Difference between revisions of "Team:Harvard BioDesign/Basic Part"

A promoter is a sequence of nucleotides that allows RNA polymerase to start transcribing mRNA from DNA. The T7 promoter is a strong, titratable promoter derived from the T7 phage. Thus, it is compatible only with cells that have T7 RNA polymerase, such as NEB’s T7 Express lysY/Iq Competent E. coli.

Source: BBa_I712074

The ribosome binding site is a sequence of nucleotides that allows a ribosome to bind to mRNA in order for the ribosome to start translating protein from the mRNA sequence. RBS 34, also known as the Elowitz RBS, is a commonly used ribosome binding site.

Source: BBa_B0034

PETase is a poly(ethylene terephthalate) degrading enzyme. It breaks down PET into ethylene glycol and terephthalic acid. Originally named ISF6_4831, PETase comes from Ideonella sakaiensis, identified in "A bacterium that degrades and assimilates poly(ethylene terephthalate)" (Yoshida et al. 2016). The sequence we used in our constructs is E. coli K12 optimized with the Codon Optimization Tool from the IDT website.

Find out more about PETase on our project description under I. Sakaiensis

Uniprot Source

Green fluorescent protein (GFP) is a common method of visualizing proteins in order to detect their presence, through basic fluorescent assays. When proteins are fused to GFP, the presence of GFP most likely indicates the presence of the proteins fused to GFP. sfGFP, or super-folded GFP, is a variant of GFP designed to help fold polypeptide chains into proteins. sfGFP also increases the solubility of fused proteins, as indicated by Wu et al. in "A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag."

Wu, Xudong, Di Wu, Zhisheng Lu, Wentao Chen, Xiaojian Hu, and Yu Ding. "A Novel Method for High-Level Production of TEV Protease by Superfolder GFP Tag." Journal of Biomedicine and Biotechnology 2009 (2009): 1-8. Web.

The ompT tag is an N-terminus secretion tag that transports a protein from the interior of an E. coli cell to the extracellular medium. ompT is an outer membrane protein naturally encoded by E. coli that is transported to the outer membrane because of its secretion tag. When you add the ompT tag to a different protein, the cell secrets the tagged protein to the outer membrane of the cell. Studies have indicated that the tagged protein becomes transported to the extracellular medium.

The pelB tag is an N-terminus secretion tag that transports a protein from the interior of an E. coli cell to the periplasm. The tag associated with pelB secretes proteins to the periplasm of the cell, the space between the interior of the cell and the extracellular medium. Although this secretion method does not allow the protein to be excreted completely, this tag has been shown to be efficient and reliable, and still allows for secretion because tagged proteins are able to diffuse from the periplasm to the extracellular medium.

Shokri, A., A. Sandén, and G. Larsson. "Cell and Process Design for Targeting of Recombinant Protein into the Culture Medium of Escherichia Coli." Applied Microbiology and Biotechnology 60.6 (2002): 654-64. Web.
Yoon, Sung, Seong Kim, and Jihyun Kim. "Secretory Production of Recombinant Proteins in Escherichia Coli." BIOT Recent Patents on Biotechnology 4.1 (2010): 23-29. Web.

The YebF “tag” actually encodes an N-terminus protein, YebF, which transports a fused protein from the interior of an E. coli cell to the extracellular medium. Although proteins fused with YebF are secreted into the extracellular medium, the exact mechanism of YebF-mediated transport is still unknown (Zhang et al. 2005). The YebF secretion method has been shown to excrete protein completely and reliably, though it should be noted that YebF may cause steric inhibition that can disrupt the function of the fused target protein.

Zhang, Guijin, Stephen Brokx, and Joel H. Weiner. "Extracellular Accumulation of Recombinant Proteins Fused to the Carrier Protein YebF in Escherichia Coli." Nature Biotechnology 24.1 (2005): 100-04. Web.

Linkers are unreactive polypeptide sequences that add space between fused proteins to facilitate proper folding . There are a variety of linkers, differing in length and amino acids. Since PETase is a novel protein, no specific linker sequence has been reported to be beneficial or inhibitory. The linkers used for this project were based on "Fusion Protein Linkers: Property, Design and Functionality," with the exception of the GGS linker, which was provided to us by our mentor, David Lips.

Chen, Xiaoying, Jennica L. Zaro, and Wei-Chiang Shen. "Fusion Protein Linkers: Property, Design and Functionality." Advanced Drug Delivery Reviews 65.10 (2013): 1357-369. Web.

A His-tag is a sequence of histidine amino acids that is used to isolate a specific protein from a solution of multiple proteins. His-tags are also used to detect the presence of a protein, such as PETase, in Western blots.