New Marker approaches
Mega
But what makes the benefit of antibotic markers, is also the dark
side:
You can't kill the germs with this antibiotic anymore. And the
ressistance genes may spread among pathogens (although farming
produces far more ressistances, we should keep this topic in mind)....
There is also the approach that some modified bacteria can't produce
an essential amino acid, and the plasmid enabels the bacteria to
produce it (and thus it makes it non-auxotroph). You put it into a
medium without this amino acid and the only surviving is the one with
the plasmid...
Yesterday I thought of a simpler(?) methode:
The lacZ gene is very commonly used for blue-white screening.
But in nature it cuts lactose.
Give e coli the lacZ gene (only this and origin of replication in a
plasmid) via transformation.
Then put them into a mixture of water, lactose and salts.
Only the lacZ+ coli will be able to cut lactose and eat it. All others
not. So after days of incubation you will have 99% lacZ colis.
(Maybe the non-transformants will take the plasmid from the
transformed to be able to grow - so yields are increased).
Zentrifuge it. Put some of the pellet into a petri dish. The major
part of them will contain lacZ (and the gene of interest if included
into the plasmid)
___
What do you say about this marker? LacZ appears in nature, so it's
absolutely no danger!!
Maybe this is yet used by some people?
Cathal Garvey
(i.e., a strain naturally lacking the Lac Operon, and therefore is
incapable of digesting Lactose).
You could do the same thing for other commonly available disaccharide
sugars, I imagine; maltose and sucrose, for example. As long as the
parent strain cannot digest them, and the plasmid provides the ability,
it should work.
The amino acid approach is very clever, but poorly suited to DIYbio;
making up broths with highly specific ingredients like pure amino acids
is very costly.
With proper strain selection or design, there's huge scope here.
Behavioural selection is another promising avenue; instead of relying on
killing those cells that don't have the gene, you try to select just the
cells that do have it and isolate them from the population.
Of course, antibiotics aren't a dead-end approach; we just need to
ensure that we don't use medically useful antibiotics, or antibiotics
related to them. After all, even if basic Penicillin G isn't useful to
us anymore, providing resistance to Penicillin G gives cells all they
need to evolve around Ampicillin, too.
Much better to focus on antibiotics that are too impractical to use in
humans anyway; bacteriocins look like a great option here. They are
protein-based, so they can't be taken as a tablet and they stimulate too
much immunity to be injected, so most of them are entirely useless for
human therapy. However, they can be pretty lethal against the specific
species and strains they affect. Further, the mechanisms of resistance
to these antibiotics are often evasive rather than degradative.
That is, while bacteria destroy ampicillin, allowing non-transformed or
plasmid-loss cells to survive alongside them, most bacteriocin
resistance systems merely protect individual cells against the
bacteriocin, without destroying it. This allows for longer culturing
times for transformed cells before plasmid loss becomes an issue, and
might even protect cultures from late-growth contamination.
With bacteriocins, you could even have your transformed cells *make* the
antibiotic, leaving the job of killing untransformed cells to the
transformed cells. That reduces your necessary ingredients from three
(bacteria, DNA, antibiotic) to two: bacteria, and DNA.
--
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Mega
the
antibiotic, leaving the job of killing untransformed cells to the
transformed cells. That reduces your necessary ingredients from three
(bacteria, DNA, antibiotic) to two: bacteria, and DNA.
And no more need to get the antibioticum from anywhere.
Yet there is the fact: If they escape to nature, the resistance gene
spreads. If LacZ or something escapes, doesn't matter. You could also
let it escape without hesitating if the gene of intereset is harmless
like gfp.
Streptococus mutans does it (tooth hole - maker), it kills other
bacteria with some bacteriocin...
Mega
Also interesting (it doesn't fit in the topic markers except that the
strain produces a mutant bacteriocin that kills wild-type s.mutans)
Cathal Garvey
technology, but I assumed it was a dead project. Have always been
planning to replicate that experiment and make my own anti-cavity
symbiote. ;)
While looking through Oragenic's stuff, I got really interested in their
pretty wide-ranging portfolio of stuff. But one thing caught my eye:
They have a weight-loss agent code-named "LPT3-04", which they claim is
part of the normal human diet, and for which they've asserted GRAS
status (i.e., an acknowledgement that their "proprietary" formula has
been eaten/used by people for a few decades at least.
I looked into their patent portfolio and found only one patent for
weight loss. Turns out their super-amazing proprietary "LPT3-04" Magic
Juice?
Glycine.
www.google.com/patents/US20060093650
Mega
Wouldn't it be best if you handle with a bacterium that CAN stand
salty agar, but doesn't need salt to grow. (facultative saline
bacteria)
Thus most contaminants - bacteria and fungi, mould - cannot grow on
the medium and you would get far less contaminants. Is there a
bacterium that fulfills that criterium?
Yeah, and it also would have to be easy to transform.
Mega
Kanamycin is considered not dangerous as a marker by the
European Food Safety Authority
.It's no longer used for human treatment and resistance is wide-spread in nature.
(German)
Nicht alle Antibiotikaresistenz-Gene sind gleich
Das GMO Panel der EFSA stuft die als Marker verwendeten Antibiotikaresistenz-Gene in drei Gruppen ein.
Gruppe 1: ABR-Gene, die in natürlich vorkommenden Mikroorganismen weit verbreitet sind. Die jeweiligen Antibiotika haben keine oder nur eine geringe Bedeutung in der Human- und Tiermedizin. Dieser Gruppe werden das nptII-Gen (Kanamycin- Resistenz) und das hph-Gen (Hygromycin) zugerechnet.
- Beispiel: nptII-Gen . Dieses seit Jahren in transgenen Pflanzen am häufigsten verwendete Markergen stammt aus einem Transposon (springendes Gen). Es vermittelt eine Resistenz gegen mehrere Antibiotika wie z.B. Kanamycin oder Neomycin. Diese werden aber nur noch selten eingesetzt, etwa bei Patienten, die andere Antibiotika nicht vertragen. Kanamycin kann zudem starke Nebenwirkungen zur Folge haben.
- Bei Markergenen dieser Gruppe wird davon ausgegangen, dass ihre Verwendung in transgenen Pflanzen nichts an der vorhandenen Verbreitung in der Umwelt ändert. Die EFSA-Experten können keine Sicherheitsgründe erkennen, die für eine Einschränkung dieser ABR-Gene sprechen. Sie empfehlen daher, gv-Pflanzen mit diesen ABR-Markergenen sowohl für Feldversuche wie für den kommerziellen Anbau weiterhin uneingeschränkt zuzulassen.
- Anfang 2007 wurde das nptII-Gen von den EFSA-Experten erneut überprüft. Anlass dafür waren neue Information der über einen verstärkten Einsatz des Antibiotikums Kanamycin und ähnlicher Wirkstoffe. Nach Ansicht der Europäische Arzneimittelagentur (EMEA), spielen diese in der Human- und Tiermedizin eine wichtige Rolle bei der Bekämpfung bestimmter Infektionskrankheiten, auch weil andere Antibiotika ihre Wirksamkeit verlieren, gegen die bakterielle Erreger Resistenzen entwickelt haben. Dennoch bekräftigten die EFSA-Experten ihre ursprüngliche Einschätzung: Ein Transfer des Gens von einer gv-Pflanze auf Bakterien ist als extrem unwahrscheinlich anzusehen ist. Außerdem sei das nptII-Gen in der Natur ohnehin weit verbreitet. Ein großer Teil der Bakterien, die etwa im Darm oder in der Umwelt anzutreffen sind, besitzt bereits eine Resistenz gegenüber Kanamycin. Eine Verwendung des nptII-Gens in gv-Pflanzen sei unbedenklich und habe keinen Einfluss auf die die Wirksamkeit von Antibiotika der Kanamycin-Gruppe.
Mega
That would surely work for chloroplasts too. The antibiotic would have to be exported out of the chloroplast, of course, so that it is not degraded wiithin the transformed plastid.
Plastid transformation is said to be quite difficult, because you have to get homoplastid-ic cells, otherwise the non-transformed chloroplasts may grow quicker than the transformesd ones and in the end there will be no transformed chloroplasts in the cell any more. With this approach however, they select theirselves!
Does anybody know by chance if there are gene clusters known that produce an antibiotic from stuff that is already present in each bacterium/plastid?
(e.g. Kanamycin, Streptomycin, .... ). I had a small google research on that, without usable results.
Unfortunately, there is no paper on the internet (I found so far) assessing the use of peptide antibiotics (like Nisin) against chloroplasts. Have to dig into the mechanism of destruction that is done by nisin, etc.