Phenotype change after transformation

[kwan]

First, i would like to say thank u so much for every replies that help me in my first post (in last month ago). all of ur suggestions are helpful for me. now i have some problem again and i need some help from all of u.

i've transformed recombinant plasmid (pHT43 vector 8 kb + DNA 1 kb) into B. subtilis ISW1214 and B.subtilis 1A751. i've got many clones from this transformation but the phenotype of colonies were change and their growth rate are very slow( in both of LB broth and LB plate contained ampicillin) and some clones were not grown after i picked and straeked them on LB+ampicliin plate.

i've ever done a genomic library by using both B. subtilis strains to transform recombinant plasmid (same vector as above) and the the phenotype of clones were change too. it has a same phenotype that i get from PCR cloning i write above.

i don't know why? it may contaminate during the step of preparing competent cell but after i streaked the competent cell,all colony have a same phenotype . it's ok and the phenotype is as same as colony before preparing competent cell. so it was not contaminated,right??? but i don't know why the phenotype of colony were change after transformation.is the size of vector or recombinant plasmid have the effect on phenotype change but i've never seen this problem in transformation of this recombinant plasmid in E.coli.


Could someone here help and explain me about this result ? what i shoud do?i'm so confuse.

Thank you so much for all your help.

Kwan

[relaxin]

It may be that your gene is toxic to the cells. Did you transform the empty vector in those cells?

[kwan]

yes.but the phenotype of colonies were change and growth rate is very slow,too.

[mchlbrmn]

Is the growth rate too slow to grow enough to purify plasmid? If they won't grow at 37, perhaps they might prefer 25 degrees?
Actually, in the past I've seen phenotype changes of bacteria growing in liquid culture after transformation, I think, a long time ago. They may become more sticky, and formed globs adhering to the tip used to innoculate them. I think it may even have varied by the insert.
If they grow on the transformation plate, but not when streaked on a new plate, perhaps some clones are not truly antibiotic resistant, but survive because of a rescue effect of other nearby colonies?

[kwan]

Thank u so much relaxin and mchlbrmn

[bluetooner]

Do the bacteria grow well in liquid without anything transformed with it? It was never confirmed, but we had problems with bacteria growing in liquid a while back, and they even grew poorly on plates tbh. It was thought it was a bad batch of ampicillin doing it, but i think it may have been someone messed up making up the plates/LB. Wrong salt concentration/pH or even concentration of antibiotic may be what is happening.

[Duke]

First, i would like to say thank u so much for every replies that help me in my first post (in last month ago). all of ur suggestions are helpful for me. now i have some problem again and i need some help from all of u.

i've transformed recombinant plasmid (pHT43 vector 8 kb + DNA 1 kb) into B. subtilis ISW1214 and B.subtilis 1A751. i've got many clones from this transformation but the phenotype of colonies were change and their growth rate are very slow( in both of LB broth and LB plate contained ampicillin) and some clones were not grown after i picked and straeked them on LB+ampicliin plate.

i've ever done a genomic library by using both B. subtilis strains to transform recombinant plasmid (same vector as above) and the the phenotype of clones were change too. it has a same phenotype that i get from PCR cloning i write above.

i don't know why? it may contaminate during the step of preparing competent cell but after i streaked the competent cell,all colony have a same phenotype . it's ok and the phenotype is as same as colony before preparing competent cell. so it was not contaminated,right??? but i don't know why the phenotype of colony were change after transformation.is the size of vector or recombinant plasmid have the effect on phenotype change but i've never seen this problem in transformation of this recombinant plasmid in E.coli.


Could someone here help and explain me about this result ? what i shoud do?i'm so confuse.

Thank you so much for all your help.

Kwan

First: What do you mean by "phenotype"? How do you detect a change in the phenotype?

What is your portocol of transformation?
Do you use the protocol similar to a E.coli Heat shock or do you prepare protoblasts?

Have you tried different media than LB? For bacillus there are some special media recommended like Spizizen medium.

As a control I would suggest tranforming empty pHT43. If you see the effect also there, it has to do either with the Bacillus strain or your protocol of transformation/growth.

[kwan]

Thank u very much bluetooner and Duke.

blutooner, your reply can explain me everything about my result becuase after i run agarose gel electrophoresis to check a concentration of plamid but i didn't see any band. too bad that it's not a real recombinant..

Duck, i have the problem same as u because my B.subtilis host can not grow in LS and HS medium. So i use another protocol to prepare a competent cell but it's still not work as i post in this topic . can u tell me the way u solve this problem?what's a protocol u use to prepare competent cell and transform?

[kwan]

ohh! sorry Duke.

[amrnegm]

i am clooning a protease gene into a bacillus subtilis strain 1012 and i have no expression so far
i have tried to make mini prep from this strain and so far no mini prep
although i can see growth
with a very tubid LB medium

[mpagel]

i am clooning a protease gene into a bacillus subtilis strain 1012 and i have no expression so far
i have tried to make mini prep from this strain and so far no mini prep
although i can see growth
with a very tubid LB medium

A protease is likely to be cytotoxic to the bacteria, as it may chew up some of their endogenous proteins, especially if expressed to a high level. So, what you may be getting is dead and dying bacteria, with another bacteria type (with your plasmid dropped out or your plasmid never present to begin with) then growing which gives you the turbid media.

I would suggest sticking the protease gene on a more strict promoter, such as one induced by anhydrotetracycline (AHT or ATC), rather than the usual leaky IPTG. I would also recommend a low copy plasmid, to control expression levels. If you're still having no luck, I'd hit the promoter binding region with a mutation to reduce specificity of ATC binding to further reduce expression.

[Onyourcase]

"phenotype change after transformation"... check it's the same bacteria!

[ngrinter]

Is it possible your plasmid is not stably inherited by the host? Have you tried growing host + plasmid without antibiotic and screening for antibiotic-sensitive colonies after a few generations growth?

[relaxin]

Without selection, the bacteria will kick out the plasmid. They will become sensitive to ampicillin. That won't prove anything.

It will be better to take a single colony of the original host bacteria, make sure it is sensitive to ampicillin, make competent cells, and try again.

[shoca]

Do you have magnesium in your medium? B. subtilis like 1-10mM MgCl2. [/quote]