|
|
| Line 1: |
Line 1: |
| − | __NOTOC__
| + | 0605 http://manualss7iy.pp.ua/bnwrwn1.html http://manualss7iy.pp.ua/xpem2.html http://documentsoex.pp.ua/tsq1.html http://documentsoex.pp.ua/kvxtcu2.html http://instructions9rxg.pp.ua/iiejn2.html http://instructions9rxg.pp.ua/lddpim4.html http://instruktsiya721.pp.ua/olkkpx2.html http://instruktsiya721.pp.ua/vrol1.html http://rukovodstnnr.pp.ua/mwcfk2.html http://rukovodstnnr.pp.ua/xuykyh3.html http://rukovodstnnr.pp.ua/juqpxs2.html http://rukovodstnnr.pp.ua/wil3.html http://instruktsiya5nd.pp.ua/mvcgo2.html http://instruktsiya5nd.pp.ua/ytjmtg3.html http://rukovodstvqk.pp.ua/tqu2.html http://rukovodstvqk.pp.ua/kjanan3.html |
| − | <partinfo>BBa_E0040 short</partinfo>
| + | |
| − | | + | |
| − | GFP (mut3b) [note that this part does not have a barcode]
| + | |
| − | | + | |
| − | ===Usage and Biology===
| + | |
| − | Un[[Help:Tag|tag]]ged version of gfp from Repressilator reporter. See the [[Part:BBa_E0040:Design | design page]] for more source information.
| + | |
| − | | + | |
| − | The original citation for GFPmut3b is as follows:
| + | |
| − | | + | |
| − | Cormack, B.P., Valdivia, R.H., and S. Falkow. FACS-optimized mutants of green fluorescent protein (GFP). Gene 173: 33-38 (1996).
| + | |
| − | | + | |
| − | Here's the link: http://www.sciencedirect.com/science/article/pii/0378111995006850
| + | |
| − | | + | |
| − | <!--
| + | |
| − | <span class='h3bb'>Sequence and Features</span>
| + | |
| − | <partinfo>BBa_E0040 SequenceAndFeatures</partinfo>
| + | |
| − | <!-- -->
| + | |
| − | <!--
| + | |
| − | ===Functional Parameters===
| + | |
| − | <partinfo>BBa_E0040 parameters</partinfo>
| + | |
| − | <!-- -->
| + | |
| − | | + | |
| − | ====Fluorescence wavelengths====
| + | |
| − | Cormack ''et al''.<cite>Cormack</cite> report the following excitation and emission data for GFPmut3 - <br>
| + | |
| − | *'''Excitation max''' - 501nm
| + | |
| − | *'''Emission max''' - 511nm
| + | |
| − | | + | |
| − | ====Latency====
| + | |
| − | Cormack ''et al''.<cite>Cormack</cite> report detectable fluorescence within 8 mins.
| + | |
| − | ''Please add maturation time data for E0040 here.''
| + | |
| − | | + | |
| − | ====References====
| + | |
| − | <biblio>
| + | |
| − | #Cormack pmid=10659856
| + | |
| − | </biblio>
| + | |
| − | | + | |
| − | ===Part Characteristics in [[Chassis/Cell-Free_Systems|Cell-Free Chassis]]===
| + | |
| − | {|border=1 width="90%" align="center"
| + | |
| − | |-
| + | |
| − | !width="20%" style="background:#CCCCFF"|Parameter
| + | |
| − | !width="80%"|Value and Description
| + | |
| − | |-align="center"
| + | |
| − | |style="background:#EEEEFF"|Calibration
| + | |
| − | |A conversion factor of 79.429 from Au to concentraion in nM
| + | |
| − | |-align="center"
| + | |
| − | |style="background:#EEEEFF"|Half-life
| + | |
| − | |33 hours in the cell-free chassis, with a degradation constant of 0.0210 (in hours)
| + | |
| − | |}
| + | |
| − | ====Purificaton====
| + | |
| − | GFPmut3b can be purified for calibration after the addition of a his-tag. The detailed [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.6 protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.6 results]for the purification can be found.
| + | |
| − | ====Calibration====
| + | |
| − | The fluorescence of purified GFPmut3B was calibrated in the [[Chassis/Cell-Free_Systems|cell-free chassis]]. The derived [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.3 calilbration curve]allows the determination of the concentration of GFPmut3b in the cell-free chassis. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.3 Detailed protocols]for generating the calibration curve are available. Other calibration curves for are also available on the results page.
| + | |
| − | [[Image:E0040_invitro_calibration.png|thumb|center|500px|Calibration curve of concentration of GFP against fluorescence. The conversion factor is 79.429.]]
| + | |
| − | ====Degradation====
| + | |
| − | The degradation of GFPmut3B in the [[Chassis/Cell-Free_Systems|cell-free chassis]] was also characterized. Purified GFPmut3B was allowed to degrade in the cell-free chassis and the fluorescence was measured over time. [http://2007.igem.org/Imperial/Wet_Lab/Protocols/Prot1.4 Detailed protocols]and [http://2007.igem.org/Imperial/Wet_Lab/Results/Res1.4 results]are attached.
| + | |
| − | [[Image:E0040 invitro degradation.png|thumb|left|500px|Degradation curve of different GFP concentrations.]]
| + | |
| − | [[Image:E0040 invitro deglog.png|thumb|none|500px|Semi-log plot of GFP degradation at different GFP concentrations]]<br clear="all">
| + | |
| − | From the semi-log plot, the degradation constant (in minutes) was derived to be 0.0003501, which is equivalent to GFPmut3b having a half-life of 33 hours in the cell-free chassis.
| + | |
| − | ====Improvements====
| + | |
| − | Yeast- and FACS optimized GFP: <html><a href="https://parts.igem.org/Part:BBa_K194001" target="_blank">BBa_K194001</a>
| + | |
| − | Yeast- and FACS optimized, fast degradable GFP: <html><a href="https://parts.igem.org/Part:BBa_K194002" target="_blank">BBa_K194002</a>
| + | |
0605 http://manualss7iy.pp.ua/bnwrwn1.html http://manualss7iy.pp.ua/xpem2.html http://documentsoex.pp.ua/tsq1.html http://documentsoex.pp.ua/kvxtcu2.html http://instructions9rxg.pp.ua/iiejn2.html http://instructions9rxg.pp.ua/lddpim4.html http://instruktsiya721.pp.ua/olkkpx2.html http://instruktsiya721.pp.ua/vrol1.html http://rukovodstnnr.pp.ua/mwcfk2.html http://rukovodstnnr.pp.ua/xuykyh3.html http://rukovodstnnr.pp.ua/juqpxs2.html http://rukovodstnnr.pp.ua/wil3.html http://instruktsiya5nd.pp.ua/mvcgo2.html http://instruktsiya5nd.pp.ua/ytjmtg3.html http://rukovodstvqk.pp.ua/tqu2.html http://rukovodstvqk.pp.ua/kjanan3.html
