Difference between revisions of "Part:BBa K1127003"

Latest revision as of 20:56, 3 October 2013

MIDAS2 gold binding peptide

MIDAS2 is a gold binding synthetic peptide. We synthesized a gene expressing this peptide with a pelB signaling sequence to be excreted out of the E. coli.

Characterization

We created four variants of the fusion between MIDAS-2 and A3. We also include the N-terminal pelB tag for secretion to the periplasm and IM9 for higher stability. Note that the tag should be removed during transportation process and potentially our peptides should be released to the extracellular space. IM9 tag is relatively small (~9.5 kDa) and should not interfere with mineralization mediated by the peptides.

For peptide activity testing , we cloned the peptides under the constitutive promoter (BBa_J23100). Characterization using Nanoparticle Tracking Analyzer (NanoSight LM10 ®). The device allows us to visualize, measure and characterize any type of nanoparticles and any size down to 10nm. The technology is based on optical properties as well as Brownian motion of the particles. Please visit http://www.nanosight.com/ for more information.

We obtained a series of recordings and several analytical reports of the particles within samples from the overnight cultures of E. coli strain DH5 alpha transformed with the hybrids. Unfortunately, the results can't provide evidence for gold bio-mineralization because changes were subtle and not significant compared to the controls (untransformed E. coli). Please visit out Protocols section for more detail [http://2013.igem.org/Team:York_UK/Notebook.html?page=protocols].

File:MIDAS2 gold binding peptide.mp4

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 78
Illegal AgeI site found at 91
1000
COMPATIBLE WITH RFC[1000]

@@ Line 5: / Line 5: @@
 ==Characterization==
-We tried to characterize the part using Nanoparticle Tracking Analyzer from NanoSight [http://www.nanosight.com]. The part was cloned into BBa_J23100 [https://parts.igem.org/Part:BBa_J23100] which allows constitutive expression of the peptide.
+We created four variants of the fusion between MIDAS-2 and A3. We also include the N-terminal pelB tag for secretion to the periplasm and IM9 for higher stability. Note that the tag should be removed during transportation process and potentially our peptides should be released to the extracellular space.  IM9 tag is relatively small (~9.5 kDa) and should not interfere with mineralization mediated by the peptides.
+For peptide activity testing , we cloned the peptides under the constitutive promoter (BBa_J23100). Characterization using Nanoparticle Tracking Analyzer (NanoSight LM10 ®). The device allows us to visualize, measure and characterize any type of nanoparticles and any  size down to 10nm.  The technology is based on optical properties as well as Brownian motion of the particles.  Please visit http://www.nanosight.com/ for more information.
+We obtained a series of recordings and several analytical reports of the particles within samples from the overnight cultures of E. coli strain DH5 alpha transformed with the hybrids. Unfortunately, the results can't provide evidence for gold bio-mineralization because changes were subtle and not significant compared to the controls (untransformed E. coli). Please visit out Protocols section for more detail [http://2013.igem.org/Team:York_UK/Notebook.html?page=protocols].
-We obtained recording (below) and several analytical reports of the particles within samples from the overnight cultures (E. coli strain DH5 alpha + BBa_K1127003/BBa_J23100 hybrid). The results can't provide evidence for gold bio-mineralization by the peptide because changes were subtle and not significant compared to the controls. Please visit our wiki page for more information [http://2013.igem.org/Team:York_UK].
 [[File:MIDAS2 gold binding peptide.mp4]]