Difference between revisions of "Part:BBa K844015"
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IMPORTANT NOTE: This part uses [https://parts.igem.org/Assembly_standard_23 Assembly Standard #23 (Silver Fusion)] scar sites, which the sequence data below will not reflect (it shows [https://parts.igem.org/Assembly_standard_10 Assembly Standard #10] scars).
 
IMPORTANT NOTE: This part uses [https://parts.igem.org/Assembly_standard_23 Assembly Standard #23 (Silver Fusion)] scar sites, which the sequence data below will not reflect (it shows [https://parts.igem.org/Assembly_standard_10 Assembly Standard #10] scars).
  
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===Usage and Biology===
 
===Usage and Biology===
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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 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K208000 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 ([https://parts.igem.org/wiki/index.php?title=Part:BBa_K208010 BBa_K208010]) used in this system was also taken from Utah State iGEM 2009.
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https://static.igem.org/mediawiki/2012/c/cc/Gfp_plates.png
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Figure 1. 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.
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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.
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https://static.igem.org/mediawiki/2012/f/fc/GFP_2.JPG
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Revision as of 04:38, 3 October 2012

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 will not reflect (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 (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 (BBa_K208010) used in this system was also taken from Utah State iGEM 2009.

Gfp_plates.png

Figure 1. 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.


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.


GFP_2.JPG


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 443
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 962
    Illegal XhoI site found at 863
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]