Difference between revisions of "Part:BBa K1813023"
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<h2>Background of <i>LacI Reversed Ptac NicF Term</i></h2>
 
<h2>Background of <i>LacI Reversed Ptac NicF Term</i></h2>
 
   
 
   
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<h4>Part Description:</h4>
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<h4>Part Description</h4><p>
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<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>nicF</i>.The enzyme coded for by <i>nicF</i> converts Maleamic Acid to Maleic Acid [5].</p>
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</p>
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<html>
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<h4>Design and Acquisition</h4><p>  
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After synthesizing a codon-optimized <i>nicE</i>, we used standard assembly to created a composite part composed of <i>nicF</i> driven by the Ptac promoter
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<html><a href="https://parts.igem.org/Part:BBa_K1813037">BBa_K1813037</a> </html> and flanked by a double terminator <html><a href="https://parts.igem.org/Part:BBa_B0014">BBa_B0014</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>nicF</i> expression cassette  was assembled behind a lacI cassette <html><a href="BBa_K1813019">BBa_K1813019</a></html> to give us the ability to control the expression of NicF.
  
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<h4>Part Origin</h4>
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All <i>nicF</i> constructs are contained within standard pSB1C3 vectors.
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<p>where part originated and <i>Genus species</i> more words if needed</p>
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<h4>Part Function</h4>
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<h4>Experience</h4><p>
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<p>part functionstuff </p>
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SDS PAGE Protein Expression for NicF
  
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<h4>Design and Acquisition</h4>
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NicF was expressed in E.Coli DH5-α.  
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<p>
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After synthesizing a codon-optimized NicF, we used standard assembly to created a composite part composed of NicF driven by the Ptac promoter (BBa_K1813037) and flanked by a double terminator (BBa_B0014). 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 NicF expression cassette was assembled behind a lacI cassette (BBa_K1813019) to give us the ability to control the expression of NicF. All nicF constructs are contained within standard pSB1C3 vectors.</p>
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<h4>Experience</h4>
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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.
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<p><p>
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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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NicF has over-expression at 20°C and 25°C at the expected size of 24kDa.
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Figure 1:
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12% SDS-PAGE gel showing expression of a protein sized about 24kDa at 20°C and 25°C. It is less strongly expressed at other temperatures, uninduced and starter cultures.
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</p>
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<h4>References</h4>
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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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<br/>
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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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<br/>
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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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<br/>
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[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
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<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

Revision as of 00:10, 19 September 2015

nicF Expression Cassette with LacI Reversed

LacI Reversed nicF


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 NgoMIV site found at 1853
  • 1000
    COMPATIBLE WITH RFC[1000]


Background of LacI Reversed Ptac NicF Term

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 nicF.The enzyme coded for by nicF converts Maleamic Acid to Maleic Acid [5].

Design and Acquisition

After synthesizing a codon-optimized nicE, we used standard assembly to created a composite part composed of nicF driven by the Ptac promoter BBa_K1813037 and flanked by a double terminator BBa_B0014.

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 nicF expression cassette was assembled behind a lacI cassette BBa_K1813019 to give us the ability to control the expression of NicF.

All nicF constructs are contained within standard pSB1C3 vectors.

Experience

SDS PAGE Protein Expression for NicF

NicF 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.

NicF has over-expression at 20°C and 25°C at the expected size of 24kDa.


Figure 1: 12% SDS-PAGE gel showing expression of a protein sized about 24kDa at 20°C and 25°C. It is less strongly expressed at other temperatures, uninduced and starter cultures.

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
[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