Difference between revisions of "Part:BBa K1065305"
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YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain (from <i>Bradyrhizobium japonicum)</i> <html><a href="#ref2" id="ret_ref2">[2]</a></html>. <BR> | YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain (from <i>Bradyrhizobium japonicum)</i> <html><a href="#ref2" id="ret_ref2">[2]</a></html>. <BR> | ||
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP production <html><a href="#ref3" id="ret_ref3">[3]</a></html>. <BR> | In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP production <html><a href="#ref3" id="ret_ref3">[3]</a></html>. <BR> | ||
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| + | For characterization tests we used our part <partinfo>Bba_K1065302</partinfo> that includes pLac upstream of the improved part whith RBS.<BR/> | ||
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<center>https://static.igem.org/mediawiki/2013/d/d8/Tn-2013Amilgfpbb.png</center> <BR> | <center>https://static.igem.org/mediawiki/2013/d/d8/Tn-2013Amilgfpbb.png</center> <BR> | ||
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: amilGFP is no longer produced <html><a href="#ref3" id="ret_ref3">[3]</a></html>. <BR> | Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: amilGFP is no longer produced <html><a href="#ref3" id="ret_ref3">[3]</a></html>. <BR> | ||
| − | <center>https://static.igem.org/mediawiki/2013/ | + | <center>https://static.igem.org/mediawiki/2013/0/09/Tn-2013Amilgfpnoo.png</center> <BR> |
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===induction test: successful improvement of the part and defined light dependent ON/OFF switch=== | ===induction test: successful improvement of the part and defined light dependent ON/OFF switch=== | ||
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| − | <center><img style="width:400px;"src=" https://static.igem.org/mediawiki/2013/ | + | <center><img style="width:400px;"src=" https://static.igem.org/mediawiki/2013/d/d7/Tn-2013_Part_improvement3.png"></center> |
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
| − | <partinfo> | + | <partinfo>BBa_K1065305 SequenceAndFeatures</partinfo> |
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| + | ===References=== | ||
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| + | <html><ol> | ||
| + | <li><a id="ref1"></a>Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.</li> | ||
| + | <li><a id="ref2"></a>Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.</li> | ||
| + | <li><a id="ref3"></a>Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416: 534: 542</li> | ||
| + | </html> | ||
<!-- Uncomment this to enable Functional Parameter display | <!-- Uncomment this to enable Functional Parameter display | ||
===Functional Parameters=== | ===Functional Parameters=== | ||
| − | <partinfo> | + | <partinfo>BBa_K1065305 parameters</partinfo> |
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Latest revision as of 22:08, 4 October 2013
Blue light sensor without inverter for the production of amilGFP
This part is an improved version of the Blue light sensor device BBa_K952003 (see details in Design notes). In the presence of blue light (470 nm) the production of the reporter amilGFP is inhibited. In the absence of blue light the device is activated, thus producing amilGFP[1].
This part was cloned and successfully improved by UNITN-Trento 2013 iGEM team. We characterized the circuit with the control of pLac promoter (to do that we created the part BBa_K1065302).
SAFETY NOTES: this part does not have safety concerns.
Usage and Biology
YF1, the blue light sensor, is a fusion protein of the LOV blue light sensor domain of Bacillus subtilis (YtvA) and FixL histidine kinase domain (from Bradyrhizobium japonicum) [2].
In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP production [3].
For characterization tests we used our part BBa_K1065302 that includes pLac upstream of the improved part whith RBS.
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: amilGFP is no longer produced [3].
induction test: successful improvement of the part and defined light dependent ON/OFF switch
To determine whether inserting an RBS after pFixK2 would actually improve the part (BBa_K952003) we characterized both our improved part and the original one, testing them under the same conditions. The improved part actually behaved as expected, producing the yellow fluorescent protein in the dark and not under illumination. Conversely, BBa_K1065300 (constituted by the original part BBa_K952003 and pLac) did not work in both cases, indicating that the original part doesn't work properly and that we effectively improved it.
Figure 1. Improved part and original part after induction: one culture of E. coli (strain NEB10β) transformed with BBa_K1065302 and one culture with BBa_K1065300, were grown until an OD of 0.7. Each culture was then split in two 10 ml samples (dark and light). Cells were kept O/N in glass culture tubes at 37°C under shaking. Cultures were then centrifuged and cell pellets were compared. A different coloration in the Bba_K1065302-transformed sample kept in the dark (1) was observed when compared to the blue light exposed control (2) and the samples without RBS (3 and 4). .
Fluorescence measurements confirms previuos testing
After induction, 10 ml of cultures were pelleted, resuspended in 2 ml of PBS, and sonicated. The supernatant was used to measure fluorescence spectra. Excitation and emission wavelengths were 503 nm and 512 nm, respectively. All measurements were taken with a Cary Eclipse Varian fluorimeter.
Figure 2. Fluorimetric spectra: Dark-induced cultures of BBa_K1065302 (purple) produced the greatest amount of chromoprotein; Cultures of BBa_K1065302 exposed to light showed a basal expression of amilGFP (green). As expected the part without RBS, BBa_K1065300 showed no activity both in the dark (blue) and under illumination (red). Our part with RBS is undeniably improved and works as expected. Cells were grown as described in figure 1.
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 562
Illegal NgoMIV site found at 634
Illegal NgoMIV site found at 724
Illegal NgoMIV site found at 742
Illegal NgoMIV site found at 1254
Illegal NgoMIV site found at 1547
Illegal NgoMIV site found at 1641
Illegal AgeI site found at 276
Illegal AgeI site found at 1422 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 1311
Illegal BsaI.rc site found at 175
References
- Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.
- Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.
- Ohlendorf, R., Vidavski, R.R., Eldar, A., Moffat, K. & Möglich, A.(2012). From Dusk till Dawn: One-Plasmid Systems for Light-Regulated Gene Expression. J. Mol. Biol., 416: 534: 542