Difference between revisions of "Part:BBa K1065305"
 
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<partinfo>BBa_K1065305 short</partinfo>
 
<partinfo>BBa_K1065305 short</partinfo>
  
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This part  is an improved version of the Blue light sensor device <partinfo>Bba_k952003</partinfo> (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.
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This part  is an improved version of the Blue light sensor device <partinfo>Bba_k952003</partinfo> (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<html><a href="#ref1" id="ret_ref1">[1]</a></html>.
 
<BR>
 
<BR>
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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 <partinfo>Bba_K1065302</partinfo>) in order to test protein production and then add an ethylene forming enzyme (EFE) after amilGFP. <BR>
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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 <partinfo>Bba_K1065302</partinfo>).
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SAFETY NOTES: this part does not have safety concerns.
 
SAFETY NOTES: this part does not have safety concerns.
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===Usage and Biology===
 
===Usage and Biology===
 
<html>
 
<html>
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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). In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP transcription. <BR>
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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>
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Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: amilGFP is no longer produced. <BR>
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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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</html>
 
 
For characterization tests we used our part <partinfo>Bba_K1065302</partinfo> that includes pLac upstream of the improved part whith RBS.<BR/>
 
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>
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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>
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<center>https://static.igem.org/mediawiki/2013/0/09/Tn-2013Amilgfpnoo.png</center> <BR>
  
  
 
===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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To understand if inserting an RBS after pFixK2 would actually improve the part (<partinfo>Bba_k952003</partinfo>) 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. <partinfo>Bba_K1065300</partinfo> (constituted by the original part <partinfo>Bba_k952003</partinfo> and pLac) instead doesn't work in both cases: that means that the original part doesn't work and we effectively improved it.
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To determine whether inserting an RBS after pFixK2 would actually improve the part (<partinfo>Bba_k952003</partinfo>) 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, <partinfo>Bba_K1065300</partinfo> (constituted by the original part <partinfo>Bba_k952003</partinfo> and pLac) did not work in both cases, indicating that the original part doesn't work properly and that we effectively improved it.  
 
<html>
 
<html>
  
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<center><img style="width:400px;"src=" https://static.igem.org/mediawiki/2013/b/b1/Tn-2013_Part_improvement.jpg"></center>
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<center><img style="width:400px;"src=" https://static.igem.org/mediawiki/2013/d/d7/Tn-2013_Part_improvement3.png"></center>
  
  
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
 
<center><p style="width:600px; margin-bottom:60px; text-align:justify">
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<b>Figure 1. improved part and not improved part after induction:</b> we grew one culture of <i> E. coli</i> (strain NEB10b) transformed with <partinfo>Bba_K1065302</partinfo> and one with <partinfo>BBa_K1065300</partinfo>, until they reached an OD = 0.7. Then we split each culture in two 10ml samples (dark and light). Cells were left in glass tubes at 37 degrees under shaking O/N. After this time lapse we centrifuged the cultures and obtained pellets to compare. We were able to observe a different color in the Bba_K1065302-transformed sample that stayed in the dark (1) when compared to the blue light exposed control (2) and the samples without RBS (3 and 4).</p></center>
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<b>Figure 1. Improved part and original part after induction:</b> one culture of <i>E. coli</i> (strain NEB10&beta;) transformed with <a href="https://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> and one culture with <a href="https://parts.igem.org/Part:BBa_K1065300">BBa_K1065300</a>, 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&deg;C under shaking. Cultures were then centrifuged and cell pellets were compared. A different coloration in the <a href="https://parts.igem.org/Part:BBa_K1065302">Bba_K1065302</a>-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).
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.</p></center>
 
<html></html>
 
<html></html>
  
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===fluorescence measurements confirms previuos testing===
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===Fluorescence measurements confirms previuos testing===
 
<html>
 
<html>
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After the induction time, 10 mL of cells culture were pelleted, resuspended in 2 mL of PBS, sonicated and the supernatant was used to measure fluorescence spectra.
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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.  
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Excitation and Emission wavelengths were 503 nm and 512 nm, respectively. All measurements were taken with a Cary Eclipse Varian fluorimeter.
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<html>
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<html><br>
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<center><img style="width:900px;"src=" https://static.igem.org/mediawiki/2013/7/79/Tn-2013Graph-amilgfp.png"></center>
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<center><img style="width:700px;"src=" https://static.igem.org/mediawiki/2013/d/d9/Tn-2013BBa_K1065302.png"></center>
 
<center><p style="width:800px; margin-bottom:60px; text-align:justify">
 
<center><p style="width:800px; margin-bottom:60px; text-align:justify">
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<b>Figure 2.fluorimetric spectra:</b> Dark-induced cultures of <partinfo>BBa_K1065302</partinfo> (red) produced the greatest amount of chromoprotein; Cultures of <partinfo>BBa_K1065302</partinfo> exposed to light showed a basal expression of amilGFP (green). As expected cultures of the part without RBS, BBa_K1065300 showed no activity at all both in the dark (orange) and with light (blue).
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<b>Figure 2. Fluorimetric spectra:</b> Dark-induced cultures of <a href="https://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> (purple) produced the greatest amount of chromoprotein; Cultures of <a href="https://parts.igem.org/Part:BBa_K1065302">BBa_K1065302</a> exposed to light showed a basal expression of amilGFP (green). As expected the part without RBS, <a href="">BBa_K1065300</a> 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.
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K1065305 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1065305 SequenceAndFeatures</partinfo>
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===References===
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<html><ol>
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<li><a id="ref1"></a>Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.</li>
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<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>
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<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>
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</html>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K1065302 parameters</partinfo>
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<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.

Tn-2013Amilgfpbb.png

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].

Tn-2013Amilgfpnoo.png


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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1311
    Illegal BsaI.rc site found at 175

References

  1. Alieva, N. O., et al. 2008. Diversity and evolution of coral fluorescent proteins. PLoS One 3:e2680.
  2. Moglich A, Ayers RA and Moffat K. (2009) Design and Signaling Mechanism of Light-Regulated Histidine Kinases. J. Mol. Bio. 385, 5, 1433-1444.
  3. 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