Coding
E coil fer

Part:BBa_K1189037

Designed by: Chris Wintersinger, Denny Hoang, Taylor Remondini   Group: iGEM13_Calgary   (2013-10-24)
Revision as of 10:34, 31 October 2013 by Cmwinter (Talk | contribs)

Ferrtin fusions

Ferritin is an protein shelled nanoparticle and is composed of a mixture of 24 light (BBa_K1189024) and heavy (BBa_K1189025) subunits. It is ubiquitous across eukaryotic and prokaryotic systems and is used to sequester intracellular iron. The 2013 iGEM Calgary used ferritin’s iron core as a reporter and its protein shell to scaffold DNA sensing TALEs (see Figure 1).

BBa_K1189037 joined with DNA sensing TALEs

Figure 1. 3D in silico rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. TALEs are used to sense DNA while the iron core is used to report its presence. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.

This particular version is a fusion of heavy and light ferritin subunits, such that nanoparticles are formed from 12 di-subunits. The rationale for this design is that it reduces the number of N-termini on ferritin to which proteins can be fused by half, which is important for lessening potential steric hindrances among fused proteins in the 3D sphere surrounding ferritin. Additionally, di-subunits ensure a 1:1 ratio of heavy and light subunits which ensures consistency in ferritin’s ability to uptake iron. Moreover, these fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (Dehal et al., 2010).

Design features

This part has an N-terminal fusion to an E coil connected to ferritin by a GS linker. The coil system is of utility to other iGEM teams because they can express K coils on their own proteins of interest, and bind them to the complementary E coil on ferritin. Such a coiled-coil linker system reduces potential for large protein fusions to harm ferritin formation, allowing user to build intricate nanoparticle devices with myriad proteins. See Figures 2 application examples.

FerriTALE Scaffold Modularity

Figure 2. Using the E and K coils in combination with ferritin as a scaffold system allows the creation of brand new FerriTALEs or protein scaffolds.

This part is identical to BBa_1189018, with the exception of a his-tag for purification.

Results

The 2013 iGEM Calgary successfully expressed and purified this protein in pSB1C3 and per this part sequence exactly using and FPLC and metal affinity purification of the his tag. See Figure 3 for an SDS-PAGE of this isolate. Please see the experience page for data on another expression vector which generated this protein with a higher yield.

GEL

This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 4 shows that this part with coiled-coils performs better as a reporter than direct fusions to TALEs (BBa_K118021). It seems that large protein fusions reduce effectiveness of ferritin as a reporter. Figure 5 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.

Creating Prussian Blue Ferritin out of our Own Ferritin

Figure 4. Measurements of the coloured substrate TMB (10 mg/mL) at 650 nm over a 600 second time period for our own Prussian blue ferritin and unmodified ferritin. Sample volume was 242 µL. Controls for this experiment include bovine serum albumin (1 mg/mL)and the substrate solution by itself. Due to limitations on the protein available only one replicate was performed. Zero time points do not have low absorbance as colour change was rapid and began before measurements started.

Recombinant Prussian Blue FerritinMole Balanced

Figure 5. Samples of our parts that were converted to Prussian Blue ferritin were mole balanced in order to ensure that the same number of effective ferritin cores are present in every sample. Additionally the ferritin-coil fusion was incubated with the TALE-coil fusion part in order to allow their binding for a separate trial. Negative controls include unconverted recombinant ferritin, bovine serum albumin and a substrate only control. Samples were incubated with a TMB substrate solution for 10 minutes at a pH of 5.6. Absorbance readings were taken at the 10 minute time-point at a wavelength of 650 nm. An ANOVA (analysis of variants) was performed upon the values to determine that there was statistical difference in the data gathered (based off of three replicates). A t-test was then performed which determined that the * columns are significantly different from the ** column (p=0.0012). Neither * column is significantly different from each other (p=0.67).

Please see the experience page page for a detailed analysis of how Prussian blue ferritin performs as a reporter.

References

  • Dehal, P. K., Livingston, C. F., Dunn, C. G., Buick, R., Luxton, R., & Pritchard, D. J. (2010). Magnetizable antibody‐like proteins. Biotechnology journal, 5(6), 596-604.
  • 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
      COMPATIBLE WITH RFC[25]
    • 1000
      INCOMPATIBLE WITH RFC[1000]
      Illegal BsaI.rc site found at 1307


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