Difference between revisions of "Part:BBa K1189037:Experience"
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<p><b>Figure 2.</b> Ferritin di-subunit fused to an E coil and his tag. The E coil allows binding to other proteins expressing a complementary K coil.</p> | <p><b>Figure 2.</b> Ferritin di-subunit fused to an E coil and his tag. The E coil allows binding to other proteins expressing a complementary K coil.</p> | ||
Latest revision as of 07:21, 2 November 2013
A fusion of two ferrtin subunits
Ferritin is a 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 (Chasteen et al., 1991). The 2013 iGEM Calgary used ferritin’s iron core as a reporter and its protein shell to scaffold DNA sensing TALEs as part of their project, the FerriTALE (see Figure 1).
Figure 1. 3D in silico rendering of BBa_K1189037 formed into functional nanoparticles bound to DNA sensing TALEs. The iron core is chemically modified and use to show when TALEs are bound to DNA. The TALEs are one specific, particular application of the ferritin E coil di-subunit fusion. This nanoparticle is the molecular basis of a DNA lateral flow strip biosensor pursued by the 2013 iGEM Calgary team.
BBa_K1189037 is a fusion of heavy and light ferritin subunits, such that ferritin 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 mandate 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 (Figure 2). 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 3 application examples.
Figure 2. Ferritin di-subunit fused to an E coil and his tag. The E coil allows binding to other proteins expressing a complementary K coil.
Figure 3. 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
Expression from pSB1C3
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 4 for an SDS-PAGE of this 42 kDa isolate. Please see the user review section for data on another expression vector which generated this protein with a higher yield.
Figure 4. FPLC purification fraction of BBa_K1189037 as express per our large scale expression protocol. Were were disappointed with the yield of this 42 kDa protein from the pSB1C3 cloning vector. Please see the user reviews section for results from a proper expression vector.
Conversion of BBa_K1189037 into a reporter
This purified protein product was successfully converted into Prussian blue ferritin, a robust colourmetric reporter. Figure 5 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 6 shows that ferritin with coiled-coils (BBa_1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers.
Figure 5. 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.
Figure 6. 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 Prussian blue ferritin Wiki page for a detailed analysis of how Prussian blue ferritin, synthesized from commercially available ferritin, performs as a reporter. This data informs how BBa_K1189037 is useful as a reporter.
References
User reviews
iGEM Calgary 2013 review
We found that expression of BBa_K1189037 was minimal when expressed in pSB1C3 cloning vector. To yield of protein in the soluble extract, we attempted expression in a different expression plasmid borrowed from Dr. Anthony Schryvers, one of the iGEM Calgary 2013 advisors. The protein coding sequence was extracted from BBa_K1189037 using PCR and placed into vector 4365 using isothermal assembly. This vector sequence placed maltose binding protein (MBP) as an N-terminal fusion with the di-subunit ferritin fusion. MBP is known to improve solubility of expressed proteins. Additionally, there is a TEV protease cleavage site between MBP and the di-subunit ferritin, which allowed us to remove MBP to generate functional ferritin. See Figure A for a diagram of the fusion genes with the new vector.
Figure A. Alternative expression vector for the coding sequence from BBa_K1189037. Proteins are fused to Maltose Binding Protein (MBP), which is useful for stabilizing expression of proteins. MBP was removed from E coil ferritin using TEV protease.
As seen in Figure B, this alternative T7 expression system significantly improved protein yield compared to the pSB1C3 protein expression. From 1L of culture, we yield approximately 8mg of E coil ferritin. Unfortunately, the Calgary team did not have time prior to finals to convert vector 4365 to the iGEM RFC10 standard. Henceforth, we were unable to share this vector in the Parts Registry. Future teams can investigate this new construct here.
Figure B. Expression of the coding sequence from BBa_1189037 in a low copy number E. coli expression vector. The purified di-subunit is approximately twice the molecular weight of the single ferritin subunit Sigma horse spleen ferritin control.
Additionally, protein expression/purification protocols were similar to the one on our Wiki. There were a few modifications including use of a glucose repression media to grow overnight cultures, use of a lactose/glucose autoinduction media, use of E. coli ER2566 instead of E. coli BL21, and two runs through an FPLC separated by incubation with TEV protease to remove MBP.
References
UNIQ497274650760bb68-partinfo-00000001-QINU UNIQ497274650760bb68-partinfo-00000002-QINU