Part:BBa_K5301005
SCSdC-mCh[1-10] is one of the components of multi-polymerized MSP.
Usage and Biology
In order to produce large nanodiscs more conveniently, we hope to flexibly extend the length of MSP according to demand, and thus propose the concept of multi-polymer MSP, which refers to large circular MSPs through end-to-end connections of multiple MSP fragments. We used NW15 as the basic MSP and selected three types of linkers (Spy/Sdy/Snoop) to achieve the connection of different MSP fragments through the formation of covalent bonds, and adopted rigorous design to prevent self-cyclization of each fragment of the multi-polymer MSP. Finally, the successful cyclization of large circular MSPs is characterized by the fluorescence of mCherry after the combination of mCherry [1-10] and mCherry [11]. SCSdC-mCh[1-10], the first component of multi-polymerized MSP, is a fusion protein composed of SpyCatcher, mCherry [1-10], NW15, and SdyCatcher, with flexible GS linkers used to connect each part.
Cultivation, Purification and SDS-PAGE
Induction
Results
Firstly, plasmids contains modified PL-promoter were constructed through PCR. During the amplification, The plasmid was divided into two parts (2.2 kb and 2.1 kb respectively) by 2 pairs of primers, and the results of agarose gel electrophoresis were shown below (Fig 1). The right position of sample DNA between makers indicated the success ligation of our PL.
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Figure 1. PL ligation through PCR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 1720
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1239
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1035
Illegal AgeI site found at 1828 - 1000COMPATIBLE WITH RFC[1000]
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