Difference between revisions of "Part:BBa K1189030"
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<p><b>Figure 4.</b> Assay showing coiled-coil interaction <i>in vitro</i>. Crude lysates from a negative control (RFP), GFP-Ecoil and His-Kcoil were combined together to investigate interaction and immunoprecipitated with GFP or an isotype control and then further probed with α-His antibody. Only in the presence of both GFP and a His tag we see a band indicating interaction. | <p><b>Figure 4.</b> Assay showing coiled-coil interaction <i>in vitro</i>. Crude lysates from a negative control (RFP), GFP-Ecoil and His-Kcoil were combined together to investigate interaction and immunoprecipitated with GFP or an isotype control and then further probed with α-His antibody. Only in the presence of both GFP and a His tag we see a band indicating interaction. | ||
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| + | <p> | ||
| + | We ordered 60mer FAM-labeled [B] (TALE B target sequence) oligos and hybridized them with their reverse complement oligo to make double stranded pieces of DNA containing the target sequence of our TALEs. Using these target sequences and following the <a href="http://2013.igem.org/Team:Calgary/Notebook/Protocols/FunctionalityAssayOnNitrocellulose" >TALE Nitrocellulose Functionality Assay</a>, we showed that TALEB binds to its target sequence. We incubated Ferritin fused to an Ecoil (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189018" >BBa_K1189018</a>) to TALE fused to a Kcoil (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189029" >BBa_K1189029</a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1189030" >BBa_K1189030</a>) to make the FerriTALE complex. The complex was then blotted on strips of nitrocellulose paper. The strips were then blocked with milk and soaked in the appropriate DNA solution. Finally, the strips were washed and imaged (figure 14 and 15). We performed a densitometery test on these results and were able to calculate the dissociation constant of the TALEs. | ||
| + | </p> | ||
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| + | <figure> | ||
| + | <img src="https://static.igem.org/mediawiki/2013/d/db/Calgary2013TALEBBlotWithKinetics.png" width="800" height="349"> | ||
| + | <figcaption> | ||
| + | <p><b>Figure 5.</b> (A) Dot blot of FerriTALE B exposed to FAM labeled DNA containing the [B] TALE B target sequence (<a href="http://2013.igem.org/Team:Calgary/Notebook/Protocols/FunctionalityAssayOnNitrocellulose" >protocol</a>). 1µg of ferritin fused to E coil was incubated with 2µg of TALE B fused to K coil for 1 hour to make the FerriTALE B complex. Subsequently the complex was blotted on the nitrocellulose strip. The blots were then exposed to 1.66 mM FAM labeled DNA from 1 to 90 minutes as indicated on the strips. The controls are to the right, with "Ftn" being ferritin only, "np" being no protein, and "D-" being no DNA exposure. The kinetics from the densitometry is shown in section B of the figure. The Kd from this plot was determined to be 66nM.</p> | ||
| + | </figcaption> | ||
| + | </figure> | ||
Revision as of 04:25, 29 October 2013
TALE-B with a his6 tag linked to a K coil under an inducible lacI promoter
For information on TALE B refer to BBa_K1189023.
E/K coils are synthetic coiled-coil domains designed specifically to bind to each other with high affinity and specificity (Litowski and Hodges, 2002) (Figure 1). They are composed of a heptad repeat that forms a coil structures that are able to interact with each other. These coils are able to interact with each other in an anti-parallel fashion that makes them useful for applications such as peptide capture, protein purification and in biosensors. For our project we decided to make use of the IAAL E3/K3 coils (BBa_K118901, BBa_K1189011) due to the balance they offer between affinity and specificity (Table 1).
Figure 1. Ribbon visualization of the E3/K3 IAAL coiled-coils.
| Coil Name | Peptide Sequence |
| IAAL E3 | NH2-EIAALEKEIAALEKEIAALEK-COOH |
| IAAL K3 | NH2-KIAALKEKIAALKEKIAALKE-COOH |
These E3/K3 coils are able to form heterodimers due to the hydrophobic residues contained within the heptad repeat. In our case these are isoleucine and leucine residues. Designated by empty arrows in the helical wheel diagram below (Figure 2) these residues form the core of the binding domain of the coils. In order to prevent the homodimerization of these coils charged residues are included in the design. The electrostatic interactions between glutamic acid and lysine residues prevent an E-coil from binding with an E-coil for example. These parts were already in the registry, however the DNA was never received, so we built, sequenced and re-submitted them.
Figure 2. A helical wheel representation of the IAAL E3/K3 coiled-coil heterodimer viewed as a cross-section based off of a similar figure created by Litowski and Hodges (2002). The peptide chain propagates into the page from the N- to the C- terminus. Hydrophobic interactions between the coils are indicated by the clear wide arrows. The intermolecular electrostatic interactions between the coils are displayed by the thin curved arrow (eg. Between Glu15 and Lys20) Letters a, b, c,and d designate the positions of IAAL repeat in the heptapeptide. The e and g positions are occupied by the charged residues.
We evaluated the binding of our coils using other constructs that make use of the E and K coil parts submitted. In the case of the coils we were interested to see if the K-coil fused to TALE proteins (BBa_K1189029, BBa_K1189030) could bind to the E-coil found on one of our Prussian blue ferritin constructs (BBa_K1189018). To complete this task we placed the TALE on the membrane, washed and blocked the membrane. The ferritin protein with the complimentary coil was then added to the membrane. If this coil successfully binds to the other coil then the ferritin will not be washed off during the next wash step. We can then see if Prussian blue ferritin is bound by adding a TMB substrate solution that will cause a colour change. To this extent we saw a blue ring in this trial indicating a positive result. This suggests that our coils are actually binding in an in vitro system.
Another interesting element of this assay is why we used two variants of the TALE K-coil negative control. A blue ring on our TALE negative control confirmed our fear that during the second protein application and wash step that some of the ferritin with coil proteins would drift over and bind to the TALE K-coils on the nitrocellulose. This did not occur for our separate negative control (Figure 3).
Figure 3. This basic qualitative assay was used to inform us whether certain elements of our system are able to bind to each other. Our TALE proteins were mounted to the membrane along with positive controls of three Prussian blue variants; two recombinant ferritins and one commercial protein. The membranes were then washed and blocked. Prussian blue ferritin with a coil was added to our TALE protein containing a coil. Prussian blue ferritin with a TALE that could bind to the DNA held by another TALE on the membrane was also added. A TMB substrate solution was added to cause a colourimetric change over 5 minutes. Positive results are indicated by dark rings of colour. Negative controls include a TALE with a coil on the same membrane and the same TALE and bovine serum albumin on separate membranes that were treated separately. Image contrast was altered to make the results more clear on a digital monitor; the same changes were applied to each element of the figure.
We also performed an immunoprecipitation assay to demonstrate the binding of the E/K coils (Figure 4).
Figure 4. Assay showing coiled-coil interaction in vitro. Crude lysates from a negative control (RFP), GFP-Ecoil and His-Kcoil were combined together to investigate interaction and immunoprecipitated with GFP or an isotype control and then further probed with α-His antibody. Only in the presence of both GFP and a His tag we see a band indicating interaction.
We ordered 60mer FAM-labeled [B] (TALE B target sequence) oligos and hybridized them with their reverse complement oligo to make double stranded pieces of DNA containing the target sequence of our TALEs. Using these target sequences and following the TALE Nitrocellulose Functionality Assay, we showed that TALEB binds to its target sequence. We incubated Ferritin fused to an Ecoil (BBa_K1189018) to TALE fused to a Kcoil (BBa_K1189029 and BBa_K1189030) to make the FerriTALE complex. The complex was then blotted on strips of nitrocellulose paper. The strips were then blocked with milk and soaked in the appropriate DNA solution. Finally, the strips were washed and imaged (figure 14 and 15). We performed a densitometery test on these results and were able to calculate the dissociation constant of the TALEs.
Figure 5. (A) Dot blot of FerriTALE B exposed to FAM labeled DNA containing the [B] TALE B target sequence (protocol). 1µg of ferritin fused to E coil was incubated with 2µg of TALE B fused to K coil for 1 hour to make the FerriTALE B complex. Subsequently the complex was blotted on the nitrocellulose strip. The blots were then exposed to 1.66 mM FAM labeled DNA from 1 to 90 minutes as indicated on the strips. The controls are to the right, with "Ftn" being ferritin only, "np" being no protein, and "D-" being no DNA exposure. The kinetics from the densitometry is shown in section B of the figure. The Kd from this plot was determined to be 66nM.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 2654
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]