Difference between revisions of "Part:BBa K844016"

Revision as of 23:57, 3 October 2012

Spider Silk Generator - 4x "B" Silk Construct with His Tag

This part was used to produce the first ever spider silk thread made using BioBrick parts.

Protein generator part for Spider Silk. Protein is a 4x fusion of spider silk "B" subunits (1x BBa_K844008 followed by 3x BBa_K844004), which were codon optimized to use a reduced set of tRNA codons (1 to 2 codons per amino acid). Contains a lactose/IPTG inducible promoter, RBS, and a 10x-His tag (BBa_K844000) for purification of protein.

IMPORTANT NOTE: This part uses Assembly Standard #23 (Silver Fusion) scar sites, which the sequence data below will not reflect (it shows Assembly Standard #10 scars).

Experience and Results

A 10x-Histidine tag was used to isolate and analyze spider silk protein produced by the 2012 Utah_State iGEM Team. Below is a Coomassie stained SDS PAGE gel showing the 4x "F" subunit spider silk protein (~25.4 kDa) that was purified using a Nickel affinity resin column. The protein in the lane is nearly pure, with only minor bands at 24 kDa and 15 kDa as contaminants. The 10x-His tag aids in producing highly pure protein samples as it binds more tightly to the column, allowing higher concentration wash steps to be used to remove contaminants from the sample. Also below is a Western blot utilizing an antibody that binds specifically to histidine tags, showing a spider silk protein band at the correct molecular weight (~25.4 kDa).

The final picture shows a fiber of spun spider silk produced from this construct. This fiber is the first thread of spider silk ever produced using BioBrick parts. The procedures for the isolation of the spider silk protein and spinning into a silk fiber is detailed on the [http://2012.igem.org/Team:Utah_State/Notebook#SpiderSilkProtocols 2012 Utah_State iGEM protocols page].

Figure 1. Coomassie blue stained SDS PAGE gel showing highly pure spider silk sample. Protein expressed is BBa_K844016. The first lane contains the cell lysate sample. The second lane is the flowthrough from the column; note the absence of spider silk band from this flowthrough, indicating the high affinity of the 10x-His tag. The third lane is the eluted spider silk band, with only very minor contaminating bands. The last lane is the Bio-Rad Precision Plus Dual Color Protein Standard, with protein sizes indicated in kDa.

Figure 2. Western Blot of Spider Silk Protein. Protein expressed is BBa_K844016. Control lane is E. coli DH5a cells without spider silk construct. Marker lane is Bio-Rad Precision Plus Dual Color Protein Standard. Primary antibody binds specifically to histidine tags. Staining was done with alkaline phosphatase attached to the secondary antibody.

Figure 3. Spider silk fiber produced from BBa_K844016 protein generator. This is the first ever spider silk thread produced from BioBrick parts. Ruler shows silk size in centimeters. Once the spider silk protein has been isolated via a nickel affinity resin column and purified, it is then freeze dried and ‘doped’ (mixed in) with Hexa-Fluoro-Isopropanol (HFIP). The [http://2012.igem.org/Team:Utah_State/Notebook#SpiderSilkProtocols 2012 Utah_State iGEM protocols page] goes into the details of this process. The silk is then spun and collected on a spool. The image above shows the silk that was collected from the BBa_K844016 construct.

Related Parts

BBa_K844000 - A 10x-His tag with double stop codons (TAATAA) which was vital to the creation of the gel and blot shown above.

BBa_K844002 - The non-optimized native spider silk subunit that K8440016 is a codon optimized and improved concatemer of.

Sequence and Features