Difference between revisions of "Part:BBa K1933001"

Revision as of 05:27, 18 October 2016

INPNC fused to 6xHis tag

INPNC codes for N- and C- terminal domain of Ice-Nucleation Protein (INP) from Pseudomonas syringae , connected by a 6xHis tag to be easily identified by Western blotting[1]. This is used as an anchoring motif for the cell surface display of passengers. This domain does not have signal peptide for anchoring on the cell surface[2]. Surface displayed passengers retain enzyme activity even when it is linked to INPNC with 6xHis tag.
For more information, please visit [http://2016.igem.org/Team:Kyoto our wiki]

Sequence and Features

Usage and Biology

Ice nucleation protein(INP) used for our surface display systems is produced by P. syringae , a famous model organism of ice nucleation active (INA) bacteria. Although the freezing point of water is 0°C, pure water can supercool to -40°C before homogeneous nucleation randomly occurs and ice forms. Generally, minimally impure water will freeze spontaneously before -15°C. However, INP produced by P. syringae causes ice nucleation as high as -2°C. By elevating the temperature at which frost forms on leaves, bacteria can cause unseasonal damage to plants, causing them to release nutrients that allow the bacteria to multiply.
INP are expressed as the ice nucleation site, expressed in the outer cell membrane. Modified INP are used as a surface expression system.

We omitted stop codon from this part to make BBa_K1933011.
We used BBa_K1933011 for construction of ☆BBa_K1933100, BBa_K1933101, ☆BBa_K1933200.

Characterization

We improved BBa_K811005 from Penn 2012!!

Western blotting

We added His-tag to BBa_K811005. This addition introduces two new functions to the fusion protein:

First, we can use anti-His tag antibody to detect their surface expression regardless of passenger protein placed downstream. We confirmed this with detections of the INPNC-His-(passenger protein) by Western blotting using anti-His tag antibody. (Fig.1(a))

Second, INPNC-His-(passenger protein) can be purified using His tag’s affinity to nickel columns even in denaturing conditions to recollect it from membrane fractions of E.coli. This would allow fusion protein detection even if the protein’s expression levels are low, further strengthening the first function. We tested this function with Western blotting. Sample was prepared by obtaining the membrane fraction from the E. coli lysate by ultracentrifugation, and solubilized them using 7M guanidine-HCl, then purification of the target protein using Nickel Sepharose. (Fig.1(b))

Figure 1: (a) whole cell Western blotting against His tag. (b) membrane fraction Western blotting against His tag.
Negative Control:BBa_K165002, BclA-His-scFv:33 kDa, INPNC-His-scFv:63 kDa, BclA-His-CBDcex:17 kDa, INPNC-His-CBDcex:47 kDa, Positive control:LPP-OmpA-scFv-His (43 kDa)

These two results confirms the enhanced functions of INPNC parts (BBa_K811005) existing in the iGEM Regisrtry.

Please see ☆BBa_K1933100, BBa_K1933101, and BBa_K1933200 for full characterization of this part.

Judging

We fulfilled criteria listed below with this part.

• Validated Part/ Validated Contribution(Silver)
• Improve a previous part or project(Gold)
• [http://2016.igem.org/Team:Kyoto/Proof Proof of concept](Gold)

Reference

[1]Yim, Sung-Kun, et al. "Surface display of heme-and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation." Journal of microbiology and biotechnology, 20.4, (2010): 712-717.

[2]Park, Tae Jung, et al. "Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis." Microbial cell factories, 12.1, (2013): 1.