Difference between revisions of "Part:BBa K2940003"

Revision as of 20:53, 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.

[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.]

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]

@@ Line 21: / Line 21: @@
+[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.''']
-[[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.''']]
@@ Line 30: / Line 27: @@
 , 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.