Part:BBa_K5136225

I0500-B0034-mnta-B0015

Biology

MntA

MntA is a membrane protein from Lactobacillus plantarumATCC 14917 which transports Cd²⁺ and Mn²⁺ into the cell. It is an effective Mn²⁺and Cd²⁺ uptake system that exhibits rapid accumulation of the two metal ions. It endows cells with sensitivity to Cd²⁺ and energy-dependent Cd²⁺ uptake activity. MntA falls into the family of P-type adenosine triphosphatases(ATPase). Its transport efficiency is affected by some factors such as other ion of high concentration and temperature(1,2).

Usage and Design

After deinking, heavy metal ions in the ink will be released into the wastewater. Thus, this wastewater needs harmless treatmentremove before discharging. Hence, we use metallothioneins (MTs) to treat wastewater to remove heavy metals. However，due to the poor competitiveness of Cd²⁺ in mixed metal ions, engineered bacteria expressing only MTs lack enough affinity and selectivity for Cd2+ to reduce Cd2+ in the wastewater to the levels required by strict government regulations. Thus, this composite part(BBa_K5136226) was constructed to express the MntA to increase the specific uptake of Cd²⁺.

Characterization

Agarose gel electrophoresis (AGE)

The composite part (BBa_K5136225) constructed was introduced into the backbone plasmid (pSB3K3) through standard assembly and transformed into E. coli BL21(DE3). The positive clones were selected, and colony PCR and gene sequencing were used to verify that the clones were correct. Target bands (3964 bp) can be observed at the position between 3000bp and 5000bp(Figure 1).

Figure 1 colony PCR of BBa_K5136225_pSB3K3 in E. coli BL21(DE3)

Reference

1. Z. Hao, S. Chen, D. B. Wilson, Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum. Appl Environ Microbiol 65, 4746-4752 (1999).
2. S. K. Kim, B. S. Lee, D. B. Wilson, E. K. Kim, Selective cadmium accumulation using recombinant Escherichia coli. J Biosci Bioeng 99, 109-114 (2005).

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal SpeI site found at 3247
  Illegal SpeI site found at 3304
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NheI site found at 2203
  Illegal NheI site found at 3388
  Illegal SpeI site found at 3247
  Illegal SpeI site found at 3304
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal SpeI site found at 3247
  Illegal SpeI site found at 3304
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal SpeI site found at 3247
  Illegal SpeI site found at 3304
  Illegal NgoMIV site found at 2039
  Illegal NgoMIV site found at 3408
  Illegal NgoMIV site found at 3481
  Illegal AgeI site found at 979
  Illegal AgeI site found at 1672
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961