Part:BBa_K2940003

Synthetic silk MaSp1 with flanking solubilizing blocks and head-to-tail assembly system

Silk proteins are composed of long stretches of monomer repeats. Major Ampullate Spidroin (MaSp1) protein is a monomer component of dragline spider silk. This BioBrick encodes a single monomer of MaSp1 methionine added with a system in place to build monomer repeats using repeated digestion and ligation (head-to-tail multimerization). This allows a simple way to build long chains of MaSp1 repeats to produce the silk protein fiber. The BioBrick also contains a polyglutamine sequence flanking the monomer repeats motif which helps to solubilize the produced protein.

Usage and Biology

• N. clavipes truncated version of MaSp1 monomer repeat for synthetic silk production.

• Head-to-tail multimerization strategy to create long monomer repeat sequences by doubling the size.

• Flanking glutamic blocks for solubilization

Additional information for successful head-to-tail multimerization

Contribution Part from Group: RUBochum, 2022; Author: Janzen Daniel

However, the use of highly repetitive modules in E.Coli also has its difficulties. The main one is the individual sequence, which requires individual consideration in the following areas. Not every E.Coli strain is suitable for sufficient plasmid and protein synthesis. Thus, at least three different strains should be chosen for optimal yield of plasmid. You can try cheap and common E. coli strains, but you should use more demanding variants. In addition, different vectors offer different possibilities for plasmid propagation and restriction and should also be checked for an optimal performance. It is recommended to amplify the gene sequence in a cloning plasmid (pbluescript ii ks +) and then later transfer it to an expression plasmid (pET28a). Finally, type II restriction enzymes offer a variety of different cutting options, with the main focus on single cut compatible sticky ends. Depending on availability and budget, care should be taken when planning. A list for selection, depending on the vectors used, can be found in Table 1, whereby it is important to ensure that the cleavage site is only contained once in the plasmid. For better ligation, we restrict ourselves to CG-containing interfaces.

Table 1. Compatible single cut restriction enzymes with a specific interface for sticky ends [1].

[1] New England Inc. (05.10.2022): Compatible Cohesive Ends and Generation of New Restriction Sites [online] https://international.neb.com/tools-and-resources/selection-charts/compatible-cohesive-ends-and-generation-of-new-restriction-sites

Characterization

Characterization using gel electrophoresis

To characterize the head-to-tail multimerization strategy for creating long monomer repeats chains, we monitor the kb size increase of the construct after each doubling round. The table 2 shows the expected sizes from each round.

Table 2. Expected size dimensions forecast of plasmid, multimerization constructs, and monomer-chain, plus construct molecular protein weight.

Results

1, 2, 4, and 8 MaSp1 monomer repeat chains were successfully constructed using this method. Figure 1 shows the sizes of the constructs corresponding each doubling round. The band sizes with 4 and 8 monomer repeats are clearly visible.

Figure 1. Electrophoresis gel digestions of plasmids with NheI and SpeI containing increasing silk monomer chain. Y-axis indicates the ladder band size marks. Superior X-axis shows the silk gene and monomer doubling round. Inferior X-axis indicates the expected band size for each monomer chain.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 128
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 20
  Illegal SpeI site found at 128
  Illegal NotI site found at 27
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 128
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 128
  
• 1000
  COMPATIBLE WITH RFC[1000]