New Basic Parts for 2021 Project

MlrA

MlrA, or microcystinase, cleaves the peptide backbone of MC-LR cyanotoxins produced by Microcystis aeruginosa cyanobacteria. This crucial step results in the peptide losing the cyclic structure, and therefore the majority of its toxicity. Further degradation continues in an enzymatic pathway by interactions with other peptidolytic enzymes from the microcystinase gene cluster. This part originally comes from the Aalto-Helsinki 2016 iGEM team (Part BBa_K1907001), but was originally codon-optimized for yeast.

Part:BBa_K3738019

MPA

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid.

Part:BBa_K3738000

MPA N-Terminal His Tag

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This maturation protein A coding region includes a histidine tag on the N-terminus, to allow for purification as a monomer for capsid assembly in vitro.

This part is a modified version of BBa_K3738000, to which an N-terminal histidine tag has been added. As maturation protein A has not been well studied for use in phage-like particles, we have designed various versions of the part for testing purposes.

Part:BBa_K3738001

MPA C-Terminal His Tag

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This maturation protein A coding region includes a histidine tag on the C-terminus, to allow for purification as a monomer for capsid assembly in vitro.

This part is a modified version of BBa_K3738000, to which an C-terminal histidine tag has been added. As maturation protein A has not been well studied for use in phage-like particles, we have designed various versions of the part for testing purposes.

Part:BBa_K3738002

MPA N-Terminal His Tag TEV

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This maturation protein A coding region includes a histidine tag on the N-terminus, to allow for purification as a monomer for capsid assembly in vitro. There is also a TEV cleavage site between the coding region and the histidine tag, in order to allow for histidine tag removal following purification if desired.

This part is a modified version of BBa_K3738000, to which an N-terminal histidine tag and TEV cleavage site have been added. As maturation protein A has not been well studied for use in phage-like particles, we have designed various versions of the part for testing purposes.

Part:BBa_K3738003

MPA C-Terminal His Tag TEV

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This maturation protein A coding region includes a histidine tag on the C-terminus, to allow for purification as a monomer for capsid assembly in vitro. There is also a TEV cleavage site between the coding region and the histidine tag, in order to allow for histidine tag removal following purification if desired.

This part is a modified version of BBa_K3738000, to which an C-terminal histidine tag and TEV cleavage site have been added. As maturation protein A has not been well studied for use in phage-like particles, we have designed various versions of the part for testing purposes.

Part:BBa_K3738004

MS2CP Dimer

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as well as one copy of maturation protein A (enter part number here), which is critical in allowing the capsid to bind to the cell and deliver its cargo. This part is a coding region comprised of 2 copies of the MS2 capsid protein, joined by a histidine tag which is flanked by linker sequences (de Martin Garrido et al., 2020). This part has been codon optimized for use in E.coli.

Part:BBa_K3738008

Improved Basic Parts for 2021 Project

MS2CP N-Terminal His Tag:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo.

The design of this part was based on BBa_K2683012, which is an MS2 capsid protein fused to a polyarginine tag cell-penetrating peptide on the N-terminus. This part has been modified from the original so as to have the polyarginine tag replaced with a histidine tag, to allow for purification as a monomer for capsid assembly in vitro. This part has been codon-optimized for use in E.coli.

Part:BBa_K3738005

MS2CP C-Terminal His Tag

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo. The design of this part was based of part BBa_K2683012, which is an MS2 capsid protein fused to a polyarginine tag cell-penetrating peptide on the C-terminus.

This part has been modified from the original (BBa_K2683012) so as to have the polyarginine tag replaced with a histidine tag, to allow for purification as a monomer for capsid assembly in vitro. It has also been codon optimized for E.coli.

Part:BBa_K3738006

MS2CP N-Terminal CPP C-Terminal His Tag:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo. The design of this part was based of part BBa_K2683012, which is an MS2 capsid protein fused to a polyarginine tag cell-penetrating peptide on the N-terminus. This part has been modified from the original (BBa_K2683012) so as to have a histidine tag placed on the C-terminus, to allow for purification as a monomer for capsid assembly in vitro. It has also been codon optimized for E.coli.

Part:BBa_K3738007

Lbu-Cas13a with an N-Terminal 6xHistidine Tag and C-Terminal Anionic Tag:

Cas13a is an enzyme originating from Leptotrichia buccalis (Lbu) which functions to cleave single-stranded RNAs (ssRNAs); particularly mRNAs. This function is achieved following protein-RNA complex formation with CRISPR RNA (crRNA) via crRNA backbone contacts with residues from the Helical-2, HEPN1, and Linker domains of Cas13a. The crRNA contains a spacer region coding for a direct repeat stem loop as well as a region complementary to target ssRNAs. Once the enzyme complex interacts with a target ssRNA, a structural conformation change occurs within the domains of the protein that permits active site formation for non-discriminate ssRNA cleavage (O’Connel et al., 2019).

Part:BBa_K3738020

Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal 6XHistidine Tag:

Cas13a is an enzyme originating from Leptotrichia buccalis (Lbu) which functions to cleave single-stranded RNAs (ssRNAs); particularly mRNAs. This function is achieved following protein-RNA complex formation with CRISPR RNA (crRNA) via crRNA backbone contacts with residues from the Helical-2, HEPN1, and Linker domains of Cas13a. The crRNA contains a spacer region coding for a direct repeat stem loop as well as a region complementary to target ssRNAs. Once the enzyme complex interacts with a target ssRNA, a structural conformation change occurs within the domains of the protein that permits active site formation for non-discriminate ssRNA cleavage (O’Connel et al., 2019).

Part:BBa_K3738021

McyH-complementary Lbu-crRNA:

Lbu-crRNA (Lbu signifying Leptotrichia buccalis, the source of the Cas13a protein and crRNA) is an RNA structure containing a direct-repeat stem loop, a recognition element for binding with the Lbu-Cas13a protein (see parts BBa_K3738020, BBa_K3738021, BBa_K3738023 and BBa_K3738024). The crRNA also contains a downstream complementary region, designed to base-pair with a target ssRNA sequence. Complex formation of crRNA-Cas13a occurs, and when the target sequence is perfectly paired with the crRNA, activation of the enzyme occurs and subsequent non-discriminate cleavage of collateral ssRNAs (O’Connell., 2019). This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway. For this reason, we have designed crRNA to target the 5’-end of McyH in order to inhibit production of microcystins.

Part:BBa_K3738022

New Composite Parts

MlrA:

MlrA, or microcystinase, cleaves the peptide backbone of MC-LR cyanotoxins produced by microcystis aeruginosa cyanobacteria. This crucial step results in the peptide losing the cyclic structure, and therefore the majority of its toxicity. Further degradation continues in an enzymatic pathway by interactions with other peptidolytic enzymes from the microcystinase gene cluster. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, MlrA coding region BBa_K3738019, and double terminator BBa_B0015. The microcystinase coding region in this part has been codon-optimized for E.coli.

Part:BBa_K3738009

MlrA and C-Terminal and Anionic Tag:

MlrA, or microcystinase, cleaves the peptide backbone of MC-LR cyanotoxins produced by Microcystis aeruginosa cyanobacteria. This crucial step results in the peptide losing the cyclic structure, and therefore the majority of its toxicity. Further degradation continues in an enzymatic pathway by interactions with other peptidolytic enzymes from the microcystinase gene cluster. This part originally comes from the Aalto-Helsinki 2016 iGEM team (Part BBa_K1907001), but was originally codon-optimized for yeast. We have modified this part by codon-optimizing it for use in E. coli. MlrA is known to be taken up into the periplasm with aid of transport protein MlrD (Saito et al., 2003), however for the mlrA sequence from Sphingomonas sp. USTB-05 the mlrD sequence is currently unavailable. Thus we have added an anionic tag for MS2-encapsulation. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with C-Terminal Anionic Tag coding region (BBa_K3738020), and double terminator BBa_B0015.The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag.

Part:BBa_K3738033

MS2 Bacteriophage Capsid Protein with Maturation Protein A:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo. The first biobrick in this composite part includes the MS2 capsid protein fused to a polyarginine tag cell-penetrating peptide (BBa_K2683012) under the control of an strong arabinose-inducible promoter (BBa_K206000), as well as the RBS BBa_B0034 and double terminator BBa_B0015. The second biobrick contains maturation protein A (BBa_K3738000) under the control of weak constitutive promoter BBa_J23114, as well as the RBS BBa_B0034 and double terminator BBa_B0015. Given the high ratio of capsid protein to maturation protein A required, this design will result in higher production of the capsid protein, which can be induced once there is sufficient cell growth, resulting in self-assembly of functional MS2 PLPs.

Part:BBa_K3738010

MPA N-Terminal His Tag:

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, maturation protein A coding region with N-Terminal histidine tag (BBa_K3738001), and double terminator BBa_B0015.

Part:BBa_K3738011

MPA C-Terminal His Tag:

Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, maturation protein A coding region with a C-terminal histidine tag (BBa_K3738002), and double terminator BBa_B0015. The maturation protein A coding region in this part has been codon-optimized for E.coli, and includes a histidine tag on the C-terminus for to allow for peptide purification.

Part:BBa_K3738012

MPA N-Terminal His Tag TEV:

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, maturation protein A coding region with a N-terminal histidine tag and TEV cleavage site (BBa_K3738003), and double terminator BBa_B0015. The maturation protein A coding region in this part has been codon-optimized for E.coli, and includes a histidine tag on the N-terminus for to allow for peptide purification. There is also a TEV cleavage site between the coding region and the histidine tag, in order to allow for histidine tag removal following purification if desired.

Part:BBa_K3738013

MPA C-Terminal His Tag TEV:

Maturation Protein A is required in order for the capsid of the MS2 phage, and subsequently MS2-derived phage-like particles (PLP), to bind to bacterial pili. It is released from the capsid upon binding, and enters the cell along with the cargo to be delivered by the MS2 capsid. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, maturation protein A coding region with a C-terminal histidine tag and TEV cleavage site (BBa_K3738004), and double terminator BBa_B0015. The maturation protein A coding region includes a histidine tag on the C-terminus for to allow for peptide purification. There is also a TEV cleavage site between the coding region and the histidine tag, in order to allow for histidine tag removal following purification if desired.

Part:BBa_K3738014

MS2CP N-Terminal His Tag:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (enter part number here), which is critical in allowing the capsid to bind to the cell and deliver its cargo. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, MS2 capsid protein coding region (BBa_K3738005), and double terminator BBa_B0015. The MS2 capsid protein coding region in this part has been codon-optimized for E.coli, and includes a histidine tag on the N-terminus for to allow for peptide purification.

Part:BBa_K3738015

MS2CP C-Terminal His Tag:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, MS2 capsid protein coding region with a C-terminal histidine tag (BBa_K3738006), and double terminator BBa_B0015. The MS2 capsid protein coding region in this part has been codon-optimized for E.coli, and includes a histidine tag on the C-terminus for to allow for peptide purification.

Part:BBa_K3738016

MS2CP N-Terminal CPP C-Terminal His Tag:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, MS2 capsid protein coding region fused to a polyarginine tag cell-penetrating peptide on the N-terminus and a histidine tag on the C-terminus (BBa_K3738007), and double terminator BBa_B0015. The MS2 capsid protein coding region in this part has been codon-optimized for E.coli, and includes a histidine tag on the C-terminus for to allow for peptide purification.

Part:BBa_K3738017

MS2CP Dimer:

The MS2 capsid protein comprises most of the capsid of the MS2 phage, as well as MS2 phage-like particles (PLPs). A single MS2 capsid requires 178 copies of the capsid protein, as one copy of maturation protein A (BBa_K3738000), which is critical in allowing the capsid to bind to the cell and deliver its cargo. This composite part includes a coding region comprised of 2 copies of the MS2 capsid protein, joined by a histidine tag flanked by 2 linker sequences (BBa_K3738008). It also contains the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, and double terminator BBa_B0015. The MS2 capsid protein coding region in this part has been codon-optimized for E.coli.

Part:BBa_K3738018

Cas13a Composite Parts:

The first biobrick in this composite part contains the coding region for the Lbu Cas13a enzyme, under control of the IPTG-inducible T7 promoter. It also includes multiple purification tags for Cas13a (BBa_K3001002), as well as the RBS BBa_B0034 and double terminator BBa_B0015. This part has already been submitted to the registry as the composite part BBa_K3001003. This plasmid will also contain a biobrick with the sequence for the Cas13a crRNA, controlled by the same T7 promoter and followed by the terminator BBa_B0015. The crRNA coding region will contain a direct repeat stem loop sequence, as well as an short sequence that is found in the cyanobacteria genome. Once the Cas13a, containing the crRNA, recognizes the same sequence in the form of mRNA inside of cyanobacteria cells, it will become activated, and begin cleaving mRNA.

Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag:

This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag coding region (BBa_K3738020), and double terminator BBa_B0015.The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag as well as the transcriptional and translational regulators for optimal overexpression and 6XHistidine tag required for nickel affinity chromatography purification.

Part:BBa_K3738023

Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal Histidine Tag:

This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag coding region (BBa_K3738020), and double terminator BBa_B0015.The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag as well as the transcriptional and translational regulators for optimal overexpression and 6XHistidine tag required for nickel affinity chromatography purification.

Part:BBa_K3738024

crRNA Composite Parts:

The first biobrick in this composite part contains the coding region for the Lbu Cas13a enzyme, under control of the IPTG-inducible T7 promoter. It also includes multiple purification tags for Cas13a (BBa_K3001002), as well as the RBS BBa_B0034 and double terminator BBa_B0015. This part has already been submitted to the registry as the composite part BBa_K3001003. This plasmid will also contain a biobrick with the sequence for the Cas13a crRNA, controlled by the same T7 promoter and followed by the terminator BBa_B0015. The crRNA coding region will contain a direct repeat stem loop sequence, as well as an short sequence that is found in the cyanobacteria genome. Once the Cas13a, containing the crRNA, recognizes the same sequence in the form of mRNA inside of cyanobacteria cells, it will become activated, and begin cleaving mRNA.

McyH complementary Lbu-crRNA:

This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway. For this reason, we have designed crRNA to target the 5’-end of McyH in order to inhibit production of microcystins.

Part:BBa_K3738025

Cas13a and crRNA Composite Parts:

The first biobrick in this composite part contains the coding region for the Lbu Cas13a enzyme, under control of the IPTG-inducible T7 promoter. It also includes multiple purification tags for Cas13a (BBa_K3001002), as well as the RBS BBa_B0034 and double terminator BBa_B0015. This part has already been submitted to the registry as the composite part BBa_K3001003. This plasmid will also contain a biobrick with the sequence for the Cas13a crRNA, controlled by the same T7 promoter and followed by the terminator BBa_B0015. The crRNA coding region will contain a direct repeat stem loop sequence, as well as an short sequence that is found in the cyanobacteria genome. Once the Cas13a, containing the crRNA, recognizes the same sequence in the form of mRNA inside of cyanobacteria cells, it will become activated, and begin cleaving mRNA.

Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag and crRNA:

This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers, each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway. For this reason, we have designed crRNA to target the 5’-end of McyH in order to inhibit production of microcystins. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with an N-terminal 6xHistidine Tag and C-Terminal Anionic Tag coding region (BBa_K3738020), and double terminator BBa_B0015, followed by a second T7 promoter (BBa_J64997), McyH complementary Lbu-crRNA (BBa_K3738025), and a second BBa_B0015 terminator. The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag as well as the transcriptional and translational regulators for optimal overexpression and 6XHistidine tag required for nickel affinity chromatography purification.

Part:BBa_K3738026

Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal 6xHistidine Tag and crRNA:

This crRNA was designed to bind with an important protein in the synthesis of harmful toxins called microcystins produced by blue-green algae (cyanobacteria) blooms. McyH is a gene in the Mcy gene cluster of Microcystic Aeruginosa and codes for a transporter protein. The protein is comprised of two homodimers, each with a hydrophobic N-terminus domain and C-terminus containing an ATPase domain. Pearson et al., 2004 experimentally examined the impacts of impairing expression of this gene, and combined with bioinformatic data, found that McyH is likely a vital exporter of harmful microcystins and essential in their biosynthetic pathway. For this reason, we have designed crRNA to target the 5’-end of McyH in order to inhibit production of microcystins. This composite part includes the IPTG-inducible T7 promoter (BBa_J64997), RBS BBa_B0034, Lbu-Cas13a with an N-terminal Anionic Tag and C-Terminal 6xHistidine Tag coding region (BBa_K3738020), and double terminator BBa_B0015, followed by a second T7 promoter (BBa_J64997), McyH complementary Lbu-crRNA (BBa_K3738025), and a second BBa_B0015 terminator. The part is codon-optimized for use in E. coli It is improved from the Lethbridge High School iGEM 2019's Parts BBa_K3001003, BBa_K3001000 and BBa_K3001002 by introducing the anionic MS2 phage-like-particle uptake anionic tag as well as the transcriptional and translational regulators for optimal overexpression and 6XHistidine tag required for nickel affinity chromatography purification.

Part:BBa_K3738027

Adding Experience to 2021 Project Parts

This year, we tested many of the constructs that we designed and ordered in the first year of our project in the wetlab. These parts include BBa_K3738005, BBa_K3738009, BBa_K3738015, BBa_K3738016, BBa_K3738018, BBa_K3738019, BBa_K3738021, BBa_K3738022, and BBa_K3738033. We have added part experience to the registry pages for these parts, which includes troubleshooting of the difficulties we had with their expression.

New Parts for 2022

For 2022, we decided to improve our project by designing parts that would make it easier to test our proof of concept in the level 1 lab that we have available to us. Additionally, we modified the design of one of our 2021 parts to improve binding affinity and target specificity, and designed a new part to take advantage of a collaboration opportunity with the Lethbridge High School Team and troubleshoot a problem we were having.

The first new parts that we designed this year were for the expression of the mcyH ATP binding cassette (ABC) transporter gene in E.coli. The mcyH gene, originally from Microcystis aeruginosa cyanobacteria, is the target for activating our CRISPR Cas13a system. Since it is unclear whether or not we would be allowed to bring potentially toxic cyanobacteria into our level 1 lab space, we designed a coding region and BioBrick to allow for the expression of this gene in E.coli; once E.coli has been transformed with this construct, it will be able to activate a version of our Cas13a system, which includes a crRNA designed to pair with the version of the mcyH sequence that has been codon-optimized for expression in E.coli.

Additionally, owing to the difficulty we had in expressing our Lbu Cas13a constructs, we collaborated with the Lethbridge High School team to acquire Lwa Cas13a, which they had expressed as a part of their 2019 project. We therefore designed mcyH-targeting crRNA to be compatible with Lwa Cas13a, for both E.coli expressing the mcyH and for cyanobacteria.

REFERENCES

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Lorenz, R. and Bernhart, S.H. and Höner zu Siederdissen, C. and Tafer, H. and Flamm, C. and Stadler, P.F. and Hofacker, I.L. "ViennaRNA Package 2.0", Algorithms for Molecular Biology, 6:1 page(s): 26, 2011
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