Part:BBa_K2888003
fused lysozyme gene with 6x His tag
Description: Our composite part within prefix and suffix consists of the most commonly used promoter, RBS, 6x His tag and terminator, as well as our functional coding sequence --- cyanophage lysozyme gene. This composite part is assembled through Gibson Assembly; then, it is fused with linearized vector, pSB1C3 backbone, via infusion. Ultimately, we successfully extract plasmids which contain our target gene from transformed bacteria. Furthermore, we purposely add 6x histag into our sequence in order to improve the efficacy and efficiency of expression and purification; it will also strengthen effects of binding to Ni-NTA column, making it possible to immobilize proteins and lyse cyanobacteria repeatedly in our prototype device.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Introduction
1. The aspartic and glutamic acid residues at the active site of the lysozyme would wrap around the glycosidic bond between 6 carbon conponent N-acetylmuramic acid （NAM）and N-acetylglucosamine (NAG) of the carbohydrate section of the peptidoglycan. The carboxyl group on the aspartic acid attacks the carbon on NAM, attaching the aspartic acid to the NAM. Meanwhile, an oxygen on the NAG would pick up the hydrogen proton on the glutamic acid, deprotonating the glutamic acid into a glutamate.
2. The lysis of lysozyme has to take place in aqueous environment. The addition of a water molecule would complete the hydrolysis reaction by protonating the glutamate back to glutamic acid; it would also deprotonate the aspartic acid from the NAM, restoring it back to its reduced form, while leaving a hydroxyl group on the end of NAM. This way, the lysozyme would keep its own structure unchanged while degrading the carbohydrate section of the peptidoglycan into the NAM/NAG components.
Design Notes
We directly copy the sequence from the previous experiment by Kunal Mehta, Niklaus Evitt and James Swartz. However, since it has the unexpected restriction sites, we have replaced them with corresponding code.
Our cp-OS lysozyme 1 is a form of bacteriophage lysozyme that exists in virus. Once the cyanophage finish replicating in cyanobacteria, they produce the cyanophage lysozyme to lyse the cyanobacterial cell wall to get out. Through protein blasting of the cyanophage lysozyme, we found its structural similarity and homogeneity with multiple other peptidoglycan binding proteins and even a type of peptidoglycan domain protein from cyanobacterium Nostoc, and this implies the working mechanism of this lysozyme is similar to other lysozyme and is somewhat specific to cyanobacterial peptidoglycan.
Application
We intend to use lysozyme gene to lyse cyanoabacteria which has unusually thick and complex cell wall. The cyanobacterium has a much thicker peptidoglycan layer and a unique external layer containing an S-layer and exopolysaccharide. In our experiment, we easily mix Bugbuster and expressed protein in order to fully lyse cyanobacteria cell wall
Our Experience
We successfully infused lysozyme gene with pSB1C3 backbone and transformed it into DH5 alpha E.coli in order to get plasmids. Then we transform extracted plasmids into BL21 E.coli in order to culture abundant E.coli and expressed proteins which contain our target gene. Moreover, we purified proteins through Ni-NTA column, as we purposely add 6x His tag into our sequence in order to improve the efficacy and efficiency of expression and purification. It will also strengthen effects of binding to Ni-NTA column, making it possible to immobilize proteins and lyse cyanobacteria repeatedly in our prototype device.
Protocol:
1. Amplify target gene and pSB1C3 backbone
2. Gel electrophoresis to verify the existence of amplified gene
2. Infusion of backbone and target gene
3. Transformation and single colony selection
4. Inoculation in liquid LB culture
5. Bacteria PCR to verify the existence of amplified gene
6. Extraction of plasmids and transformation into BL-21 E.coli
7. Growth of E.coli
8. Protein Purification
Functional Test:
1. Cyanobacterial bacteria lysis reaction.
2. In a microfuge tube, take 300uL cyanobacteria with a OD660nm absorbance of 1.5.
3. Centrifuge and take out all the supernatant.
4. Add 30uL Bugbuster, 30uL of pH buffer(dependent on pH), 60uL of enzyme solution(made previously according to different protein concentration requirement), and 180uL of water.
5. React different tubes in shaker under different temperatures(according to different temperature requirement) for a 0.5,1,1.5 or 2 hours(depends on different reaction time requirement.
6. Take out the microfuge tube, centrifuge for 10 minutes at 14500 rpm.
7. Take out 270uL of the supernatant to test the OD660nm light absorbance.
Result
1. By applying the lysozyme to a system（300ml in a microfuge tube reacting under different temperature) with bugbuster, cyanobacteria, pH buffer and water, we are able to observe cleavage effect illustrated by the OD660nm green light absorptance across large temperature and pH range. And through a series of mathematical computation and modeling, we are able to identify that the ideal pH is around 6.9 and temperature is at 37 °C.
2. After finding the ideal pH and temperature for the lysozyme, we successfully applied these elements to a new set of function test with different protein concentration and reaction time, and we are also able to, through PSO algorithm and 3D modeling in matlab, find out the optimal reaction time being 0.5h( quite different from the paper) and the protein concentration being 18 ug/mL.
Source
This cyanophage lysozyme is discovered by Heideburg et al in Yellowstone National Park {Heideburg:2009hb} among 29 lysozyme family.
Reference
Jensen HB, Kleppe K: Effect of ionic strength, pH, amines and divalent cations on the lytic activity of T4 lysozyme. Eur J Biochem 1972, 28:116–122.
Kunal Mehta, Niklaus Evitt, James Swartz: Chemical Lysis of Cyanobacteria. Journal of Biological Engineer, 9/10/2015
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