Part:BBa_K1995024

IHG (INP-N-GFP-HG)

Based on BBa_K1995000 (BHG), we added INP-N and GFP to the upstream of BHG. Using INP-N, we anchored GFP and BHG out of the outer membrane of E.coli in order to increase the binding efficiency and using GFP to detect the concentration of cupric and mercuric ions. Ice nucleation protein (INP)

 We used INP to anchor GFP and BHG out of the outer membrane of E. coli, firstly increasing the probability of the meeting of BHG and heavy metal ions and secondly using the fluorescence quenching of GFP to detect the concentration of heavy metal ions.

Ice nucleation protein (INP) is an excretion surface protein. It is widely used for establishing germ surface display system. It contains an internal repeated domain (IRD) and an N-terminal anchoring domain (fig.1). Studies have shown that decrease the time of repetition of IRD can also get a better-off rate of displaying. So we decreased the time of repetition of IRD to reduce pressure of synthesis of E. coli. File:24file1.png Fig.1 Ice nucleation protein (INP) and its passenger GFP reporter GFP is an excellent reporter in synthetic biology. We add GFP into IHG for following functions: ① Initial survey of the expression of IHG. ② Detect the binding efficiency and other features of IHG. ③ Detect and measure the concentration of cupric and mercuric ions.

Fig.2 (A) Fluorescence of IHG (B) Fluorescence intensity of IHG

Fig. 3 (A) Fluorescence spectra of IHG (OD600=1.3) in the presence of Hg2+ with varying concentrations. (B) Fluorescence responses of IHG to Hg2+ with varying concentrations.

Fig. 4 (A) Selectivity of the IHG sensing system. Fig.4 (B) Fluorescence response of IHG (OD600=1.3) in the presence of Cu2+ and various additional metal ions. The black bars represent the addition of an excess of the appropriate metal ion (50 uM) to a bacteria solution. The red bars represent the subsequent addition of 100 uM CuSO4 to the solution. Excitation wavelength: 488 nm. Binding heavy metals We detect the binding efficiency of IHG and other parts and get the following chart. (Fig. 5)

Fig.5 Cu2+ binding efficiency of IH, IHG, HG, PHG, PH and 2H. (B) Zn2+ binding efficiency of IH, IHG, HG, PHG, PH and 2H.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Unknown
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 429
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1181
  Illegal SapI.rc site found at 238