how do lysis of bacillus to extract protein

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how do lysis of bacillus to extract protein

Postby jin » Jun 15 2004 12:27 pm

hi,

i've been trying to detect the protein in bacillus with antibody. but when i do lysis of bacillus and run sds-gel, i saw very little protein on gle and i cannot detect the protein i want. i use SDS and lysozyme (1-5mg/ml)to do lysis and extraction.

i'd ever express the gene in E. coli and putrified the protein and it is easy to purify the protein. Anybody know how to extract protein from bacillus? thank you very much!!
helping is always appreciated.
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Postby Dynein » Jun 16 2004 9:25 am

Depending on how much bacteria you want to lyse, you should be able to spin the bugs down and add a volume of SDS Laemmli sample buffer and boil them at 100 degrees for 10-20 min. After boiling, spin the samples at 14,000 rpm in a microfuge and take the sup for use on a gel. I have used this method successfully for lysing E.coli but I think it should also work for bacillus.
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Postby jin » Jun 17 2004 9:38 pm

yes, i can do cell lysis for E. coli. but i cannot do cell lysis for Bacillus subtilis with the same method as in E.coli
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Postby Dynein » Jun 21 2004 9:18 am

Just out of my curiosity can I ask why not?
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Postby Ziggy » Jun 22 2004 5:22 am

How do you do lysis of E. coli and B. subtilis? E. coli I would normally lyse on ice; the cell wall is rather thin, and is degraded quickly (in the presence of Tris and EDTA). B. subtilis, however, has a much thicker cell wall. You will need to incubate with lysozyme at 37 oC, and leave it a bit longer than you would with E. coli.

However, if it is for just analysing samples on gel, it should be OK to dissolve the pellet in water (or some TE buffer or whatever), resuspend the cells properly (no clumps visible), add equal volume of SDS-Page loading buffer, and boil the sample for 3 min.
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Postby jin » Jun 22 2004 2:18 pm

hi ziggy,

i used the buffer consisting of tris, PMSF and SDS to dissolve the pellet then i add lysozyme and incubate 30 min in ice. then i boil them 10 mins. after finishing these porcedures, i still see pellet after centrifuge(usually, this kind pellet do not happen to E. coli).. on SDS-page gel, i just see very very faint band. i think the the cell were not lysied well. i also tried commercial protein extraction buffer for yeast to lyse bacillus, it could not work yet.

thank you for your replying!!

Jin
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Postby Ziggy » Jun 23 2004 9:34 am

If cells don't lyse properly, you can make protoplasts as described [URL]http:// molecularbiology.forums.biotechniques.com/forums/viewtopic.php?t=7020&highlight=[/URL], and then add loading buffer.
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thanks

Postby jin » Jul 26 2004 4:26 pm

Hi ziggy,

thanks. with the method suggested by you, i can lyse bacillus.

now i want to ask you other question. i knocked out two genes individually in bacillus. now my professor want me to knock out the two gene together. i looked for some reference, but i did not find. do you have some clues on how to do double knockout in bacillus?


thanks a lot!!!

Jin
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Postby Ziggy » Jul 27 2004 3:16 am

If you need to knock-out two individual genes, you have 2 options. First, you can use different plasmids for integration with different antibiotic markers. So clone an internal fragment of each gene in a different integration vector.

Alternatively, delete the genes in such a way that no marker is left. So you can delete the first gene, and then the second gene with the same system. An example of such a system is described in:

Leenhouts et al (1996) A general system for generating unlabelled gene replacements in bacterial chromosomes. Mol Gen Genet. 253: 217-224.
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Postby jin » Oct 04 2004 11:30 am

Hi ziggy,

have you ever grow wild type bacillus subtillis ?

i am growing wild type bacillus subtillis 168 strain to compare the its growth with my mutants' growth. but i find it is difficult to get repeatable results on growth rate. i am using LB medium. i doubt the unrepeatability is caused by contamination. anyone has good experience to culture wild type baceria without antibiotics. would you please tell me how to avoid the contamination? thank you very much!

Jin
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Postby Ziggy » Oct 12 2004 3:14 am

I have grown B. subtilis 168 without problem. How do you start your cultures? From -80 oC stocks or plates? Sometimes you get different lag phases, which can make it difficult to make comparisons. To compare growth rates it is best to make a preculture first (just an overnight culture). Then, dilute cultures to similar optical density, e.g OD 600 = 0.01 and follow growth during the day.
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Postby jin » Oct 18 2004 4:39 pm

Hi,

thank you for your replying. i firstly grow it on plate from -80 stock. then i make preculture 5 ml from plate. then i usually dilute the preculture to OD 0.07 at 600. grow the diluted medium at different temperature. but it is difficult to get repeated result.

Jin
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which vector is better

Postby jin » Nov 02 2004 11:33 am

Hi Zyggy,

have you ever used other integration vector except for pMutin4 vector? which integration vector is better except for pMutin4?

Thanks!

Jin
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Postby Ziggy » Nov 02 2004 11:58 am

I have actually never used pMutin4; it's been quite a while since I did any Bacillus work, and I have only used an earlier version of the plasmid, pMutin2. That has basically been the only plasmid that I used for knock-outs.
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Re: how do lysis of bacillus to extract protein

Postby meggie » Aug 16 2016 3:54 pm

I am also doing extract protein from bacillus subtilis. My band on SDS-PAGE gel is very faint. Could anyone give me some suggestions? Later I need detect target protein(one is in vegetative cells, the other is on the spore surface) by western blot. Only sonication does not work for me. Should I use lysozyme? What temperature and how long should I try? Thanks!
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