Part:BBa_K844015
lac/IPTG inducible Spider Silk 1x "F" Subunit fused to GFP
This part is a spider silk 1x "F" subunit (BBa_K844007) with GFP (BBa_K208000) fused to its C-terminus. This generator is lactose/ITPG inducible.
IMPORTANT NOTE: This part uses Assembly Standard #23 (Silver Fusion) scar sites, which the sequence data below and on composite parts using this part will not reflect this scar sequence (it shows Assembly Standard #10 scars).
Usage and Biology
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 (BBa_K208003) is possible in E. coli a single spider silk gene was tagged with GFP at the C-terminus. By tagging the C-terminus, only when full length constructs of the silk are produced will the GFP marker be attached. This production is not expected to be an issue with the 1x "F" subunit, but as the number of silk subunits fused together increases, eventually the cell will be unable to keep the ribosome bound to the mRNA through the entire silk construct. When it falls off early, no GFP is fused and expressed, so GFP fluorescence can be used as an indirect measure of the amount of full-length spider silk proteins in the cell.
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.
Figure 1. Fluorescent E. coli cultures containing the fused 1x "F" spider silk and GFP proteins. The bacterial cells harboring BBa_K844015 were spread on LB plates containing chloramphenicol and IPTG. The figure shows that the GFP construct is functional and expressing both 1x "F" spider silk protein and GFP.
Fluorescent Microscopy
Figure 2. E. coli cells expressing the fused spider silk and GFP protein. This demonstrates that the bacteria fluoresce 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).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 443
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 962
Illegal XhoI site found at 863 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |