Difference between revisions of "Part:BBa K1813020"
 
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__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K1813020 short</partinfo>
 
<partinfo>BBa_K1813020 short</partinfo>
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LacI Reversed nicC
 
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<partinfo>BBa_K1813020 parameters</partinfo>
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<h2>Background</h2>
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<h2>Background of <i>LacI Ptac NicC Term</i></h2>
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<h4>Part Description</h4><p>
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6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid degradation that is both toxic to bees [1], and a persistent environmental contaminant [2].The conversion from 6-CNA to 6-HNA, a well studied intermediate in nicotine degradation [3], is catalyzed by 6-chloronicotinic acid chlorohydrolase (<i>cch2</i>), a chlorohydrolase from SG-6C ''Bradyrhizobiaceae'' [4]. 6-HNA can be further degraded into Fumaric Acid using the following pathway, which includes <i>nicC</i>. The enzyme coded for by <i>nicC</i> is a 6-HNA monooxygenase that converts 6-Hydroxynicitinoic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) [5].</p>
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https://static.igem.org/mediawiki/2015/e/ec/Pathway.png
  
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<h4>Part Description:</h4><p>
 
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6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid degradation that is both toxic to bees (1), and a persistent environmental contaminant (2).The conversion from 6-CNA to 6-HNA, a well studied intermediate in nicotine degradation (3), is catalyzed by 6-chloronicotinic acid chlorohydrolase (cch2), a chlorohydrolase from SG-6C ''Bradyrhizobiaceae'' (4). 6-HNA can be further degraded into Fumaric Acid using the following pathway, which includes <i>nicC</i>. The enzyme coded for by nicC is a 6-HNA monooxygenase that converts 6-Hydroxynicitinoic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) (5).</p>
 
 
<html>
 
<html>
 
<h4>Design and Acquisition</h4><p>  
 
<h4>Design and Acquisition</h4><p>  
 
After synthesizing a codon-optimized <i>nicC</i>, we used standard assembly to created a composite part composed of <i>nicC</i> driven by the Ptac promoter  
 
After synthesizing a codon-optimized <i>nicC</i>, we used standard assembly to created a composite part composed of <i>nicC</i> driven by the Ptac promoter  
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(<html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1813037">BBa_K1813037</a> </html>) and flanked by a double terminator (<html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1813014">BBa_K1813014</a> </html>).  
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<html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1813037">BBa_K1813037</a> </html> and flanked by a double terminator <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1813014">BBa_K1813014</a> </html>.  
  
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The tac promoter contains a lac operator sequence that can be bound by LacI,  the lac repressor protein, allowing inducible expression by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Our <i>nicC</i> expression cassette  was assembled behind a lacI cassette (<html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1813019">BBa_K1813019</a> </html>) to give us the ability to control the expression of <i>nicC</i>.  
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The tac promoter contains a lac operator sequence that can be bound by LacI,  the lac repressor protein, allowing inducible expression by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Our <i>nicC</i> expression cassette  was assembled behind a lacI cassette <html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1813019">BBa_K1813019</a> </html> to give us the ability to control the expression of <i>nicC</i>.  
  
 
All <i>nicC</i> constructs are contained within standard pSB1C3 vectors.
 
All <i>nicC</i> constructs are contained within standard pSB1C3 vectors.
  
 
<h4>Experience</h4><p>
 
<h4>Experience</h4><p>
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SDS PAGE Protein Expression for <i>nicC</i>
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<b>SDS PAGE Protein Expression for <i>nicC</i>:</b>
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</p><i>nicC</i> was expressed in E.Coli DH5-α.
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Transformed E.Coli was grown at 37°C until an OD600 of 0.6 to 0.8. They were then induced with IPTG and grown overnight at 16°C, 20°C, 25°C, 30°C, 37°C to discern which temperature resulted in optimal protein expression. The samples were prepared for SDS page gel via the SDS page sample preparation protocol and SDS page gel protocol (reference). </p>
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<i>nicC</i> has over-expression at 25°C and 30°C at the expected size of 52kDa, however there is a significant amount of insoluble fraction of the gene product.</p>
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</p><i>nicC</i> was expressed in E.Coli DH5-α. Transformed E.Coli was grown at 37°C until an OD600 of 0.6 to 0.8. They were then induced with IPTG and grown overnight at 16°C, 20°C, 25°C, 30°C, 37°C to discern which temperature resulted in optimal protein expression. The samples were prepared for SDS page gel via the SDS page sample preparation protocol and SDS page gel protocol <html><a href="https://static.igem.org/mediawiki/2015/b/b8/Preparation_of_Samples_to_run_SDS_UBC.pdf">here</a></html> and <html><a href="https://static.igem.org/mediawiki/2015/2/2c/SDS_Gel_Prep_UBC.pdf">here</a></html> respectively.
  
  
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https://static.igem.org/mediawiki/2015/c/c6/NicC_UBC.png
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https://static.igem.org/mediawiki/2015/f/fe/UBC_igem_TABLE_THINGY_6CNA.png
  
 
<p>Figure 1: 12% SDS-PAGE gel run with <i>nicC</i>. No discernible expression is seen at the expected size of <i>nicC</i>, 43kDa. </p>
 
<p>Figure 1: 12% SDS-PAGE gel run with <i>nicC</i>. No discernible expression is seen at the expected size of <i>nicC</i>, 43kDa. </p>
  
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<h5>References:</h5><p>
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<h5>References</h5><p>
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1. Nauen, R., Ebbinghaus-Kintscher, U. and Schmuck, R. (2001) Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae) Pest. Manag. Sci. 57 (7) DOI: 10.1002/ps.331 </p>
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[1] Nauen, R., Ebbinghaus-Kintscher, U. and Schmuck, R. (2001) Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae) Pest. Manag. Sci. 57 (7) DOI: 10.1002/ps.331  
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<p>2. Rouchaud J, Gustin F, Wauters A (1996) Imidacloprid insecticide soil metabolism in sugar beet field crops. Bull Environ Contam Toxicol 56: 29–36. doi: 10.1007/s001289900005</p>
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<br/>
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<p>3.Tang, H., Yao, Y., Wang, L., Yu, H., Ren, Y. et al. (2012) Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Scientific Reports 2, Article number: 377 doi:10.1038/srep00377</p>
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[2] Rouchaud J, Gustin F, Wauters A (1996) Imidacloprid insecticide soil metabolism in sugar beet field crops. Bull Environ Contam Toxicol 56: 29–36. doi: 10.1007/s001289900005
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<p>4.Shettigar M, Pearce S, Pandey R, Khan F, Dorrian SJ, et al. (2012) Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C. PLoS ONE 7(11): e51162. doi: 10.1371/journal.pone.0051162</p>
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<br/>
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<p>5.Jiménez, J., Canales, A., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J.,  Díaz, E.(2008) Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440 1Proc Natl Acad Sci 05(32): 11329–11334.  doi: 10.1073/pnas.0802273105
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[3] Tang, H., Yao, Y., Wang, L., Yu, H., Ren, Y. et al. (2012) Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Scientific Reports 2, Article number: 377 doi:10.1038/srep00377
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PMCID: PMC2516282</p>
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<br/>
 +
[4] Shettigar M, Pearce S, Pandey R, Khan F, Dorrian SJ, et al. (2012) Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C. PLoS ONE 7(11): e51162. doi: 10.1371/journal.pone.0051162
 +
</br>
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[5] Jiménez, J., Canales, A., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J.,  Díaz, E.(2008) Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440 1Proc Natl Acad Sci 05(32): 11329–11334.  doi: 10.1073/pnas.0802273105 PMCID: PMC2516282

Latest revision as of 02:15, 19 September 2015

nicC Expression Cassette with LacI Reversed

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 105
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1658
  • 1000
    COMPATIBLE WITH RFC[1000]


Background

Part Description

6-chloronicotinic acid (6-CNA) is an intermediate in imidacloprid degradation that is both toxic to bees [1], and a persistent environmental contaminant [2].The conversion from 6-CNA to 6-HNA, a well studied intermediate in nicotine degradation [3], is catalyzed by 6-chloronicotinic acid chlorohydrolase (cch2), a chlorohydrolase from SG-6C Bradyrhizobiaceae [4]. 6-HNA can be further degraded into Fumaric Acid using the following pathway, which includes nicC. The enzyme coded for by nicC is a 6-HNA monooxygenase that converts 6-Hydroxynicitinoic acid (6-HNA) to 2,5-dihydroxypyridine (2,5-DHP) [5].

Pathway.png

Design and Acquisition

After synthesizing a codon-optimized nicC, we used standard assembly to created a composite part composed of nicC driven by the Ptac promoter BBa_K1813037 and flanked by a double terminator BBa_K1813014 .

The tac promoter contains a lac operator sequence that can be bound by LacI, the lac repressor protein, allowing inducible expression by Isopropyl β-D-1-thiogalactopyranoside (IPTG). Our nicC expression cassette was assembled behind a lacI cassette BBa_K1813019 to give us the ability to control the expression of nicC.

All nicC constructs are contained within standard pSB1C3 vectors.

Experience

SDS PAGE Protein Expression for nicC:

nicC was expressed in E.Coli DH5-α. Transformed E.Coli was grown at 37°C until an OD600 of 0.6 to 0.8. They were then induced with IPTG and grown overnight at 16°C, 20°C, 25°C, 30°C, 37°C to discern which temperature resulted in optimal protein expression. The samples were prepared for SDS page gel via the SDS page sample preparation protocol and SDS page gel protocol here and here respectively.


NicC_UBC.png UBC_igem_TABLE_THINGY_6CNA.png

Figure 1: 12% SDS-PAGE gel run with nicC. No discernible expression is seen at the expected size of nicC, 43kDa.

References

[1] Nauen, R., Ebbinghaus-Kintscher, U. and Schmuck, R. (2001) Toxicity and nicotinic acetylcholine receptor interaction of imidacloprid and its metabolites in Apis mellifera (Hymenoptera: Apidae) Pest. Manag. Sci. 57 (7) DOI: 10.1002/ps.331
[2] Rouchaud J, Gustin F, Wauters A (1996) Imidacloprid insecticide soil metabolism in sugar beet field crops. Bull Environ Contam Toxicol 56: 29–36. doi: 10.1007/s001289900005
[3] Tang, H., Yao, Y., Wang, L., Yu, H., Ren, Y. et al. (2012) Genomic analysis of Pseudomonas putida: genes in a genome island are crucial for nicotine degradation. Scientific Reports 2, Article number: 377 doi:10.1038/srep00377
[4] Shettigar M, Pearce S, Pandey R, Khan F, Dorrian SJ, et al. (2012) Cloning of a Novel 6-Chloronicotinic Acid Chlorohydrolase from the Newly Isolated 6-Chloronicotinic Acid Mineralizing Bradyrhizobiaceae Strain SG-6C. PLoS ONE 7(11): e51162. doi: 10.1371/journal.pone.0051162 </br>

[5] Jiménez, J., Canales, A., Jiménez-Barbero, J., Ginalski, K., Rychlewski, L., García, J., Díaz, E.(2008) Deciphering the genetic determinants for aerobic nicotinic acid degradation: The nic cluster from Pseudomonas putida KT2440 1Proc Natl Acad Sci 05(32): 11329–11334. doi: 10.1073/pnas.0802273105 PMCID: PMC2516282