Based on BBa_K1995004 (BC), we added INP-N and GFP to the upstream of BC. Using INP-N, we anchored GFP and BC 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 BC out of the outer membrane of E. coli, firstly increasing the probability of the meeting of BC 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.
GFP is an excellent reporter in synthetic biology. We added GFP into IC for following functions:
① Initial survey of the expression of IC.
② Detect the binding efficiency and other features of IC.
③ Detect and measure the concentration of cupric and mercuric ions.
In different strains of E. coli
We tested the binding efficiency of Hg2+ with IC using E. coli and engineering E.coli. (Fig. 4)
Binding heavy metals in fish gut
We detect the binding efficiency of IC in fish gut by feeding fishes with IC metal catcher and get the following chart. (Fig. 5)
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25INCOMPATIBLE WITH RFCIllegal AgeI site found at 429
- 1000INCOMPATIBLE WITH RFCIllegal BsaI.rc site found at 1181
Illegal SapI.rc site found at 238