Part:BBa_K5382120:Design
InaK-linker-Im7_Cell membrane anchoring protein and immune protein complex
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1801
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 723
Illegal NgoMIV site found at 963
Illegal NgoMIV site found at 1131
Illegal AgeI site found at 518 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 693
Illegal BsaI.rc site found at 1183
Illegal SapI.rc site found at 327
Design Notes
After constructing the plasmid, we transferred it into EcN for expression for subsequent operations, so that it can have a better expression level under the premise of ensuring the operation standard, so as to improve the binding rate and stability of the above membrane surface display system.
Experimental result
We transferred pET23a-CL7-sfGFP into Escherichia coli BL21 and purified it to meet the use requirements. The results were as follows:
Figure 1. EcN engineered bacteria induced expression of InaK-Im7 fusion protein. 1, 3, 5 are before induction, 2, 4, 6 are after induction with IPTG.
It can be seen that the purified protein concentration and purity are high, and subsequent incubation binding and fluorescence confocal experiments can be conducted to verify the binding of Inak-Im7 and CL7-sfGFP (see the engineer and result section of wiki for the results).
Source
Inak is an ice nucleated protein from Pseudomonas syringae KCTC1832. linker is composed of Gly and Ser. Im7 is an immune protein derived from E. coli. The source of the composite parts is artificially constructed plasmids containing Inak and Im7 genes (pCold-Inak-Im7).