Difference between revisions of "Part:BBa K1065302"

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<partinfo>BBa_K1065302 short</partinfo>
 
<partinfo>BBa_K1065302 short</partinfo>
  
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This part consists of a Blue light sensor device, Bba_k952003 that was improved by 2013 UNITN-Trento iGEM team with an RBS sequence. When blue light (470 nm) is present production of the reporter amilGFP is inhibited. In the dark the device is activated. Everything is under the control of a constitutive promoter pLac.
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This part includes  the promoter pLac and <partinfo>BBa_K1065305</partinfo>, the 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>. The whole system is under the control of a constitutive promoter pLac. <BR>
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<BR>
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This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team 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, improved and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein transcription and then add an ethylene forming enzyme (EFE) after amilGFP. <BR>
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Unlike Bba_K1065310, we built this part to have Ethylene production in the dark. The purpose is to demonstrate that the presence of the inverter cassette in Bba_K1065310 could be one of the reasons of the imperfect behavior of the switch. <BR>
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The part used is from 2012 Columbia-Cooper-NYC iGEM team.<BR> <BR>
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SAFETY NOTES: this part does not have safety concerns.
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SAFETY NOTES: this part does not present safety issues. <BR>
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<center>https://static.igem.org/mediawiki/2013/0/09/Tn-2013Amilgfpnoo.png</center> <BR>
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<center>https://static.igem.org/mediawiki/2013/d/d8/Tn-2013Amilgfpbb.png</center> <BR>
 
===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). <BR>
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YF1, the blue light sensor, is a fusion protein made of the LOV blue light sensor domain of <i>Bacillus subtilis</i> (YtvA) and FixL histidine kinase domain from Bradyrhizobium japonicum <html><a href="#ref2" id="ret_ref2">[2]</a></html>. In the dark, YF1 autophosphorylates and successively phosphorylates FixJ, its Response Regulator, which in turn activates the pFixK2 promoter <html><a href="#ref3" id="ret_ref3">[3]</a></html> allowing amilGFP transcription . <BR>
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In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP transcription. <BR>
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Under constant illumination with blue light, net kinase activity is strongly repressed, resulting in the inactivation of pFixK2: amilGFP is no longer produced <html><a href="#ref1" id="ret_ref1">[1]</a></html>.  
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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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<BR>
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The functioning of the device is due to the presence of an RBS inserted after pFixK2, that was missing in the original part.
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We characterized this part in E. coli using cells NEB10b. <BR/></html>
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We characterized this part in <i>E. coli</i> using cells NEB10&beta;. <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 we characterized both our new part and the original one, testing them under the same conditions. Infact we saw an impressive difference between the 2 of them: the improved part actually behaved as expected, producing the yellow fluorescent protein at dark and not under illumination. Bba_K952003 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 E. coli (strain NEB10b) transformed with Bba_K1065302 and one with Bba_K952003, until they reached an OD = 0.7. Then we splitted each culture in 2 5ml samples ( at dark and under illumination). We let them grow in glass tubes at 37 degrees under shaking O/N. After this time lapse we centrifuged the cultures and obtained pellets to compare. AmilGFP gives a yellow shade to the cells so it was a little difficult to get a difference in color. Nevertheless we could see a different color in the Bba_K1065302-transformed sample that stayed in the dark.</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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===fluorometer measurements confirms previuos testing===
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===Fluorescence measurements confirms previuos testing===
 
<html>
 
<html>
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After the induction time, we took some fluorimetric measurement since amilGFP is a fluorescent reporter that emits at 512 nm and absorbs at 503 nm.
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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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<html>
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<html><br>
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<center><img style="width:600px;"src=" https://static.igem.org/mediawiki/2013/2/22/Tn-2013_part_improvement_plot.jpg"></center>
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<center><img style="width:700px;"src=" https://static.igem.org/mediawiki/2013/d/d9/Tn-2013BBa_K1065302.png"></center>
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<center><p style="width:600px; margin-bottom:60px; text-align:justify">
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<center><p style="width:800px; margin-bottom:60px; text-align:justify">
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<b>Figure 2.fluorimetric spectra:</b> samples were excited with 503 nm wavelenght radiation, so we were able to see the emission peak at 512 nm, that confirmed the presence of amilGFP. As shown in the picture the sample with RBS activated by dark (purple) produced the gratest amount of chromoprotein; light worked as an inhibitor of the transcription (green). As expected both samples without RBS (red and blue) showed no activity at all.
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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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<partinfo>BBa_K1065302 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1065302 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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</ol></html>
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 14:07, 28 October 2013

Blue light sensing device without inverter for the production of amilGFP

This part includes the promoter pLac and BBa_K1065305, the 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]. The whole system is under the control of a constitutive promoter pLac.
This part was cloned and successfully characterized by UNITN-Trento 2013 iGEM team in order to test protein production and then add an ethylene forming enzyme (EFE) after amilGFP.


SAFETY NOTES: this part does not present safety issues.

Tn-2013Amilgfpnoo.png

Tn-2013Amilgfpbb.png

Usage and Biology

YF1, the blue light sensor, is a fusion protein made of the LOV blue light sensor domain of Bacillus subtilis (YtvA) and FixL histidine kinase domain from Bradyrhizobium japonicum [2]. In the dark, YF1 autophosphorylates and successively phosphorylates FixJ, its Response Regulator, which in turn activates the pFixK2 promoter [3] allowing amilGFP transcription .
Under constant illumination with blue light, net kinase activity is strongly repressed, resulting in the inactivation of pFixK2: amilGFP is no longer produced [1].

We characterized this part in E. coli using cells NEB10β.

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 770
    Illegal NgoMIV site found at 842
    Illegal NgoMIV site found at 932
    Illegal NgoMIV site found at 950
    Illegal NgoMIV site found at 1462
    Illegal NgoMIV site found at 1755
    Illegal NgoMIV site found at 1849
    Illegal AgeI site found at 484
    Illegal AgeI site found at 1630
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1519
    Illegal BsaI.rc site found at 383

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