Difference between revisions of "Part:BBa K1065203"
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Cells were induced with 0.5 mM of IPTG. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).<br/> | Cells were induced with 0.5 mM of IPTG. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).<br/> | ||
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+ | Cells were induced with 1% of xylose. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).<br/> | ||
+ | <br/> | ||
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+ | Ethylene production was tested by Gas Chromatography as we previoulsy did for <html><a href="https://parts.igem.org/Part:BBa_K1065001">BBa_K1065001</a></html>. The experiment was performed from both fresh plates and from dry spores. | ||
+ | We did not observe any production of ethylene after 4 hours, nor after overnight induction. | ||
+ | At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips. Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip: | ||
<html><img src="https://static.igem.org/mediawiki/2013/6/6f/Tn-2013_Pspac_zolfo.jpg"/width="300"></br></br></br></html> | <html><img src="https://static.igem.org/mediawiki/2013/6/6f/Tn-2013_Pspac_zolfo.jpg"/width="300"></br></br></br></html> | ||
<html> | <html> | ||
<span style="text-align:justify;"class="tn-caption center"> Detection of sulfur compounds. <i>B. subtilis</i> 168 transformed with <a href="https://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate (see <a href="http://2013.igem.org/Team:UNITN-Trento/Project/Bacillus">our wiki</a> for more informations. </span></html> | <span style="text-align:justify;"class="tn-caption center"> Detection of sulfur compounds. <i>B. subtilis</i> 168 transformed with <a href="https://parts.igem.org/Part:BBa_K1065203">BBa_K1065203</a> were grown until O.D. 0.9 was reached. At this O.D. one sample was then supplemented with 1 mM IPTG. Cells were left to grow overnight into vials containing a lead acetate strip. The day after, transformed and induced samples showed a darker strip indicating the presence of sulfur compounds. The non trasformed cells supplemented with the inducer did not show that precipitate (see <a href="http://2013.igem.org/Team:UNITN-Trento/Project/Bacillus">our wiki</a> for more informations. </span></html> | ||
+ | The induced sample darkened much more than the not induced indicating the presence of sulfur compounds (the part <html><a href= "https://parts.igem.org/Part:BBa_K1065204">BBa_K1065204</a> behaved similarly</html>). | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
Latest revision as of 09:54, 4 October 2013
Efe+Bba_B0015 in pSpac (BBa_K823026)
2-oxoglutarate oxygenase/decarboxylase is an Ethylene Forming Enzyme (EFE) from Pseudomonas Siringae pv, that catalyzes Ethylene biosynthesis from 2-oxoglutarate. The enzyme was inserted in the IPTG inducible vector pSPAC constructed by the LMU Munich iGEM team 2012 for expression in Bacillus subtilis. This part was cloned by the iGEM Trento 2013 team for the creation of an aerobically engineered pathway for the control of fruit ripening. Further information about this part and its characterization can be found in the iGEM Trento 2013 wiki. If interested in this part please contact us.
NOTE: We used a modified WORKING version of the pSpac vector that has been sent to us from the LMU Munich 2012 team.
Toxicity and sulfur compounds tests
We measured the optical density of cells induced and non induced:
Cells were induced with 0.5 mM of IPTG. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).
Cells were induced with 1% of xylose. The graphic shows that the induced samples (blue trace) grow slightly slower than the controls (red trace).
Ethylene production was tested by Gas Chromatography as we previoulsy did for BBa_K1065001. The experiment was performed from both fresh plates and from dry spores. We did not observe any production of ethylene after 4 hours, nor after overnight induction. At this point we are not able to confirm that EFE was correctly expressed under these conditions. Surprisingly, induced culture had a strong smell of methane and mercapto compounds. The presence of sulfur compounds was confirmed by exposing the culture to lead acetate paper strips. Hydrogen sulfide and other mercapto compounds react with lead-acetate to form lead(II) sulfate, a black insoluble precipitate that darkens the white strip:
BBa_K1065204 behaved similarly).
The induced sample darkened much more than the not induced indicating the presence of sulfur compounds (the partUsage and Biology
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 9491
Illegal suffix found in sequence at 1224 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 9491
Illegal SpeI site found at 1225
Illegal PstI site found at 1239
Illegal NotI site found at 1232
Illegal NotI site found at 9497 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 9491
Illegal BglII site found at 319
Illegal BglII site found at 7085
Illegal BamHI site found at 2601 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 9491
Illegal suffix found in sequence at 1225 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 9491
Illegal XbaI site found at 9506
Illegal SpeI site found at 1225
Illegal PstI site found at 1239
Illegal NgoMIV site found at 17
Illegal NgoMIV site found at 4148
Illegal AgeI site found at 1070
Illegal AgeI site found at 6696
Illegal AgeI site found at 7658
Illegal AgeI site found at 8333 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 3972
Illegal BsaI.rc site found at 5411
Illegal BsaI.rc site found at 7927
Illegal SapI site found at 2889
Illegal SapI.rc site found at 6909