through GoldenGate cloning methods.
Expression cassette for outer membrane expression of DeHa type II via Lpp OmpT.
This section describes a modification of the intracellular expression cassette BBa K5109001 , enhanced by integrating a sequence that encodes the anchoring motif BBa K5109004 , resulting in the creation of a display system. This expression cassette enables the surface presentation of an intracellular protein, specifically Dehalogenase type II, by fusing it to an Lpp-OmpT carrier. The component utilizes the coding sequence of Dehalogenase derived from the Deha2 structure: by assembling part BBa_K5109016 with the carrier BBa K5109004, a surface display system is established.
The design incorporates various restriction sites positioned both upstream and downstream of the carrier and dehalogenase sequences, thereby facilitating its application in multiple projects. Notably, the Lpp-OmpT sequence can be excised using NdeI, allowing for conversion of the cassette to an intracellular expression format or enabling the substitution of the carrier. This substitution could facilitate comparative studies on the efficacy of Lpp-OmpT as a carrier in contrast to alternative anchoring motifs. The passenger protein can also be exchanged via digestion with BsaI and BamHI.
To optimize the design strategy presented, we have modified the restriction enzyme sites compared to BBa K5109001: specifically, two BsaI restriction sites located downstream of the passenger were removed, and a BsaI restriction site was introduced upstream of the sequence encoding the passenger. This modification enhances the manipulation of the surface expression cassette, enabling the modification of the passenger protein irrespective of the presence of the carrier in the expression cassette.
The structure of this component is repeated in three additional basic parts that adopt the same design but feature different coding sequences, thereby providing a surface display system for the enzymes Alfa/beta hydrolase, ECOL laccase, and Multicopper Oxidase.
This collection of composite parts is intended to evaluate the functional performance of the various enzymes under consistent experimental conditions and settings. Moreover, it allows for the assessment of the synergistic activity of the enzymes on identical substrates by employing two distinct cell lines, each expressing one construct, within the same environment.
This section describes a modification of the intracellular expression cassette BBa K5109001 , enhanced by integrating a sequence that encodes the anchoring motif BBa K5109004 , resulting in the creation of a display system. This expression cassette enables the surface presentation of an intracellular protein, specifically Dehalogenase type II, by fusing it to an Lpp-OmpT carrier. The component utilizes the coding sequence of Dehalogenase derived from the Deha2 structure: by assembling part BBa_K5109016 with the carrier BBa K5109004, a surface display system is established.
The design incorporates various restriction sites positioned both upstream and downstream of the carrier and dehalogenase sequences, thereby facilitating its application in multiple projects. Notably, the Lpp-OmpT sequence can be excised using NdeI, allowing for conversion of the cassette to an intracellular expression format or enabling the substitution of the carrier. This substitution could facilitate comparative studies on the efficacy of Lpp-OmpT as a carrier in contrast to alternative anchoring motifs. The passenger protein can also be exchanged via digestion with BsaI and BamHI.
To optimize the design strategy presented, we have modified the restriction enzyme sites compared to BBa K5109001: specifically, two BsaI restriction sites located downstream of the passenger were removed, and a BsaI restriction site was introduced upstream of the sequence encoding the passenger. This modification enhances the manipulation of the surface expression cassette, enabling the modification of the passenger protein irrespective of the presence of the carrier in the expression cassette.
The structure of this component is repeated in three additional basic parts that adopt the same design but feature different coding sequences, thereby providing a surface display system for the enzymes Alfa/beta hydrolase, ECOL laccase, and Multicopper Oxidase.
This collection of composite parts is intended to evaluate the functional performance of the various enzymes under consistent experimental conditions and settings. Moreover, it allows for the assessment of the synergistic activity of the enzymes on identical substrates by employing two distinct cell lines, each expressing one construct, within the same environment.
Expression cassette for outer membrane expression of alfa/beta Hydrolase via Lpp OmpT.
Expression cassette for outer membrane expression of ECOL laccase via Lpp OmpT.
Expression cassette for outer membrane expression of Multicopper Oxidase via Lpp OmpT.
