Part:BBa_K2027007

Bacterial Collagen with Homotrimeric Coiled-Coil Domain

To be added before 2016 deadline.

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
  COMPATIBLE WITH RFC[1000]

Characterization

After induction of our liquid cultures, we extracted the proteins from our constructs. We kept all protein washes to assure that our desired protein product did not get extracted earlier than expected (we expected the protein to be in the final elution buffer washes). After confirming its presence in the final elution washes (shown in Figure 6), we ran the extracts against each other. To verify the size of the protein, we ran our protein extractions in SDS-PAGE gels. We mostly used Ni columns to purify our protein extract. Since our construct has a Lumio-Flag-His tag, we also used Anti-FLAG magnetic beads, with the help of our mentor Kosuke, to examine which protocol was most efficient in extracting protein.

<figure class="fig"><img src="" class="img-R"><figurecaption>Figure 6: SDS-PAGE gel of all washes of extracted proteins using Ni-NTA resin columns and Lumio staining. AF stands for Anti-FLAG, and represents samples that were extracted using Anti-FLAG magnetic beads.</figurecaption></figure>

<figure class="fig"><img src="" class="img-L"><figurecaption>Figure 7: SDS-PAGE gel of extracted C2 and CN proteins (lanes 2-5 and lanes 6-8 respectively) using Lumio stain</figurecaption></figure>

In Figure 7, lanes 2-5 represent the Lumio stained protein extract of the BioBrick <a href=https://parts.igem.org/Part:BBa_K2027005>BBa_K2027005</a> or construct 2 (C2). Lanes 2,3, and 4 are respectively first, second, and third elution buffer washes using the His-tag nickel resin columns. Lane 5 is also C2's extracted protein, however it was extracted using a different protocol, Anti-FLAG. Lanes 6-8 contains corresponding elution buffers of the natural construct (CN), <a href=https://parts.igem.org/Part:BBa_K2027007>BBa_K2027007</a>. CN was extracted using His-tag nickel resin columns.

Figure 8 also represents an SDS-PAGE gel containing the protein extracts of <a href=https://parts.igem.org/Part:BBa_K2027003>BBa_K2027003</a> (natural construct without the elastin cross-linking domain or SN) in lanes 2-4 and <a href=https://parts.igem.org/Part:BBa_K2027002>BBa_K2027002</a> in lanes 5-7. BBa_K2027002 is the fully assembled heterotrimeric coiled-coil collagen fiber monomer, consisting of the domains BBa_K2027004, BBa_K2027005, and BBa_K2027006. Because the fully assembled construct contains three domains, we expected the protein produced would be at least three times larger than the proteins with singular constructs. However, as indicated by the gel in Figure 2, BBa_K2027002 did not produce this size, which suggested that the assembly of all parts may have introduced errors in the protein synthesis.

<figure class="fig"><img src="" class="img-L"><figurecaption>Figure 8: SDS-PAGE gel of extracted SN and full construct proteins (lanes 2-4 and lanes 5-7 respectively) using Lumio stain</figurecaption></figure>

We also explored the possibility of cross-linking the proteins. Our plan was to cross-link the proteins and compare the results with un-processed protein in an SDS-PAGE. Initially, we wanted to test small samples of cross-linked C1, C2, and C3 and S1, S2, and S3 against non-cross-linked samples. We expected cross-linked proteins to have a higher molecular weight than proteins that weren't. Since this was our first time attempting to cross-link proteins, we wanted to test our protocol on only one sample before proceeding on a large-scale cross-linking experiment.

In this experiment, we used pure pyrroloquinoline quinone (PQQ) as a catalyst for the cross-linking reaction for sample construct CN. PQQ was diluted in a copper (II) solution and mixed with the purified protein. Since the purified protein exists in a phosphate buffer (extract was purified from the Ni-NTA resin columns), we maintained the pH of the reaction solution and kept it close to pH 7 while adding excess copper because the precipitation of copper(II) phosphate causes an increase in acidity of the buffer. When the solution became basic, we found that the solution was mostly clear and the precipitate was a light green-blue color, likely copper hydroxide. When the solution was nearly pH 7 and borderline acidic, the solution turned a clear blue and produced a fine blue copper(II) phosphate precipitate, but copper remained in solution. We incubated the two different sample solutions at room temperature for 24 hours. After 24 hours, we found that the solutions separated into two layers. We weren't sure if the cross-linked protein would exist in the precipitate-like layer or the supernatant so we ran both against the original untreated CN in a gel.

Tubes containing the cross-linking reaction solution. Left tube is pH 8 and right tube is pH is 6.8.

SDS-PAGE with Lumio stain of non-cross-linked and cross-linked CN protein. Lane 2 is the untreated CN protein. Lanes 3 and 4 are the pH 6.8 tube's precipitate and supernatant, respectively. Lanes 5 and 6 are the pH 8 tube's precipitate and supernatant, respectively.

The SDS-PAGE gel results showed that cross-linking did not occur. It is difficult to read the gel because the protein may have run off early on. In this experiment, we used a different gel percentage, 8%, because we ran out of our usual gel type. This is the third iteration of running our SDS-PAGE gel, because we had trouble figuring out the optimal run conditions. Even though the gel was run at 200 V for 10 minutes, the samples were mostly at the bottom. This may have contributed to the production of a messy gel. Due to time constraints and material limitations, we could not rerun more gels to further optimize our protocol. Despite the difficulty in gauging the exact results of the gel, we estimated our results of failure based on the location of the untreated CN band. The bands in the other lanes were approximately around the same location, suggesting that the treated proteins did not change in size. We found it peculiar that the lanes 3 and 4 had additional bands of smaller molecular weight and are unsure what it is.