Data Section

Data section stuff goes here, Asif!

Cloning

This year Utah State iGEM team has created 64 different spider silk BioBrick parts and has submitted 10 of these parts to the registry. We are the first iGEM team to create a collection of BioBrick parts and demonstrated that spider silk can be produced from these parts. Furthermore, from this work we have successfully demonstrated that the methods of standard assembly can be used to create these functioning spider silk BioBricks.

The figure below shows the 64 different spider silk BioBricks that we have created. All the 64 parts are in pSB1C3 so different parts can be assembled together to create different repeating spider silk units. An example of this would be to take the part ‘patgB8’ (which is an 8 x B unit with a promoter and rbs) and combine it with ‘B6HT’ (6 x B unit with 10 Histag) to produce ‘patgB14HT’.

After cloning separate spider silk units (U, W, F, and B) into pSB1C3 we wanted to show the range of spider silk subunits available to produce different spider silk. We decided to make ‘spider steps’ or ‘spider ladder’ out of the ‘B’ and ‘F’ constructs. These are shown in the agarose gel in the figure below. From this figure there is an insert and a vector band (pSB1C3) and the insert size increases with increased number of repeating subunits. Also, at the high level of repeating unit the insert is larger than the vector backbone. In addition to showing spider silk units, we believe that this is a classic demonstration of the standard assembly which is an excellent tool to teach a new person the methods of standard assembly.

Using the spider silk BioBrick parts we have run different DNA agarose gels to show the various sizes of these BioBricks. One such gel we ran to make the letter U, S, and U which stands for Utah State University (USU). This gel is shown below with the legend directly below it showing the different silk BioBrick units used. The first ‘U’ consists entirely of ‘F’ BioBrick units, the second ‘U’ consists of only ‘B’ BioBrick units and the ‘S’ is a mix of both ‘F’ and ‘B’.

GFP Expression

Green fluorescent protein (GFP) has been used by various iGEM teams to demonstrate expression and functionality of a BioBrick system. In order to demonstrate that the expression of a BioBrick spider silk gene (F1) is possible in E.coli a single spider silk gene was tagged with GFP at the C terminus to demonstrate silk protein expression. The GFP that was chosen for this study was taken from Utah State iGEM 2009 ( BBa_K208000 ) as it demonstrated high levels of GFP expression. This GFP protein has an excitation wavelength of 395nm and an emission wavelength of 509nm. The lac promoter and ribosome binding site ( BBa_K208010 ) used in this system was also taken from Utah State iGEM 2009. A plasmid map demonstrating this construct is shown below and this plasmid was transformed into DH5α.

The bacterial cells harboring the patgF1GFP plasmid was spread on LB plates containing chloramphenicol and isopropyl-β-D-1-thiogalactopyranoside (IPTG). The figure below shows that the GFP construct is functional and expressing both spider silk (F1) and GFP.

GFP Fluorescent Microscopy

To show that bacteria fluorescence at the cellular level the E. coli were heat fixed and observed using an inverted microscope (Nikon Eclipse Ti-U, Melville, NY) equipped with a B-2A Longpass Emission filter set, a Photometrics® CoolSNAP HQ2 high-resolution camera, and a 100X oil immersion objective was used along with 10X oculars. GFP was exposed for 1.5 s and a DAPI filter was used for GFP imaging. Images were taken using NIS-Elements software (Nikon, Melville, NY). An image demonstrating the expression of both F1 spider silk protein and GFP in DH5α is shown below.

Protein Expression

This 10x-Histidine tag (BBa_K844000) 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 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). This demonstrates that the 10x-histidine tag is functioning and is compatible with staining and antibody techniques used with 6x-histidine tags.

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.

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.

Silk Production

This is the first ever spider silk thread produced from BioBrick parts.

Once the spider silk protein has been isolated via the nickel column and purified it is then freeze dried and ‘doped’ (mixed in) with Hexa-Fluoro-Isopropanol (HFIP). The protocols page goes into the details of this process. The silk is then spun and collected on a spool. The image below shows the silk that was collected from the patgB4HT construct.