Difference between revisions of "Part:BBa K2940003"

(→‎Characterization)
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[Image:T--Edinburgh OG-- Table MaSp1.png|400px|thumb|Table 1. '''Expected size dimensions forecast of plasmid, multimerization constructs, and monomer-chain, plus construct molecular protein weight.''']
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[[Image:T--Edinburgh OG-- Table MaSp1.png|400px|Table 1. '''Expected size dimensions forecast of plasmid, multimerization constructs, and monomer-chain, plus construct molecular protein weight.''']]
  
  
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[[Image:T--Edinburgh OG-- Figure2 MaSp1.png|400px|thumb|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.''']]
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[[Image:T--Edinburgh OG-- Figure2 MaSp1.png|400px|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.''']]
  
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Revision as of 20:56, 20 October 2019


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

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 1 shows the expected sizes from each round.


Table 1. 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]