Bacillus: An alternative for E coli in DIYbio?

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Koeng

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Jan 1, 2014, 6:16:45 PM1/1/14
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Hey guys!

Recently I have begun using Bacillus subtilis a lot, and I gotta say... it is a LOT easier then E coli. For example I did one experiment with a strain of Bacillus that over expressed comK under a xylose inducible promoter... all I did was get the cells in growth phase, give them some DNA, grow them for 45 minutes then plate. I got transformation efficiency of 6x 10^3 with a circular integration plasmid. That is even easier then Cathal's Bacillus transformation protocol (no offense (:  ), which is one of the simplest out there. I can easily get better expression of comK by optomizing the RBS and I can make this work under  a sucrose inducible promoter! (Working on that right now) In addition to this, I have found a way to negatively select in Bacillus (I think it is the mazF gene from e coli), meaning that I could possibly make a strain which all you need to do is transform them, no selection method needed! 

Plus Bacillus can take pure PCR fragments... meaning that using homologous recombination and negative selection you could literally PCR an interesting protein from lets say, some cheek cells, then integrate it into bacillus. The integration could then be expressed, secreted, and then purified (using a variety of tags already in the bacillus, given that the gene has no introns/exons) Skip plasmids and e coli entirely! (of course... I need to make these strains)

Anyone have anything to add or conflicting views?

-Koeng

Mega [Andreas Stuermer]

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Jan 2, 2014, 4:43:32 PM1/2/14
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Sounds awesome! But how do you surpress the protein feom killing the cell befire transformation? You gotta amplify it first carrying the lethal gene.

Maybe incubating them with some inhibitor?

Koeng

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Jan 2, 2014, 6:07:00 PM1/2/14
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The sacB promoter, which is activated by sucrose, would express lets say, cro. The cro would then repress the mazF gene. Once you cut the bacteria's sugar rations, cro no longer gets expressed, so mazF could be expressed

The sacB promoter would also control comK, which would make it so you make comp cells in the presence of sucrose. Double whammy!

elegans

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Jan 2, 2014, 9:24:09 PM1/2/14
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Sure, all that sounds good. Plasmids are useful though if you want to use bacteria to produce DNA for you to use in other downstream applications. For example, many people would use bacteria to produce plasmids that are then transfected into cell culture.

Geoff Ivison

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Jan 3, 2014, 12:43:28 AM1/3/14
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Never done cloning with Bacillus before, but I'd think its tendency to form spores could make cross contamination an issue. You definitely have some interesting benefits there!

Nathan McCorkle

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Jan 3, 2014, 1:18:51 PM1/3/14
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Bacillus subtilis can discard pieces of your DNA if the transformed
size if too large. An old source says:

"During transformation of B.subtilis, plasmid DNA is linearized as it
is transported into the cell. One strand of the entering duplex is
degraded, and resynthesized at a later step within the cell. Once
inside the cell, chromosomal DNA would recombine with homologous
sequences on the recipients chromosome. Plasmid DNA, however, has no
such homologous sites. Unless it is able to recircularize, plasmid DNA
is eventually degraded. No natural mechanism for rejoining linearized
plasmids has been described. Thus the only possibility for
recircularization is a self-recombination event between homologous
parts of a single plasmid or a multimeric plasmid."

"E. coli does not have a requirement for multimeric plasmids but will
generate multimers through the normal course of replication and
recombination."

"Therefore it is possible to initially transform E. coli with a
ligation mix and, on the following day, extract plasmid from the
transformed culture and use it to transform B. subtilis. A liability
with this strategy is the B.subtilis tends to randomly reduce the size
of large plasmids. Thus plasmids approaching about 10kb are likely to
suffer rearrangements and reductions."

pulled from:
Cloning Methodology Chapter 1a
Biotechnology Vol 7b 1989
http://people.rit.edu/rhrsbi/GEPages/LabManualPDF5ed/Cloning%20Methodology%20.pdf
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Koeng

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Jan 3, 2014, 1:33:56 PM1/3/14
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I think if you have the resources to transfect cells that it would be easy to get plasmids

@nathan Thats for plasmids though. I am talking about homologous recombination into the genome. It is actually more stable then any other large DNA cloning method, given that you do it properly. It can stably hold 3Mbps, and most likely more. Also, using plasmids in Bacillus for homologous recombination would be rather unstable, since 1 untransformed plasmid could destroy your experiment

Nathan McCorkle

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Jan 3, 2014, 3:49:18 PM1/3/14
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Hmm, one of the references there is:
http://diyhpl.us/~nmz787/pdf/DNA_Shuttling_Between_Plasmid_Vectors_and_a_Genome_Vector_Systematic_Conversion_and_Preservation_of_DNA_Libraries_Using_the_Bacillus_subtilis_Genome_BGM_Vector.pdf

which does actually mention multiple month growth and heat cycling and
spore induction with tests before and after.

I found that they use BReT to get the DNA back out, but couldn't find
a nice overview of that which didn't require me to read too much more.
> https://groups.google.com/d/msgid/diybio/72542ffa-89f4-4ddc-b2ed-08a54176c1c9%40googlegroups.com.

Josiah Zayner

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Jan 3, 2014, 8:10:51 PM1/3/14
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It is great to try new things but the problem with starting with a completely new species of bacteria as a model genetic tool is that you are starting over or nearly as I am sure some research work has been done with Bacillus. I assume the strains of Bacillus are not endA- and recA-(meaning your DNA and the DNA of the host have a chance of being screwed up) and you probably cannot do blue white screening with them, a useful genetic tool. 
Say you want to express a protein with a T7 or T5 promoter are you just going ask someone for the prophage and lysogenize these strains? Genetic editing is probably much more difficult without Warner strains and such. Do you know if Bacillus can produce comparable amount of protein compared to E. coli using T7/lac/IPTG? Does Bacillus restrict non-methylated DNA? Would make working from PCR/synthesized products difficult. Seems like an interesting hobby to test these things but Scientists probably will not use Bacillus just because it is slightly better than E. coli as E. coli has been used and tested and strains have been perfected for 10s/100s of years? Maybe Bacillus would have been the correct choice to start with cloning 100 years ago but  it is kind of too late. Remember you are fighting against years and years of research and optimization(probably more than a single lifetime) with the thousands/millions of strains of E. coli it doesn't seem like the _best_ choice. Though I am sure it could have some advantages maybe it would be good to focus on those.

Josiah

Koeng

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Jan 3, 2014, 10:26:11 PM1/3/14
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Bacillus subtilis is not a "new species"... It is the second most studied bacterium in the world and has more information on its genetic structure then E coli. It has the most complete minimal gene set of any living organism

First of all, remember that plasmids are NOT being used. So yes, they are endA- and recA-. The purpose of Bacillus is to engineer its genome, not plasmids. E coli can't even compare to its ability to put stuff into its genome, even engineered strains. Genetic editing is WAY easier then in E coli, where you must usually have to use complicated tactics to genome edit. In fact, a group of researchers have made more then 3000 strains of bacillus, each with a single gene in its genome knocked out, which got added on to the already huge database of bacillus strains at the Bacillus genetic stock center, http://www.bgsc.org/_catalogs/Catpart1.pdf . If you HAVE to have a screen in Bacillus, just use the amyE locus with iodine, which is even cheaper then XGal. Or, put GFP there instead and just replace that. 

Bacillus subtilis may restrict some non-methylated DNA, but in literature it has been shown to be more effective to use PCR products then it is to use recombination vectors (given that they aren't linearized). 

For protein production, if you wanted to express a protein with T7 or T5 promoter, just PCR the gene for each polymerase out of one of your e coli strains and replace, lets say, an integrated phage gene. Hell, you wouldn't even need to add a promoter or RBS, it would already be there if you do it correctly. Bacillus are also a lot more efficient at secreting enzymes then E coli, since they only have 1 membrane. Secreting the enzymes produced can be the difference between killing your cells with too high expression and keeping them alive

So rather, I would think that working with Bacillus is not fighting against but rather with years and years of research and optimization (tools for competence in Bacillus have been more explored then E coli, you can even "express competence" under a promoter with the protein comK)

Mega [Andreas Stuermer]

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Jan 4, 2014, 4:05:34 AM1/4/14
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If the problem with plasmids is that they need to recombine, why don't you give them homologous recombination sites?

I imagine some 300 bp each side should be sufficient.
There has to be a restriction site between them, though. And maybe a lethality gene for e coli that gets a promoter when it recombines to ensure stability during cloning?

Josiah Zayner

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Jan 4, 2014, 10:54:57 AM1/4/14
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E. coli has so many powerful genetic tools it is a pity not to use it.

I have seen multiple single papers with more edits in the E. coli genome than that whole catalog for Bacillus. 

How about MAGE?

If one is going to "express" recombination competence one can do that in E. coli also. And use "PCR products" to recombine into the host genome. See Wanner strains (http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement)

Expressing proteins to purify is not so simple as you say, just secrete them from cells. And overexpressing proteins rarely kills cells. Though I am sure it might actually happen this is rarely/never a case Scientists worry about. Protein folding/aggregation and intrinsic toxicity is of way more importance. Expressing something like a restriction enzyme that would damage the host DNA would be a case in which B. subtilis would be useful but general protein expression I have my doubts. Though I have no evidence for or against.

Your enthusiasm is great but if B. subtilis is so amazing why don't most Scientists and companies use it as their genetic tool of choice? Have the majority of Scientists in the world for the past 50? years been wrong and you and Cathal are correct? 

If I had to guess why people don't use B. subtilis I would say:

1. B. subtilis might be autocompetent but homologous recombination from what I read requires overlapping regions of > 500bp(http://mic.sgmjournals.org/content/154/9/2562.full) (if you could point me to a paper that shows less is required without a complicated strain of complex manipulations that would be cool). This adds complicated work to any design and multiple cloning steps. E. coli homologous recombination can be done with ~40bp which means it can be added to the end of a cheap primer and the gene created in a single PCR step. Further, needing to express comK is similar to needing to express a protein in E. coli for homologous recombination making it no more useful.

2. Under optimal growth conditions the doubling time of B. subtilis is always slower than E. coli. B. subtilis is measured to be somewhere around 50-120min(http://jb.asm.org/content/167/1/219.full.pdfhttp://jb.asm.org/content/180/3/547.full). While E. coli is consistently measured to be 20 minutes or less(http://jb.asm.org/content/180/3/547.full). This is very significant considering it would take triple the amount of time for the ~same cell mass.

3. Transformation efficiency as you have stated and as I have read in other papers is staggeringly low in B. subtilis, on the order of 10^5 /ug of DNA at the extreme high end(http://www.ncbi.nlm.nih.gov/pubmed/22387342) and usually <=10^3. E. coli on the other hand is around 10^10 or greater /ug of DNA generically. When creating genetic libraries or performing simple transformations this is highly significant and greatly restricts the use of B. subtilis. A typical ligation reaction I perform has around <=100ng of final product that would yield <=1 colony. I know regularly when cloning I often only receive a few colonies of E. coli DH5a or DH10B cells when transforming a quickchange or a ligation, this would likely yield zero colonies in B. subtilis and cause much more work.

4. According to the NCBI database there are being worked on or have been completed 237 projects to sequence E. coli genomes(http://www.ncbi.nlm.nih.gov/genome/?term=escherichia+coli) and over 75 have been completed (http://ecoliwiki.net/colipedia/index.php/Sequenced_E._coli_Genomes) B. subtilis currently has 23 projects total (http://www.ncbi.nlm.nih.gov/genome/?term=bacillus+subtilis). Though logically one could argue that this might not mean an awful lot it shows how much more Scientists know/want to know about E. coli genetics/metabolism &c. This makes the longterm viability of E. coli much more inviting. It is like using an open source package that has 23 versus 237 people working on it. Which one do you think will have all the snazzy new features and still be around in 1, 2, 5, 10 years? Further, according to Google Scholar in 2013 ~190,000 papers contain the term "e coli" and ~26,000 contain the term "b subtilis"(no quotes used in actual queries). 
 
5. Many proteins don't unfold/refold well, as occurs during secretion. Many protein have cofactors or require chaperones not found outside cells. This makes secretion only a slightly useful tool. Searching for optimal protein expression yield I couldn't find data for E. coli and B. subtilis in which they used the same protein and conditions so I don't know which is better for protein expression. 

I agree B. subtilis has its positives. This discussion has even made me consider using it for one of my projects that requires protein secretion but I don't think you are going to convince me or most Scientists that it is better than E. coli as a genetic or synthetic biology tool. 

I am not saying to stop your work I am just suggesting that if you want to use B. subtilis you should focus your work on the advantages it has over E. coli instead of attempting to compete with an already established genetic/synthetic biology tool.

Cheers,
     Josiah Zayner, Ph.D. 
     Synthetic Biology Group
     NASA Ames Research Center

John Griessen

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Jan 4, 2014, 12:49:41 PM1/4/14
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On 01/04/2014 09:54 AM, Josiah Zayner wrote:
> Have the majority of Scientists in the world for the past 50? years been wrong and you and Cathal are correct?

This kind of logic is frequent from you, but it is not backed with relevant to the original idea
of alternatives to e. coli...

That statement seems like peer pressure and justifying your position
as Scientist, (you use a capital S.), and hinting that non e. coli users are opposed to capital S science,
or even not a member of the capital S. set?

b. subtilis *not* being part of many existing patented researches, is part of the reason to use it
in a free-published way.

Josiah Zayner

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Jan 4, 2014, 1:18:15 PM1/4/14
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Interesting how that single sentence is what you took away from my whole email. And there is still no answer to my question. Why do we need alternatives to E. coli when it is such a powerful tool?

Creating a new patent/MTA free B. subtilis strain probably requires similar effort as to creating an E. coli strain. So I don't see that as a valid argument. 

It is not peer pressure. It is experience. I used to always have similar ideas, as to how to change something that has been done the same way for many years. What I learned is that Scientists are smart. They really are and I would say the vast majority of the time Scientists have good reason for what they do. What I learned is that Science is not about bucking the system, that requires so much effort for very little gain. I gave many reason in my email about things that would hinder B. subtilis from being an accepted genetic tool, which the original post asked for. I think that instead in Science instead of attempting to "reinvent the wheel" it is better to focus on having interesting and creative ideas that could contribute alot. Maybe I wrong, which happens frequently, this is just my advice. I am 32 and don't have an infinite amount of time so I like to focus on things that have a higher probability of making a difference/being cool/being beautiful. If you are starting a new project why not pick the one with the best/coolest/most beautiful outcome?

I was just attempting to give advice. I wasn't trying to put people down.

I always capitalize Science and Scientist. It is because I cherish the words and they are very important to me. I meant no disrespect.
 




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Alexey Zaytsev

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Jan 4, 2014, 1:30:47 PM1/4/14
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If the growth rate is so much slower, this alone could be a good
reason to choose coli, especially for DIY. We don't always work in
sterile conditions, but E.coli is often able to outgrow contaminants.

I started as DIY, and didn't have much problems working with them in
my kitchen - I would eventually get some mold growing on the plates,
but by that time E.coli would long have formed nice colinies.
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John Griessen

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Jan 4, 2014, 2:04:54 PM1/4/14
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On 01/04/2014 12:18 PM, Josiah Zayner wrote:
> If you are starting a new project why not pick the one with the best/coolest/most beautiful outcome?

If it is genuinely new, innovative, the outcome will not be knowable at the start.

> I think that instead in Science instead of attempting to "reinvent the wheel" it is better to focus on having interesting and
creative ideas that could contribute alot.

Alternatives like b. subtilis may be so free thinking and creativity supporting as to
go beyond what you consider Science,
and be great for mere tech development.
Development of things like bioplastics, biopaper, bioconcrete, artificial wood, biofilms that filter
or osmote, bio-electric-critters, bio-PV-cells, etc.

After the bugs get some good mods and start being useful to many,
there will be new unforseeable discoveries that would not happen with e. coli. Unforseeable outcomes
are just normal for diverging from the main group.

Don't worry, it won't cost you a bit. You won't be involved from the way you talk of it.

Koeng

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Jan 4, 2014, 2:51:20 PM1/4/14
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@Mega I honestly have not looked into plasmids for Bacillus, I didn't really see any reason to use them over. Just use that 300bp to recombine into the genome

@Alexey there is not a noticeable difference between the 2 growth times unless you are measuring it. It still grows very very fast

Remember that I am not trying to replace E coli, but rather have a complement bacterium for all of its faults. If a guy wants to work with plasmids, use E coli. If you want to get into genome engineering, use Bacillus. And I have to defend the Bacillus genetic stock center... that is just one of their catalogs, I have never found a strain of Bacillus that wasn't there (excluding anthrax of course). They also never denied to ship me plasmids and strains 

1) Expression of comK is only to make it easier to transform. Cathal's protocol, for example, uses sporulation to get the cells competent. For overlaps required, I know several papers that use PCR, but just for example this one ( http://mic.sgmjournals.org/content/154/9/2562.full ).

2) I can't argue because Bacillus IS slower, but at the same time in my experience overnight growth is more then enough to get saturated cultures. Since i always do things overnight, I have never seen a difference

3) Maximum transformation efficiency is low, but the effort it takes to get there is much lower. (10^5 with PCR products, http://onlinelibrary.wiley.com/doi/10.1111/j.1751-7915.2010.00230.x/full ) Remember the transformation efficiency 10^10 is with plasmids. I see no reason to NOT use E coli in anything that needs plasmids or library prep. Anyway, in my experience a microliter of minipreped plasmid is more then enough for the cells.

4) Personally I think that a paper search will naturally be against Bacillus. Someone transfecting cells, using CRISPR in mammals, studying human viruses, or even working with zebrafish will use E coli for their cloning. Not all the papers will be on techniques for E coli. But i think that nearly ALL papers with Bacillus are working on something with physiology, since in the past that is what you did with Bacillus. In addition the reason there is so many sequenced e coli is because most of them cause disease, which

5) If someone like me doesn't go out and try and make a new technology like with Bacillus, who is? Who knows, if no one tries we may never discover a great new technology.

MAGE's idea can EASILY be ported to bacillus

I agree this has been a very interesting discussion, just the thing that i came here for :). Some of these links are making me consider actually working and making the technologies in both of these organisms (however doubling the amount of my personal money I spend....)

Anyway, on the topic of first trying a project with "best/coolest/most beautiful outcome"... People need to make technology that allows others to do that as well! I am 14 years old and work at UCI in a research lab with my own project, and so I have plenty of leftover time to explore my world and make new technology for the benefit of not only me but a whole range of people. When I got into biology in 7th grade I couldn't make plasmids very well... hell even a transformation was very difficult. I don't really see anyone making bacterial strains or plasmids for DIYbio, but it would be great if they where there for a beginner

-Koeng

Mega [Andreas Stuermer]

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Jan 4, 2014, 5:03:40 PM1/4/14
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What may be nice for beginners would be a highly bioluminescent strain of Bac.subtilis. When you bring your GOI into it, it will stop being luminescent (therefore grow faster btw).

One day, when gene synthesis is cheap enogh, I'll do that :)

Cathal Garvey

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Jan 6, 2014, 8:28:31 AM1/6/14
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I'd like to address some of your points, but first I'd like to suggest
that saying "don't do that, everyone else is doing this" is a poor
reason to ward someone off of a project. E.coli is awesome, it's true,
but B.subtilis has long-recognised advantages in areas where E.coli lags.

So, if I want to do libraries and sequencing, I'll take E.coli. If I
want a high-risk project where I need my variables as minimally-variable
as possible, I'll take E.coli.

If I want long-term shelf-life or easily shipped, or simply tolerant of
abuse and forgetfulness, I'll take B.subtilis. If I want something
that's more accessible to an E.coli-averse public, I'll take B.subtilis.
If I want protein export from the cell (which is sometimes necessary for
correct protein folding, I'll point out), I'll take B.subtilis, because
E.coli protein export beyond the periplasm is pretty damned awkward.

This is biology; relative species advantage is the name of the game, and
for some things B.subtilis is more attractive than E.coli.

For that reason, long back I advocated B.subtilis for amateur biotech
because it's easier to handle and grow (I don't notice a *practical*
difference in growth rate btw; it's there but is rarely an issue), it
survives real life amateur conditions (forgotten, mishandled, stored in
a shoebox for years), and it's naturally competent. As a store of
genetic material, it's great in theory; without a -80, E.coli stocks can
be very unreliable, but B.subtilis can be stored on card on a shelf.
Again, for practicality, B.subtilis is great for amateur use.

Since then, I've found protocols that help make E.coli more practical,
too; better transformation systems for amateur environs, but B.subtilis
still has advantages.

My attempt to make a B.subtilis plasmid that would be practical for
amateurs was a partial success, but I didn't have the time or money to
make it a full success, so it's shelved for now. For reasons you
suggest, I am working on a lower-risk project right now in E.coli, but
that doesn't diminish my interest in providing a B.subtilis plasmid
someday, perhaps soon.

Anyway, by heading:

> 1. *B. subtilis* might be autocompetent but homologous recombination
> from what I read requires overlapping regions of > 500bp [...]

Yes. Homologous recombination in most species requires long tracts, and
this is no shorter AFAIK in E.coli or Yeast. The advantage of B.subtilis
is that it has an *active uptake and recombination* system, rather than
passive, so actually you can make do with shorter tracts and get
reasonable efficiency. This was never a target of mine so I don't
remember how low the homology can go.

Remember also that for gene erasure or replacement, the overlapping
regions are part of the target region, so it's not so burdensome. If you
want to introduce new DNA, then having to add up to 500bp extra is
harsh. For deleting DNA if all you need is 500bp, that's cheap. More on
this below, where deletion is relevant to strain improvement..

> 2. Under optimal growth conditions the doubling time of
> *B. subtilis* is always slower than *E. coli*. *B. subtilis*
> is measured to be somewhere around 50-120min

This is true, and I'm not disputing that for the same *cell mass* this
is a problem. However, I can only say that in my experience, this rarely
impinges upon the ease of working with B.subtilis. Part of the reason is
probably that colonies in B.subtilis are more sparse, so if you're only
seeking colonies and not a large cell mass, B.subtilis effectively
competes with E.coli because colonies as fast by volume if not mass.

Mass is important for protein or DNA yield, so for miniprep DNA yield,
this is still an issue, yes.

Bottom line is that for my purposes, which probably overlap well with
Mega's and many others', growth rate was rarely an issue when working
with Bacilli, for me at least.

> 3. Transformation efficiency as you have stated and as I have read in
> other papers is staggeringly low in *B. subtilis*, on the order of
> 10^5 /ug of DNA at the extreme high end

Pretty sure I recall seeing it up at 10^6/7 sometimes, but yes; getting
high efficiency is harder than with E.coli. IIRC (and I probably don't),
those protocols I saw were chemical methods that involved making
spheroplasts, so let's chalk those up as "not applicable".

So yes, as you well point out, you don't want to do libraries for
sequencing or in-vitro mutagenesis in Bacilli; go with E.coli. Without
taking away from that fact, consider that for sequencing, libraries are
of decreasing relevance, and for mutagenesis there are in-vivo
alternatives if you really want to work with Bacilli.

If you only have a tiny amount of DNA with which to transform, for
example with a quickligate or what-have-you, then I'm with you here;
don't risk your tiny amount of DNA on B.subtilis if you can't afford to
lose it.

However, again on the practicalities; most use-cases of a competent
bacterium, I find, do not call for 10^6/7/8/9. 10^3 might be a bit on
the low side, but frankly I'm impressed that just throwing DNA at
growing cells worked out for Andreas! B.subtilis 168 (the "type" lab
strain) is widely known to be extremely easy to hack, and I had someone
say to me that you can practically throw DNA at it but I thought they
were kidding..

Anyways, I never quantified my transformational efficiency because of
the particulars of the selection system I was trialling, which was
positive rather than negative selection. I ordered a conventional
negative selection plasmid but failed to transform it into an E.coli
stock prior to use in Bacillus and lost the DNA. So, deduct a point from
E.coli, I guess. Takeaway point; I got plenty of transformants using my
sporulate-then-germinate-with-added-DNA method. Bonus; that's the
protocol, it's really that simple. No steps or nothin', just add spores
and DNA and plate. :)

> 4. According to the NCBI database there are being worked on or have
> been completed 237 projects to sequence *E. coli* genomes

Forgive me, but isn't this an unfair comparison? E.coli genomes will
outnumber probably every other species, so you could use this argument
to suggest that E.coli is a more important organism than Humans, for
example.

That said, let's address a non-obvious factor you ought to consider even
if you apply weighting by publication count, genome size, etc. to figure
out the relative sequence coverage of Bacillus: species-level balkanization.

This was a really annoying issue for me when I was designing my plasmid,
because one of my design constraints (which may help account for your
questions as to my bizarre methodology) was that all genetic material on
the plasmid should be of B.subtilis origin only. This is because EU law
regards cisgenics as "not Genetically Modified", in a very explicit way.
So if my plasmid were useful and only consisted of B.subtilis, I could
sell it to unlicensed people in the EU and they could legally use it.

I discovered that, whereas E.coli is a pretty diverse species name,
B.subtilis had been diced up into lots of separate species in the
nineties. Being honest with you, I never bothered to align E.coli
genomes and pre-balkanisation B.subtilis genomes to see whether they
were justifiably different, but it struck me that there was a
fashionable trend of renaming B.subtilis strains as new species for a
while there, and it hindered my work because some of the more pigmented
strains became legally "new species" and therefore pigments were
off-limits on my plasmid. Boo. Funny how humans can so quickly rewrite
Nature, right? Who needs to actually do biohacking to make new species
when you can just join a classification committee and make new species
with the stroke of a key? :)

> 5. Many proteins don't unfold/refold well, as occurs during
> secretion. Many protein have cofactors or require chaperones
> not found outside cells. This makes secretion only a slightly
> useful tool.

Here I'm going to mostly agree with you. On the one hand, you can't take
the average protein and just shove it out of the cell and expect good
results without some trial and error, mostly error. On the other hand,
B.subtilis' ability to export functional protein is oversold, because
the average strain also exports loads of proteases in late growth/early
stationary phase.

Here's where I catch my above reference to strain improvement. There is
prior work in removing the protease genes in order to improve export
capability of Bacilli, and to add in copies or modify-in-place the
export machinery to improve throughput (usually just by increasing the
amount of export machinery in the cell to remove bottlenecks). Without
these improvements, the advantage of export isn't as big a selling point
over periplasmic export in E.coli followed by cell rupture.

However, it's a *nice feature* to have. E.coli is awful for export, as I
learned during my ill-fated protein purification project. There are two
options for periplasmic export, but no single option for export beyond
that; E.coli proteins that are fully exported seem to each have their
own system for export. Some of these systems appear somewhat modular,
but from what I could see the modularity was hit-and-miss. Some proteins
could be exported with a given protein's machinery, but not others.

If, for some reason, export from the cell is critical to your project
(and it might well be!), B.subtilis seems to have the upper hand, and
there's not much in E.coli's toolbox that can compete. This isn't
because B.subtilis is amazeballs at export, it's simply because they
have the same export machinery in common, but E.coli wrapped a periplasm
around it all. So B.subtilis' homologues of E.coli periplasmic export
just drop the protein into the extracellular environment.

> 6. <You mentioned plasmid instability in a prior email I think>

Here's a virtual point I want to address, a partial myth. Yes:
B.subtilis is known to have issues with plasmid stability in the
literature. However, some reading on this yielded that this instability
can be traced to a consensus sequence believed to be associated with
TopoI in B.subtilis. Excluding that consensus sequence from your designs
is easy with tools that accept IUPAC notation (PySplicer, for example!),
and should address the instability problem. RecA homologues etc. may be
a problem for some projects but I hear little complaint about B.subitlis
168's stability outside of the TopoI issue.

And again, I worked with B.subtilis for only one major cloning project,
so I can't pretend to be an expert. Just pointing at the TopoI
connection as an example of a problem remaining more widely known than
its solution.

On 04/01/14 15:54, Josiah Zayner wrote:
> *E. coli* has so many powerful genetic tools it is a pity not to use it.
>
> I have seen multiple single papers with more edits in the* E. coli* genome
> If one is going to "express" recombination competence one can do that in *E.
> coli* also. And use "PCR products" to recombine into the host genome. See
> Wanner strains (
> http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement
> )
>
> Expressing proteins to purify is not so simple as you say, just secrete
> them from cells. And overexpressing proteins rarely kills cells. Though I
> am sure it might actually happen this is rarely/never a case Scientists
> worry about. Protein folding/aggregation and intrinsic toxicity is of way
> more importance. Expressing something like a restriction enzyme that would
> damage the host DNA would be a case in which *B. subtilis* would be useful
> but general protein expression I have my doubts. Though I have no evidence
> for or against.
>
> Your enthusiasm is great but if *B. subtilis* is so amazing why don't most
> Scientists and companies use it as their genetic tool of choice? Have the
> majority of Scientists in the world for the past 50? years been wrong and
> you and Cathal are correct?
>
> If I had to guess why people don't use *B. subtilis* I would say:
>
> 1. *B. subtilis* might be autocompetent but homologous recombination from
> what I read requires overlapping regions of > 500bp(
> http://mic.sgmjournals.org/content/154/9/2562.full) (if you could point me
> to a paper that shows less is required without a complicated strain of
> complex manipulations that would be cool). This adds complicated work to
> any design and multiple cloning steps. *E. coli* homologous recombination
> can be done with ~40bp which means it can be added to the end of a cheap
> primer and the gene created in a single PCR step. Further, needing to
> express comK is similar to needing to express a protein in *E. coli* for
> homologous recombination making it no more useful.
>
> 2. Under optimal growth conditions the doubling time of *B. subtilis* is
> always slower than *E. coli*. *B. subtilis* is measured to be somewhere
> around 50-120min(http://jb.asm.org/content/167/1/219.full.pdf,
> http://jb.asm.org/content/180/3/547.full). While *E. coli* is consistently
> measured to be 20 minutes or less(http://jb.asm.org/content/180/3/547.full).
> This is very significant considering it would take triple the amount of
> time for the ~same cell mass.
>
> 3. Transformation efficiency as you have stated and as I have read in other
> papers is staggeringly low in *B. subtilis*, on the order of 10^5 /ug of
> DNA at the extreme high end(http://www.ncbi.nlm.nih.gov/pubmed/22387342) and
> usually <=10^3. *E. coli* on the other hand is around 10^10 or greater /ug
> of DNA generically. When creating genetic libraries or performing simple
> transformations this is highly significant and greatly restricts the use of *B.
> subtilis*. A typical ligation reaction I perform has around <=100ng of
> final product that would yield <=1 colony. I know regularly when cloning I
> often only receive a few colonies of *E. coli* DH5a or DH10B cells when
> transforming a quickchange or a ligation, this would likely yield zero
> colonies in *B. subtilis *and cause much more work.
>
> 4. According to the NCBI database there are being worked on or have been
> completed 237 projects to sequence *E. coli* genomes(
> http://www.ncbi.nlm.nih.gov/genome/?term=escherichia+coli) and over 75 have
> been completed (
> http://ecoliwiki.net/colipedia/index.php/Sequenced_E._coli_Genomes) *B.
> subtilis* currently has 23 projects total (
> http://www.ncbi.nlm.nih.gov/genome/?term=bacillus+subtilis). Though
> logically one could argue that this might not mean an awful lot it shows
> how much more Scientists know/want to know about E. coli
> genetics/metabolism &c. This makes the longterm viability of *E. coli* much
> more inviting. It is like using an open source package that has 23 versus
> 237 people working on it. Which one do you think will have all the snazzy
> new features and still be around in 1, 2, 5, 10 years? Further, according
> to Google Scholar in 2013 ~190,000 papers contain the term "e coli" and
> ~26,000 contain the term "b subtilis"(no quotes used in actual queries).
>
> 5. Many proteins don't unfold/refold well, as occurs during secretion. Many
> protein have cofactors or require chaperones not found outside cells. This
> makes secretion only a slightly useful tool. Searching for optimal protein
> expression yield I couldn't find data for *E. coli* and *B. subtilis* in
> which they used the same protein and conditions so I don't know which is
> better for protein expression.
>
> I agree *B. subtilis* has its positives. This discussion has even made me
> consider using it for one of my projects that requires protein secretion
> but I don't think you are going to convince me or most Scientists that it
> is better than *E. coli *as a genetic or synthetic biology tool.
>
> I am not saying to stop your work I am just suggesting that if you want to
> use *B. subtilis* you should focus your work on the advantages it has over *E.
> coli* instead of attempting to compete with an already established
> genetic/synthetic biology tool.
>
> Cheers,
> Josiah Zayner, Ph.D.
> Synthetic Biology Group
> NASA Ames Research Center
>
>
>
> On Friday, January 3, 2014 9:26:11 PM UTC-6, Koeng wrote:
>>
>> Bacillus subtilis is not a "new species"... It is the second most studied
>> bacterium in the world and has more information on its genetic structure
>> then E coli. It has the most complete minimal gene set of any living
>> organism
>> http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page<http://www.google.com/url?q=http%3A%2F%2Fsubtiwiki.uni-goettingen.de%2Fwiki%2Findex.php%2FMain_Page&sa=D&sntz=1&usg=AFQjCNE3QeH9cJULnJgx4WWzN_Vgg1qRyA>
>>
>> First of all, remember that *plasmids are NOT being used*. So yes, they
>>> *Bacillus*. I assume the strains of* Bacillus* are not endA- and
>>> recA-(meaning your DNA and the DNA of the host have a chance of being
>>> screwed up) and you probably cannot do blue white screening with them, a
>>> useful genetic tool.
>>> Say you want to express a protein with a T7 or T5 promoter are you just
>>> going ask someone for the prophage and lysogenize these strains? Genetic
>>> editing is probably much more difficult without Warner strains and such. Do
>>> you know if *Bacillus* can produce comparable amount of protein compared
>>> to* E. coli* using T7/lac/IPTG? Does *Bacillus* restrict non-methylated
>>> DNA? Would make working from PCR/synthesized products difficult. Seems like
>>> an interesting hobby to test these things but Scientists probably will not
>>> use *Bacillus* just because it is slightly better than *E. coli* as *E.
>>> coli* has been used and tested and strains have been perfected for
>>> 10s/100s of years? Maybe *Bacillus* would have been the correct choice
>>> to start with cloning 100 years ago but it is kind of too late. Remember
>>> you are fighting against years and years of research and
>>> optimization(probably more than a single lifetime) with the
>>> thousands/millions of strains of *E. coli* it doesn't seem like the
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Cathal Garvey

unread,
Jan 6, 2014, 8:32:35 AM1/6/14
to diy...@googlegroups.com
Look at that, Koeng wrote a better, more terse rebuttal already. I
should really get into the habit of reading everything prior to
responding. :)
>> *E. coli* has so many powerful genetic tools it is a pity not to use it.
>>
>> I have seen multiple single papers with more edits in the* E. coli*genome than that whole catalog for Bacillus.
>> If one is going to "express" recombination competence one can do that in *E.
>> coli* also. And use "PCR products" to recombine into the host genome. See
>> Wanner strains (
>> http://openwetware.org/wiki/Recombineering/Lambda_red-mediated_gene_replacement
>> )
>>
>> Expressing proteins to purify is not so simple as you say, just secrete
>> them from cells. And overexpressing proteins rarely kills cells. Though I
>> am sure it might actually happen this is rarely/never a case Scientists
>> worry about. Protein folding/aggregation and intrinsic toxicity is of way
>> more importance. Expressing something like a restriction enzyme that would
>> damage the host DNA would be a case in which *B. subtilis* would be
>> useful but general protein expression I have my doubts. Though I have no
>> evidence for or against.
>>
>> Your enthusiasm is great but if *B. subtilis* is so amazing why don't
>> most Scientists and companies use it as their genetic tool of choice? Have
>> the majority of Scientists in the world for the past 50? years been wrong
>> and you and Cathal are correct?
>>
>> If I had to guess why people don't use *B. subtilis* I would say:
>>
>> 1. *B. subtilis* might be autocompetent but homologous recombination from
>> what I read requires overlapping regions of > 500bp(
>> http://mic.sgmjournals.org/content/154/9/2562.full) (if you could point
>> me to a paper that shows less is required without a complicated strain of
>> complex manipulations that would be cool). This adds complicated work to
>> any design and multiple cloning steps. *E. coli* homologous recombination
>> can be done with ~40bp which means it can be added to the end of a cheap
>> primer and the gene created in a single PCR step. Further, needing to
>> express comK is similar to needing to express a protein in *E. coli* for
>> homologous recombination making it no more useful.
>>
>> 2. Under optimal growth conditions the doubling time of *B. subtilis* is
>> always slower than *E. coli*. *B. subtilis* is measured to be somewhere
>> around 50-120min(http://jb.asm.org/content/167/1/219.full.pdf,
>> http://jb.asm.org/content/180/3/547.full). While *E. coli* is
>> consistently measured to be 20 minutes or less(
>> http://jb.asm.org/content/180/3/547.full). This is very significant
>> considering it would take triple the amount of time for the ~same cell mass.
>>
>> 3. Transformation efficiency as you have stated and as I have read in
>> other papers is staggeringly low in *B. subtilis*, on the order of 10^5
>> /ug of DNA at the extreme high end(
>> http://www.ncbi.nlm.nih.gov/pubmed/22387342) and usually <=10^3. *E. coli*on the other hand is around 10^10 or greater /ug of DNA generically. When
>> creating genetic libraries or performing simple transformations this is
>> highly significant and greatly restricts the use of *B. subtilis*. A
>> typical ligation reaction I perform has around <=100ng of final product
>> that would yield <=1 colony. I know regularly when cloning I often only
>> receive a few colonies of *E. coli* DH5a or DH10B cells when transforming
>> a quickchange or a ligation, this would likely yield zero colonies in *B.
>> subtilis *and cause much more work.
>>
>> 4. According to the NCBI database there are being worked on or have been
>> completed 237 projects to sequence *E. coli* genomes(
>> subtilis* currently has 23 projects total (
>> http://www.ncbi.nlm.nih.gov/genome/?term=bacillus+subtilis). Though
>> logically one could argue that this might not mean an awful lot it shows
>> how much more Scientists know/want to know about E. coli
>> genetics/metabolism &c. This makes the longterm viability of *E. coli*much more inviting. It is like using an open source package that has 23
>> versus 237 people working on it. Which one do you think will have all the
>> snazzy new features and still be around in 1, 2, 5, 10 years? Further,
>> according to Google Scholar in 2013 ~190,000 papers contain the term "e
>> coli" and ~26,000 contain the term "b subtilis"(no quotes used in actual
>> queries).
>>
>> 5. Many proteins don't unfold/refold well, as occurs during secretion.
>> Many protein have cofactors or require chaperones not found outside cells.
>> This makes secretion only a slightly useful tool. Searching for optimal
>> protein expression yield I couldn't find data for *E. coli* and *B.
>> subtilis* in which they used the same protein and conditions so I don't
>> know which is better for protein expression.
>>
>> I agree *B. subtilis* has its positives. This discussion has even made me
>> consider using it for one of my projects that requires protein secretion
>> but I don't think you are going to convince me or most Scientists that it
>> is better than *E. coli *as a genetic or synthetic biology tool.
>>
>> I am not saying to stop your work I am just suggesting that if you want to
>> use *B. subtilis* you should focus your work on the advantages it has
>> over *E. coli* instead of attempting to compete with an already
>> established genetic/synthetic biology tool.
>>
>> Cheers,
>> Josiah Zayner, Ph.D.
>> Synthetic Biology Group
>> NASA Ames Research Center
>>
>>
>>
>> On Friday, January 3, 2014 9:26:11 PM UTC-6, Koeng wrote:
>>>
>>> Bacillus subtilis is not a "new species"... It is the second most studied
>>> bacterium in the world and has more information on its genetic structure
>>> then E coli. It has the most complete minimal gene set of any living
>>> organism
>>> http://subtiwiki.uni-goettingen.de/wiki/index.php/Main_Page<http://www.google.com/url?q=http%3A%2F%2Fsubtiwiki.uni-goettingen.de%2Fwiki%2Findex.php%2FMain_Page&sa=D&sntz=1&usg=AFQjCNE3QeH9cJULnJgx4WWzN_Vgg1qRyA>
>>>
>>> First of all, remember that *plasmids are NOT being used*. So yes, they
>>>> *Bacillus*. I assume the strains of* Bacillus* are not endA- and
>>>> recA-(meaning your DNA and the DNA of the host have a chance of being
>>>> screwed up) and you probably cannot do blue white screening with them, a
>>>> useful genetic tool.
>>>> Say you want to express a protein with a T7 or T5 promoter are you just
>>>> going ask someone for the prophage and lysogenize these strains? Genetic
>>>> editing is probably much more difficult without Warner strains and such. Do
>>>> you know if *Bacillus* can produce comparable amount of protein
>>>> compared to* E. coli* using T7/lac/IPTG? Does *Bacillus* restrict
>>>> non-methylated DNA? Would make working from PCR/synthesized products
>>>> difficult. Seems like an interesting hobby to test these things but
>>>> Scientists probably will not use *Bacillus* just because it is slightly
>>>> better than *E. coli* as *E. coli* has been used and tested and strains
>>>> have been perfected for 10s/100s of years? Maybe *Bacillus* would have
>>>> been the correct choice to start with cloning 100 years ago but it is kind
>>>> of too late. Remember you are fighting against years and years of research
>>>> and optimization(probably more than a single lifetime) with the
>>>> thousands/millions of strains of *E. coli* it doesn't seem like the
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Cathal Garvey

unread,
Jan 6, 2014, 8:39:18 AM1/6/14
to diy...@googlegroups.com
Spores are resistant to alcohol, but very easily destroyed by light
bleach. I recommend the "so oxygen wow" brands of surface disinfectants,
which usually use a mild peroxide for their "oxygen wow" powers.

I never had contamination problems working with B.subtlis 168, either
between bacillus plates or to my E.coli plates, but I did treat my bench
with oxygen-wow and alcohol to be sure.
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Cathal Garvey

unread,
Jan 6, 2014, 8:44:30 AM1/6/14
to diy...@googlegroups.com
Indeed, here. As I mentioned, stability of successfully transformed DNA
is compromised by the TOPOI site, but at the point of transformation is
a different issue entirely, and there is research suggesting (as you
cite) that plasmid recircularisation requires overlapping tracts.

This isn't something you usually have to worry about, because any given
miniprep will contain partially replicated multimeric plasmids, and
because several plasmids can cross-combine to form a single working
plasmid. However, I recall research that found that more multimeric
plasmids meant more transformants, which helps account for the
apparently low transformation rates with miniprepped DNA; it's a
minority of the DNA that's responsible for the transformation.

In fact, it suggests that, mg for mg, if you used PCR'd plasmid with
some overlap, you'd get drastically higher transformation rates than
using miniprepped plasmid. If you're working with circular template,
this is easy; use the reverse complement of your plasmid primers and use
double the extension time. Although that's excessive, you're better off
only copying ~200bp extra to either side of your chosen midpoint..
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Josiah Zayner

unread,
Jan 6, 2014, 10:55:04 AM1/6/14
to diy...@googlegroups.com
Very nice and thorough response Cathal. Glad to hear your first hand experience with it. Really adds alot to this discussion. 

Cathal Garvey

unread,
Jan 6, 2014, 11:08:10 AM1/6/14
to diy...@googlegroups.com
And I'm sure gratifying for you to hear that I'm working with E.coli
these days! ;)

Ah, Bacilli, my lost love. Had I no responsibilities, I'd probably still
be shaving that yak, but I need to shave lower-risk yaks for now. I'll
return to Bacilli in time, I hope.
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Ian Leith

unread,
Jan 18, 2014, 5:59:58 PM1/18/14
to diy...@googlegroups.com, cathal...@cathalgarvey.me
http://link.springer.com/article/10.1007/BF00538711
According to the abstract: B. subtilis requires 70bp of homology for plasmid to chromosome. I wouldn't think it would be different for non-plasmid DNA but I'm still new at this...

Anyone have full access to the article?

Other articles suggest that there is a logarithmic relationship between recombination and differences in sequences so I would think the larger your synthetic gene, more homologous bps are needed.

Koeng

unread,
Jan 20, 2014, 12:07:43 PM1/20/14
to diy...@googlegroups.com
I believe that 70bp is required for plasmids, but linear DNA is always more efficient. However 70bp might be more efficient.

Ironic part is different papers cite different lengths...

Mega [Andreas Stuermer]

unread,
Jan 21, 2014, 3:32:48 PM1/21/14
to diy...@googlegroups.com

In chloroplasts, at least, I read usually you use 500-1000 bp used. Also 300 bp would be ok, but at worse efficiency. But if you do PCR you don't care how long the product is - primers cost the same. 

So, you could say that if synthesize it, it would be better too keep it short. Does efficiency matter that much actually? If they grow on kanamycin, you know that recombination must have occured. 

W. Estell

unread,
Jan 21, 2014, 4:19:46 PM1/21/14
to diy...@googlegroups.com
As far as reading the literature on Bacillus, I suggest focusing your attention to the work of scientists at Genencor (currently DuPont) and Novozymes. The scientists at these companies have thousands of patents regarding Bacillus enzyme and genetic engineering. I will now use this space to plug one such scientist, Eugenio Ferrari, and point those interested to US5,387,521 as a starting point, and US8,124,399 for a more current example.

Cathal Garvey

unread,
Jan 22, 2014, 6:49:25 PM1/22/14
to diyb...@diybio.eu, diybio
On 22/01/14 22:32, Rüdiger Trojok wrote:> PS: may I ask: what has come
of your approach with B.subtilis?
> Would you still recommend its usage as a DIYbio model organism?

Good question, for those with a good memory. :)

So, I've used the name "IndieBB" before, and in private correspondance
with many other biohackers I've referred to the previous project, which
was based in Bacillus. For some perspective on Bacillus itself as a
host, see below forwarded message from a discussion on the DIYbio
Googlegroup: I still believe Bacillus can be a great model organism.

I think I'll write a blogpost during the campaign (because this deserves
a proper blogpost, both for exposition, for images and link-citations,
and to have a public record of full disclosure) about my experiences and
outcomes working on an "IndieBB" for bacillus. To cut me some slack, it
was my first project, and it was a moonshot. Rudiger correctly surmised
that it was too ambitious, and indeed it didn't work fully
first-time..and I never ordered a second prototype.

But, it *did* partially work, and the core mechanism of the Bacillus
plasmid which was intended to permit antibiotic-free initial selection
after transformation (not bacteriocin-based, totally crazy and new)
*did* work. It was simply the case that after initial selection, the
plasmid was apparently instantly lost. I reckon that this problem was
caused by leaky expression through an inducible promoter-repressor
element I was using for the system which I'll describe in the blogpost.
Suffice to say, I think I could still get it to work.

So why am I doing this in E.coli instead first? Because I'm sick of "I
think I could". We all know biology takes a long time, and is error
prone, and unpredictable, and I've been living that reality for 3 years
now. I'm planning to return to IndieBB-mark-zero in Bacillus, but for
now I want to get something into the real world and see it used by other
people, and IndieBB (present-day) is a much better bet. If I don't see
my work in the real world, I'll burn out and go broke at the same time.
This way, I get the satisfaction of seeing fruits of my labour, while
earning funding to continue developing great things.

Hope that helps! ;)
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