Team:NEFU China/Parts
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Parts
01. Mms13 BBa_K1947000
This part is the coding sequence of Mms13. Mms13 is a particle protein, which was found on the membrane of Magnetosome in Magnetospirillum magneticum AMB-1. Our purpose is to display a protein (in our case it is Spycatcher) on the membrane of Magnetosome by fusing the protein with Mms13.
02. Pmsp3 BBa_K1947001
This part is a promoter that can be used in Magnetospirillum magneticum AMB-1 to promote the target gene transcription in strain AMB-1. This promoter is one of the strongest promoters in strain AMB-1, the gene drived by this promoter will express continuously.
03. intein protein BBa_K1947002
This sequence encodes a short polypeptide which can be specifically recognized and spliced by DL-Dithiothreitol (DTT). In our case we add this short sequence between the recombinant protein which we want to purify and the linker protein Spytag. After adding DTT the recombinant protein will be separated from the Magnetosome.
04. SPA Z domain BBa_K1947003
The SPA Z domain is a polypeptide mutated from the B domain of Protein A of Staphylococcus aureus. As the molecular weight of this polypeptide has been well characteristic, we used this part to test the stability of our system with different molecular weight by replacing the recombinant protein with SPA Z domain in different copy repeats.
05. Pmsp3 + EGFP BBa_K1947016
This part is constructed for testing of the Pmsp3 promoter activity. This is a coding region part for EGFP to determine whether the Pmsp3 promoter can drive the expression of a coding sequence in strain AMB-1.
06. STA (Spytag+tev+Amilcp) BBa_K1947023
This part serves as a prey system which was expressed in E. coli. There is a highly specific recognition and covalent conjugation between Spytag (a peptide with 13 amino acids) and Spycatcher (a peptide with 116 amino acids). A protein of interest with a Spytag at its N- or C-terminus is expressed in E. coli and present in bacterial lysate. Extensive and stringent washing can be applied to remove proteins of nonspecific binding then the protein of interest can be released by cleavage of TEV protease.
07. MGS (Mms13+GS linker+Spycatcher) BBa_K1947025
This part serves as a catch system expressed in strain AMB-1. Mms13 is a protein that bound to Magnetosome directly and tightly on a bilayer phospholipid membrane of the bacterial magnetic particles (BMPs). GS linker is used to construct the fusion protein to prevent the two proteins from influencing each other. And there is a highly specific recognition and covalent conjugation between Spytag (a peptide with 13 amino acids) and Spycatcher (a peptide with 116 amino acids). The Spycatcher-Mms13-linked Magnetosome can specifically and covalently conjugate to the Spytag-tagged protein in the bacterial lysate and they can be simply co-purified by a magnet.
08. AIS (Amilcp+intein+Spytag) BBa_K1947029
This part is used to improve the protein purification instead of TEV site. TEV protease may bring some impure proteins in the final elution of the recombinant protein. We use a self-cleavable sequence, intein, to replace the TEV site. The Mycobacterium Xenopi GyrA intein provides a paradigm for a minimal protein splicing element. Intein can be cleaved by DTT. DTT as reducing agent protects the free thiol groups of the protein from oxidation and it doesn’t bring foreign substance. As a result, intein can achieve a better effect of purification than TEV protease.
09. 3 x SPA Z domain
This is our smallest gradient protein and this part will be used to test the binding rate of Spytag and Spycatcher with the protein we want to purify with different molecular weight due to the simplicity of adjusting the size of SPA Z domain. The data will be used to build mathematical model of our project.
10. 6 x SPA Z domain
This is our smaller gradient protein and this part will be used to test the binding rate of Spytag and Spycatcher with the protein we want to purify with different molecular weight due to the simplicity of adjusting the size of SPA Z domain. The data will be used to build mathematical model of our project.
11. 9 x SPA Z domain
This is our medium gradient protein and this part will be used to test the binding rate of Spytag and Spycatcher with the protein we want to purify with different molecular weight due to the simplicity of adjusting the size of SPA Z domain. The data will be used to build mathematical model of our project.
12. 12 x SPA Z domain
This is our bigger gradient protein and this part will be used to test the binding rate of Spytag and Spycatcher with the protein we want to purify with different molecular weight due to the simplicity of adjusting the size of SPA Z domain. The data will be used to build mathematical model of our project.
13. 15 x SPA Z domain
This is our biggest gradient protein and this part will be used to test the binding rrate of Spytag and Spycatcher with the protein we want to purify with different molecular weight due to the simplicity of adjusting the size of SPA Z domain. The data will be used to build mathematical model of our project.
14. Pmsp3+Mms13+EGFP
In this part, we want to verify the expression effect of Mms13. Mms13 and EGFP are fused together to produce a fusion protein. Then it can prove whether Mms13 and EGFP can be successfully expressed in strain AMB-1 by the detection of EGFP florescence.
