Part:BBa_K2638200

OprC (TonB dependent copper transport porin)

Accumulation Results

Short Summary

To test whether our accumulation system with the importers oprC, hmtA, copC and copD works as intended, we conducted several experiments analyzing Cu(II) ion uptake. We conducted growth experiments and observed hindered cell growth when expressing our transporter proteins, which indicates import of toxic copper into the cell. Furthermore, we measured membrane permeability thereby, showing the ion channel nature of the transporter proteins in the outer membrane. Finally we showed the proteins specifity for taking up Cu(II).

Toxicity Assays

As intracellular copper leads to toxic effects in the cell (read more on our toxicity) page, an increased uptake of Cu(II) ions should slow down and exacerbate cell growth. Therefore, we examined the growth of E. coli KRX expressing oprC in lysogeny broth (LB) media with 30 ng/µL chloramphenicol supplemented with different concentrations of CuSO₄ (0 mM, 1 mM, 2 mM, 3 mM, 4 mM, 8 mM) by measuring the optical density at a wavelength of 600 nm (OD600). As a control we decided to use the pSB1C3 null vector under same conditions as well. The measurement was performed with the Infinite® 200 PRO in a 24 wellplate with flat bottom (Greiner®). For expression of the BioBricks either a T7 promoter and a ribosome binding site (RBS) (BBa_K525998) or a combination of the pBAD/araC promoter (BBa_I0500) and a RBS (BBa_B0030) (RBS) were cloned upstream of (oprC (BBa_K2638200).

All toxicity measurements were performed by inoculating E. coli KRX cultures harbouring the plasmid of interest with a starting OD600 of 0.01 from an overnight culture. Afterwards cells were grown (37 °C, 350 rpm) for 3 h and were induced either with 0.1 % rhamnose and 0.1 mM IPTG for the T7 constructs or 1 % arabinose for the arabinose constructs. The OD600 of the cultures were measured every hour for up to 12 hours.

The growth of E. coli KRX expressing oprC (BBa_K2638201) after induction in comparison showed lower maximum cell densities, even in the culture without added Cu(II). This effect is a consequence of the high metabolic burden bearing on the cells due to strong expression of the T7 promoter controlled genes. When growing in copper-containing media the cells expressing oprC show a stronger growth inhibition. This effect gets stronger with increasing Cu(II) concentrations. Especially interesting is the growth curve progression at a concentration of 2 mM Cu(II) (figure 1). The optical density does not only increase at a reduced rate, it even decreases after approximately 220 minutes, which indicates cell death. Both growth inhibitions cannot be observed with E. coli carrying pSB1C3.

The NPN assay is a easy and fast method to measure membrane permeability. In both strains which expressed the composite parts BBa_2638201 and BBa_K2638204 a higher increase in fluorescence than the pSB1C3 controll strain was measurable.

An orhto-Nitrophenyl-β-galactoside (ONPG) assay was performed with eight different pSB1C3 constructs to show that our copper transport Biobricks do not unspecifically take up different substrates. In both strains which expressed the composite parts BBa_2638201 and BBa_K2638204 a higher increase in fluorescence than the pSB1C3 controll strain was measurable.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 252
  Illegal NgoMIV site found at 583
  Illegal NgoMIV site found at 787
  Illegal NgoMIV site found at 1831
  Illegal NgoMIV site found at 2032
  Illegal AgeI site found at 246
  
• 1000
  COMPATIBLE WITH RFC[1000]