Difference between revisions of "Part:BBa K1189021:Experience"
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<h3>iGEM Calgary 2013</h3> | <h3>iGEM Calgary 2013</h3> | ||
| − | <p>We expressed and purified this protein from the pSB1C3 cloning vector and isolated a limited amount of product using metal affinity purification and an FPLC (see the <a href="http://2013.igem.org/Team:Calgary/Notebook/Protocols/LargeScaleProteinExpressionAndPurification">large scale expression protocol</a>. We suggest that future teams attempt switching the protein sequence into a low copy number expression vector to improve yield as per our attempt with <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_K1189037#User reviews</a>. | + | <p>We expressed and purified this protein from the pSB1C3 cloning vector and isolated a limited amount of product using metal affinity purification and an FPLC (see the <a href="http://2013.igem.org/Team:Calgary/Notebook/Protocols/LargeScaleProteinExpressionAndPurification">large scale expression protocol</a>. We suggest that future teams attempt switching the protein sequence into a low copy number expression vector to improve yield as per our attempt with <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189037">BBa_K1189037:Experience#User reviews</a>. |
<br></br> | <br></br> | ||
Revision as of 17:52, 1 November 2013
Fusion of ferrtin subunits linked to a DNA detector
Ferritin is a protein shelled nanoparticle and is composed of a mixture of 24 light (BBa_K1189024) and heavy (BBa_K1189025) subunits (see Figure 1). 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 a DNA sensing TALE (BBa_K1189022) as part of their project, the FerriTALE. The TALE sequence was based off of BBa_K782004 from the 2012 Slovenia iGEM team. The Calgary team used this TALE as a proof of concept for their DNA detector.
Figure 1. Ribbon visualization of a fully assembled ferritin protein.
Design features
BBa_K1189021 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 the fused TALE in the 3D sphere surrounding ferritin. Additionally, di-subunits mandate a 1:1 ratio of heavy and light subunits, ensuring consistency in ferritin’s iron uptake dynamics. Moreover, these di-subunit fusions have been shown stable in engineered applications with other proteins scaffolded to ferritin (Dehal et al., 2010).
TALE A (BBa_K1189022) is attached to the N-terminus of the ferritin di-subunit. Given that the C-terminus of ferritin is oriented toward the core of ferritin, and that we wanted proteins to be displayed on the outside of the ferritin sphere, we selected the N-terminus (Luzzago et al., 1989). TALE A is connected to ferritin by a flexible GS linker (BBa_1189022). The iGEM Calgary team made this decision because spatial modelling suggested that TALEs require freedom of movement to allow for DNA binding. See Figure two for a diagram of BBa_K1189021.
Figure 2. TALE A is directly fused via a GS linker to a di-hybrid ferritin fusion. This construct will scaffoled 12 TALE A to the outside of ferritin.
Results
Performance of BBa_K1189021 as a reporter
BBa_K1189021 was expressed in E. coil BL21 and purified using metal affinity chromatography on an FPLC. The purified protein was chemically modified into Prussian blue ferritin. Output from this TALE-ferritin fusion (BBa_K1189021) was compared to output from ferritin/TALEs bound with a coiled-coil linker (BBa_K1189037).
Purified K_1189021 was successfully converted into Prussian blue ferritin. However, Figure 5 shows that the TALE ferritin fusion is a less effective reporter than ferritin and TALEs connected with coiled-coils linkers (BBa_K1189037). It seems that large protein fusions reduce effectiveness of ferritin as a reporter. Figure 6 shows that ferritin with coiled-coils (BBa_K1189037) maintains reporter functionality when TALEs are scaffolded using coiled-coil linkers. Therefore, the 2013 iGEM Calgary team has elected to use BBa_K1189037 as their FerriTALE DNA detector.
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.
User reviews
iGEM Calgary 2013
We expressed and purified this protein from the pSB1C3 cloning vector and isolated a limited amount of product using metal affinity purification and an FPLC (see the large scale expression protocol. We suggest that future teams attempt switching the protein sequence into a low copy number expression vector to improve yield as per our attempt with BBa_K1189037:Experience#User reviews.
References
User Reviews
UNIQ7f9c49caa192536c-partinfo-00000001-QINU UNIQ7f9c49caa192536c-partinfo-00000002-QINU