Difference between revisions of "Part:BBa K1065302"

Revision as of 16:35, 24 September 2013

Blue light sensing device 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. Everything is under the control of a constitutive promoter pLac.
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.

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). In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP transcription.
Under constant illumination with blue light net kinase activity is strongly suppressed, consisting in a consequent inactivation of pFixK2: amilGFP is no longer produced.
We characterized this part in E. coli using cells NEB10b.

induction test: successful improvement of the part and defined light dependent ON/OFF switch

To understand if 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 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.

Figure 1. improved part and not improved part after induction: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 two 5ml 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 when compared to the controls.

fluorescence measurements confirms previuos testing

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. 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 cultures of the part without RBS, BBa_952003 showed no activity at all (red and blue).

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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1065302 short</partinfo>
-This part  is an improved version of the Blue light sensor device Bba_k952003 (see details in Design notes).  When blue light (470 nm) is present production of the reporter amilGFP is inhibited. In the absence of blue light the device is activated, thus producing amilGFP. Everything is under the control of a constitutive promoter pLac.
+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. Everything is under the control of a constitutive promoter pLac.
 <BR>
 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>
@@ Line 12: / Line 12: @@
 ===Usage and Biology===
 <html>
-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>
+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>
-In the dark, the autophosphorylated YF1 phosphorylates FixJ, its Response Regulator, which activates the pFixK2 promoter allowing amilGFP transcription. <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. <BR>
-The functioning of the device is due to the presence of an RBS inserted after pFixK2, that was missing in the original part.
 We characterized this part in E. coli using cells NEB10b. <BR/></html>
 ===induction test: successful improvement of the part and defined light dependent ON/OFF switch===
-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.
+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 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.
 <html>
@@ Line 27: / Line 27: @@
 <center><p style="width:600px; margin-bottom:60px; text-align:justify">
-<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>
+<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 two 5ml 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 when compared to the controls. </p></center>
 <html></html>
-===fluorometer measurements confirms previuos testing===
+===fluorescence measurements confirms previuos testing===
 <html>
-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.
+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.
+Excitation and Emission wavelengths were 503 nm and 512 nm, respectively. All measurements were taken with a Cary Eclipse Varian fluorimeter.
 <html>
 <center><img style="width:600px;"src=" https://static.igem.org/mediawiki/2013/2/22/Tn-2013_part_improvement_plot.jpg"></center>
 <center><p style="width:600px; margin-bottom:60px; text-align:justify">
-<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.
+<b>Figure 2.fluorimetric spectra:</b> 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 cultures of the part without RBS, BBa_952003 showed no activity at all (red and blue).