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Team:Utah State/Results
From 2012.igem.org
Cloning
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 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 used in this system was also taken from Utah State iGEM 2009 (BBa_K208010, http://partsregistry.org/wiki/index.php?title=Part:BBa_K208010). 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
Silk Production
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. This is the first ever spider silk thread produced from BioBrick parts.
