Part:BBa_K4439007

mSA-N[AS]4C-CBD-10xHis

Abstract

To complete

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]

Protein Characterization

Usage and Biology

• Silk proteins demonstrate interesting mechanical properties such as toughness, strength, lightweight, biodegradability and the possibility to produce different morphologies (fibers, foams, capsules, films). In addition to this, silk proteins comprise a high percentage of the amino acids glycine, serine and alanine which have an intermediate hydrophobicity.

• Green lacewing insects produce two types of silk: one produced by the larvae (cocoon) and the other by adult females (egg-stalk). The adult produced silk acts as a protective shelter and structural support for egg stalks, which are two ideal properties for a waterproof coating for our aerogel.

• In green lacewings, two serine- and glycine-rich silk proteins (Ma1XB1 and Ma1XB2) have been identified, both with highly repetitive core domains and small terminal domains. The core domain’s structure is rich in β-sheets with an approximative sheet-length of four amino acids between turns. These form repeating structural units constituting β-helices which have a significant positive correlation with the proteins’ surface hydrophobicity. A consensus motif for the core domain of Ma1XB2 (named [AS])had already been generated. Furthermore, a recombinant protein constituted by 8 repetitions of this [AS] module had also already been expressed in E. coli.

Modeling

Figure 8 | AlphaFold2 prediction for 01a. (A) First rank 3D model prediction of 01a protein; the iteration who got the highest score in the modeling. (B) Different correlation graphs between the query sequence of 01a and the predicted one, for each proposed 5 models. (C) Figure of sequence coverage of 01a and indices on alignment with other sequences in the mSA. (D) IDDT graph for 01a per residue to get an idea of the confidence of the model in predicting the geometry.

• Analysis : According to (fig. 8, A), we could identify the structure of the different single chains of interest easily. The blue slightly smaller helicoidal structure represents the mSA protein that would be attached to the silk protein, the elongated green structure and finally we would get the CBD sequence that is displayed in red. The (fig.8, B) confirms the hypothesis of knowing very efficiently the three domains and having trouble distinguishing the in-between linkage which is totally normal since we designed those linking segments to be able to keep the structure of the three main elements. The same graph showed that few iterations of the prediction lacked to characterize the silk segment, which could be an issue in the experiments. However both (fig. 8, C) and (fig. 8, D), confirmed the reasoning that our proteins would keep a structure preserving their initial aim. The linkage would give in reality more freedom of placement of those chains. The IDDT score helped to identify the percentage of correctly predicted and true structure that could be superimposed. In general, the higher this score the better the model is considered.

Experiments

Figure 1 | Protein Purification Protocol using Ni-NTA Beads

• For more information, check out our Protocols page on our wiki.

Figure 2 | Diagram of Procedure for Silk Biofilm Fabrication

• For more information, check out our Protocols page on our wiki.

Lab's Tips and Tricks

• Purification might prove difficult : elution with EDTA works better but might damage the protein more.

• The silk biofilm can be created even if the recombinant protein is in liquid form.

Results

Expression

To complete

Purification

To complete

Biofilm Fabrication

To complete

Hydrophobicity tests

To complete