Difference between revisions of "Part:BBa K2888000:Experience"
Yinchizhou18 (Talk | contribs) (→User Reviews) |
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<p>7. Growth of E.coli</p> | <p>7. Growth of E.coli</p> | ||
<p>8. Protein Purification</p> | <p>8. Protein Purification</p> | ||
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| + | <img src="https://static.igem.org/mediawiki/2018/4/4a/T--SBS_SH_112144--Cyanobacteria12_.jpg" width=600 height=500 | ||
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<h4>Functional Test:</h4> | <h4>Functional Test:</h4> | ||
<p>1. Cyanobacterial bacteria lysis reaction.</p> | <p>1. Cyanobacterial bacteria lysis reaction.</p> | ||
Latest revision as of 17:49, 17 October 2018
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how you used this part and how it worked out.
Applications of BBa_K2888000
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
User Reviews
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. Finally, we used our expressed proteins in cyanobacteria lysis reaction
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.