Part:BBa_K1813020

nicC Expression Cassette with LacI Reversed

LacI Reversed nicC

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 of LacI Ptac NicC 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 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).

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.</p>

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 (reference). </p>

nicC 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>

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