Part Collections
Our unique NisQ and PHB part collections
NisQ Part Collection
Expression of NisQ in E. coli
Our nisin part list consists of 1 new basic part and 3 composite parts. All parts relate to the expression of NisQ, thus forming a collection. To read more about how our parts were used in our project, head to our Preservation - Nisin.
Derived from Lactococcus lactis, nisin is a food-safe, antimicrobial peptide (AMP) that targets a wide range of gram positive bacteria by binding to lipid II on the pathogens membrane, creating a pore, and causing cell death. Literature suggests that nisin Q (NisQ) demonstrates greater antimicrobial and antioxidant activity against pathogens compared to other variants of nisin, such as NisA (BBa_K1365000). Nisin’s optimal pH stability is between 2 and 7 but can maintain its antibacterial activity up to a pH of 12, and can also retain its antimicrobial activity at temperatures of 120oC.
A fusion protein with NisQ tagged with a 6XHis-tag on the N-terminus end for protein purification from E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for coherent design with BBa_K4437002.
A fusion protein with both N-utilizing substance A (NusA, BBa_K4015004) and a 6X His tag on the N-terminus of NisQ for protein purification from E. coli. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification.
A novel fusion protein with 6XHis-tagged Glutathione S-transferases (GST), NusA, and a 6XHis-tag fused to the N-terminus of NisQ. GST was incorporated into BBa_K4437002 due to its presence within the Xpress expression vector (BBa_K3945014), which we used to successfully produce NisQ protein. GST is a solubility tag which is useful for expressing difficult proteins. NusA is a solubility factor to help increase production and solubility of a desired peptide in E. coli. Double enterokinase cut sites are included for removal of the 6XHis-tag to isolate NisQ alone. A TEV protease site is included for removal of NusA, revealing the 6XHis-tag for subsequent NisQ purification. Using GST and NusA increases the expected band size from 7kDa (NisQ alone) to 91kDa, which is easier to visualize on SDS-PAGE gels.
PHB Part Collection
Fine-tuning PHB Production
Our PHB parts list consists of 1 new basic part and 4 new composite parts. All of these parts relate to the expression of phasin, an integral protein in the PHB secretion process. To read more about how these parts were used, please visit the Protection - PHB.
The DeadRBS is an RBS library synthesized by Kosuri et al. (2013) in a study which assessed how translational efficiency changes depending on the strength of the RBS used in E. coli (1). The DeadRBS was used as a negative control, or point of reference for the other RBS, as it has the least amount of protein translated (1). Although the DeadRBS (BBa_K4437500) has experimentally demonstrated to have a low affinity for the ribosome, it still is capable of producing trace amounts of protein, which is a tool that can be leveraged by future teams that want to produce trace amounts of certain genes in an operon.
A fusion protein with FlagTM-tagged phasin, and a hemolysin A (HlyA) tag for secretion in E. coli. This part design used the University of Calgary 2017 team’s BBa_K2260002 part as a template, but the BBa_B0034 RBS was exchanged for the our DeadRBS (BBa_K4437500) as a negative control for phasin expression experiments. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression.
A fusion protein with FlagTM-tagged phasin, and a hemolysin A (HlyA) tag for secretion in E. coli. This part design used the University of Calgary 2017 team’s BBa_K2260002 part as a template, but the BBa_B0034 RBS was exchanged for the our B0030 RBS (BBa_B0030), a relatively stronger RBS than B0034, as a test for phasin expression experiments. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression.
A fusion protein with FlagTM-tagged phasin, and a hemolysin A (HlyA) tag for secretion in E. coli. This part design used the University of Calgary 2017 team’s BBa_K2260002 part as a template, and included the original BBa_B0034 RBS positive control for phasin expression experiments, and as a means to compare expression levels to potentially demonstrate improvement upon this part. The restriction enzyme cut sites for BstAPI and BstBI were again added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression.
A fusion protein with FlagTM-tagged phasin, and a hemolysin A (HlyA) tag for secretion in E. coli. This part design used the University of Calgary 2017 team’s BBa_K2260002 part as a template, but the BBa_B0034 RBS was exchanged for the our B0035 RBS (BBa_B0035), a relatively weaker RBS than B0034, as a test for phasin expression experiments. The restriction enzyme cut sites for BstAPI and BstBI were also added, making the part compatible for digestion and ligation into the Tokyo Tech 2012 team’s BBa_K934001 plasmid for PHB expression.
