Difference between revisions of "Part:BBa K1813023"
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__NOTOC__
 
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<partinfo>BBa_K1813023 short</partinfo>
 
<partinfo>BBa_K1813023 short</partinfo>
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LacI Reversed nicF
 
  
  
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<h4>Design and Acquisition</h4><p>  
 
<h4>Design and Acquisition</h4><p>  
 
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  
 
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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<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>. 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. All <i>nicF</i> constructs are contained within standard pSB1C3 vectors.
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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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All <i>nicF</i> constructs are contained within standard pSB1C3 vectors.
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<h4>Experience</h4><p>
 
<h4>Experience</h4><p>
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SDS PAGE Protein Expression for NicF
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<b>SDS PAGE Protein Expression for <i>NicF</i>:</b>
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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 <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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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 <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. NicF has over-expression at 20°C and 25°C at the expected size of 24kDa.
  
 
https://static.igem.org/mediawiki/2015/1/11/Nic_F_igem.png
 
https://static.igem.org/mediawiki/2015/1/11/Nic_F_igem.png

Revision as of 01:29, 19 September 2015

nicF 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 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.

Nic_F_igem.png UBC_igem_TABLE_THINGY_6CNA.png

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