Parts
Our constructs were designed specifically for the pDB.His.x series plasmids and the pET-28 derived plasmid pTHST, which contains the SUMO protein tag. We chose to use each of these plasmids because they are IPTG inducible, since they have lacI, the lac repressor gene. Once transformed into E. coli, expression can be induced by adding IPTG. After pelleting and lysing cells, it is possible to purify our protein. Before cleavage, our protein has a 6xHis and GST, MBP, or SUMO tag. These tags can be removed using TEV protease, which cleaves between the protein expression tags and our bacteriocins.Using the plasmids above, we designed tagged and untagged constructs. Our tagged constructs were cut using NdeI and XhoI, so our inserts are between the TEV cleavage site and a 6xHis tag that is not expressed. We designed untagged inserts, cut with BglII and XhoI, which removed the protein expression tag from our construct, but these constructs were difficult to work with and we abandoned them. In order to make our inserts BioBrick compatible, we used primers with the BioBrick prefix and suffix and PCRed our inserts.
Aureocin A53
This part was synthesized by Integrated DNA Technologies and contains the coding sequence for the bacteriocin Aureocin A53. Aureocin A53 is synthesized by Staphylococcus aureus A53 and has antimicrobial properties towards MRSA and S. aureus strains. This part is designed to be inserted into a vector containing an RBS and a protein tag, so that it can be expressed in E. coli, cleaved from a protein tag, and purified. We hope that it will be beneficial to use this part as a possible antimicrobial agent against other pathogens.
Epidermicin NI01
This part was synthesized by Integrated DNA Technologies. It codes for the bacteriocin Epidermicin NI01, which can be used to target various pathogenic strains of bacteria including Staphylococcus aureus and MRSA. It was designed to be produced in a plasmid that already contains a ribosome binding site and a protein tag. This will be beneficial in its production in E. coli, and purification. We hope that teams will use this part to test its antimicrobial properties against other pathogens.
Lacticin Z
Lacticin Z
This part was synthesized by Integrated DNA Technology, and produces the bacteriocin, Lacticin Z. Lacticin Z is known to have antimicrobial properties against a variety of bacteria, including Methicillin-resistant Staphylococcus aureus and other Staphylococcus aureus strains. This part is designed to be used in a plasmid that already contains a ribosome binding site and a tag that will be used to purify the protein. We hope that other teams will be able use this part to test its antimicrobial properties on other pathogenic bacteria.
Lacticin Z-Aureocin A53 Hybrid
This part was synthesized by Integrated DNA Technologies. It codes for the hybrid bacteriocin Aureocin A53 and Lacticin Z, joined by a triglycine linkage. The bacteriocins were joined at the N-terminus of Aureocin A53 and the C-terminus of Lacticin Z. Since both bacteriocins are effective against MRSA and other Staphylococcus aureus strains, it is possible that the hybridization of the two will have some antimicrobial properties too. This part is designed to be inserted into a vector containing an RBS and a protein tag, so that it can be expressed in E. coli, cleaved from a protein tag, and purified. We hope that teams will be able to test the antimicrobial properties of this hybrid bacteriocin compared to the individual bacteriocins on their own.
Lacticin Z-Epidermicin NI01 Hybrid
This part was synthesized by Integrated DNA Technologies. It codes for the hybrid bacteriocin Epidermicin NI01 and Lacticin Z, joined by a triglycine linkage. The bacteriocins were joined at the N-terminus of Epidericin NI01 and C-terminus of the Lacticin Z. Since both of the bacteriocins are effective against MRSA and other Staphylococcus aureus strains, it is possible that the hybridization of the two will have some antimicrobial properties as well. This part is designed to be inserted into a vector containing an RBS and a protein tag, so that it can be expressed in E. coli, cleaved from a protein tag, and purified. We hope that teams will be able to test the antimicrobial properties of this hybrid bacteriocin compared to the individual bacteriocins on their own.
Lacticin Z-Lacticin Q Hybrid
This part was synthesized by Integrated DNA Technologies. The coding sequence of this part was codon-optimized for production in E.coli using the proprietary codon optimization program on the Integrated DNA Technologies website. It codes for the hybrid bacteriocin Lacticin Q and Lacticin Z, joined by a triglycine linkage. They were joined at the N-terminus of Lacticin Q and the C-terminus of Lacticin Z. Since both of the bacteriocins are effective against MRSA and other Staphylococcus aureus strains, it is possible that the hybridization of the two will have some antimicrobial properties too. This part is designed to be inserted into a vector containing an RBS and a protein tag, so that it can be expressed in E. coli, cleaved from a protein tag, and purified. We hope that teams will be able to test the antimicrobial properties of this hybrid bacteriocin compared to the individual bacteriocins on their own.
