Difference between revisions of "Part:BBa K844015"

Latest revision as of 16:18, 10 May 2013

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

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]

@@ Line 4: / Line 4: @@
 This part is a spider silk 1x "F" subunit ([https://parts.igem.org/Part:BBa_K844007 BBa_K844007]) with GFP ([https://parts.igem.org/Part:BBa_K208000 BBa_K208000]) fused to its C-terminus. This generator is lactose/ITPG inducible.
-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 and on composite parts using this part will not reflect this scar sequence (it shows [https://parts.igem.org/Assembly_standard_10 Assembly Standard #10] scars).
@@ Line 23: / Line 23: @@
-Figure 2. ''E. coli'' cells expressing the fused spider silk and GFP protein. This demonstrates that the 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).
+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).