Any yeast culturing and/or gen transfer protocol, simple enough to do at a simple laboratory and sourcing yeast?

[AKS]

Hi friends

I want to learn some biology experiment at home for fun. I wonder
is there any yeast culturing protocol that can be done without a
research grade laboratory but a simple bench, a light microscope,
micro pipette and syringe, etc. May I know where are the potential
sources where can I get them? Thanks.

Sincerely

Aung

[Mega]

I have seen a yeast integrative plasmid + a strain for it. It costed some 300-600 Dollars. I don't remember the number exactly, but I remember I was shocked by the price for 'just' a plasmid and some baker's yeast  :)

All you additionally need is restriction enzyme and some eukaryontic genes to insert into the yeast plasmid.

[Avery louie]

You might want to look into bacterial transformation (adding dna) its cheaper, and easier!

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[AKS]

Hi

Thanks for the tip but are there any cheaper ways?

Sincerely

Aung

[Daniel C.]

If all you want to do is culture yeast, that's dead easy. People have
been doing it for about as long as we've been planting crops. You
just sprout some barley, roast it to stop the sprouting process, crush
the now-malted barley, soak it in hot water and leave the liquid out
in the open air. Yeast will grow.

You can also drink this. It's called beer. I wouldn't recommend the
above protocol because you'll get things other than yeast growing in
it, which can lead to some pretty nasty flavors. A better result can
be had by boiling some malt extract for ~30 minutes, then adding yeast
specifically for brewing.

Of course if you want to do something besides grow more yeast, you'll
need additional equipment. I personally am pretty ok with just
growing yeast and drinking the byproduct. Hell, I don't even
(usually) save the yeast.

-Dan

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[Avery louie]

The cheapest way to get started with genetic engineering in bacteria is probably this:

https://groups.google.com/forum/?fromgroups#!searchin/diybio/free$20na/diybio/cX8mleshcvM/qLgwCo5NUowJ

--A

[Avery louie]

Dan does make a good point though.

[shamrock]

Hi Brian,

Bakers yeast (Saccharomyces cerevisiae) is just as easy to work with as E. coli, keeping in mind a few things. One minor complication is that they grow slower and most antibiotics don't work as selections. What that means from a practical standpoint is that contamination becomes a slightly greater problem then when working with E. coli, and the media is a bit more complex to make. However if you keep a clean environment and use good aseptic techniques you shouldn't have much of a problem.

 

The background yeast strain used for most genetic experiments is one known as S288C. Other strains and backgrounds are used for a variety of industrial, brewing and baking purposes but S288C is the one most often used in genetic experiments. It has the advantage that it does not have a lot of chromosomal duplications and gene amplifications that are found in most industrial strains, plus a wide variety of different strain types are avaiilable.

 

There are a number of repositories that have collections of yeast strains-alas I do not know how willing they would be to send something to a home biologist. You could search on the internet for biological repositories or if you have access the latest issue of journal of industrial microbiology and biotechnology has a review on yeast culture collections (Boundy-Mills, K. 2012. Yeast culture collections of the world: meeting the needs of industrial researchers. JIMB 39:673).

 

Like I said earlier working with yeast is just as easy as E. coli with a few caveats. First of all because most antibiotics don't work the most often used selections are nutritional selections. What this means is that the starting strain is unable to make a particular nutrient (usually an amino acid or a nucleotide precursor) because of a mutation in a single gene. The selectable marker carried on the plasmid has an unmutated copy of the same gene. Therefore cells carrying the plasmid can grow in the absence of the nutrient but cells that do not have the plasmid cannot-just like E. coli cells with a plasmid can grow in the presence of ampicillin but cells that don't have the plasmid cannot. From a practical standpoint it means that the media used is a bit more complex so instead of growing cells in rich media (which is relatively simple) you need to select for recombinant cells on media that lacks only that single nutrient. You can but the media known as drop-out media or you can make it yourself but it is more complex to make from scratch. Cells are transformed similar to E. coli cells although the method used to make the cells competent to take up DNA is different. With yeast Li salts (Li acetate for cerevisiae and LiCl for Pichia ) are used. You can also electroporate yeast like you can E. coli, and there is a method based on digesting away the outer cell wall.

A number of different plasmid vectors exist for use in yeast and a number of different inducible promoters are available as well. So you can make use of high oor low copy episomal vectors or you can use integrating vectors. Promoters, such as the GAL promoter, can be turned on just by shifting the carbon source from glucose to galactose (making sure the glucose is used up). One really cool thing about yeast is that it is relatively easy to make genome modifications either by knocking out certain genes or adding new ones. Plus you can do some nice genetic experiments through simple mating and sporulation.

Working with yeast is fun, pretty easy to do, and they smell a lot better than E. coli!

[Cathal Garvey]

Ages back on the list we discussed Yeast transformation without lithium, because lithium's hard to get (at least over here), probably because of its medicinal uses.

Turns out you can do a *really* inefficient yeast transformation with PEG 3350 (available as a laxative, brandnames "GoLytely" and "Miralax" should be easy to find online) and table salt. I'll dig up the protocol when I get a chance, but it ought to be in the list archives, too.

If you have access to an enzyme that digests Yeast cell walls, you can do more high-efficiency transformations; I have a plasmid that produces an enzyme called "Lyticase" that does just that. I've never tested it..

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[shamrock]

I'll have to try the Lithium free transformation, sounds interesting.

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[Andreas Sturm]

Lithium is no problem. have a look at

http://www.youtube.com/watch?v=BliWUHSOalU

Be sure not to hurt yourself by doing so.



( Sorry for OT, @Cathal, do you think, PEG works also with agrobacterium? )

2012/6/24 shamrock <thmsb...@gmail.com>

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[AKS]

Hi Shamrock

Thanks for the comprehensive and informative answer. Cheers!

[Cathal Garvey]

Don't know, sorry! :)

On 25/06/12 18:46, Andreas Sturm wrote:
> Lithium is no problem. have a look at
>
> http://www.youtube.com/watch?v=BliWUHSOalU
>
> Be sure not to hurt yourself by doing so.
>
>
>
> ( Sorry for OT, @Cathal, do you think, PEG works also with agrobacterium? )
>
>
>
> 2012/6/24 shamrock <thmsb...@gmail.com>
>
>> I'll have to try the Lithium free transformation, sounds interesting.
>>
>> On Sunday, June 24, 2012 3:56:03 PM UTC-4, Cathal wrote:
>>
>>> Ages back on the list we discussed Yeast transformation without lithium,
>>> because lithium's hard to get (at least over here), probably because of its
>>> medicinal uses.
>>>
>>> Turns out you can do a *really* inefficient yeast transformation with PEG
>>> 3350 (available as a laxative, brandnames "GoLytely" and "Miralax" should
>>> be easy to find online) and table salt. I'll dig up the protocol when I get
>>> a chance, but it ought to be in the list archives, too.
>>>
>>> If you have access to an enzyme that digests Yeast cell walls, you can do
>>> more high-efficiency transformations; I have a plasmid that produces an
>>> enzyme called "Lyticase" that does just that. I've never tested it..
>>>
>>> On 24 June 2012 19:58, shamrock <thmsb...@gmail.com> wrote:
>>>

>>>> Hi Brian, ******

>>>>
>>>> Bakers yeast (Saccharomyces cerevisiae) is just as easy to work with as
>>>> E. coli, keeping in mind a few things. One minor complication is that they
>>>> grow slower and most antibiotics don't work as selections. What that means
>>>> from a practical standpoint is that contamination becomes a slightly
>>>> greater problem then when working with E. coli, and the media is a bit more
>>>> complex to make. However if you keep a clean environment and use good

>>>> aseptic techniques you shouldn't have much of a problem. ****

>>>>
>>>>
>>>>
>>>> The background yeast strain used for most genetic experiments is one
>>>> known as S288C. Other strains and backgrounds are used for a variety of
>>>> industrial, brewing and baking purposes but S288C is the one most often
>>>> used in genetic experiments. It has the advantage that it does not have a
>>>> lot of chromosomal duplications and gene amplifications that are found in
>>>> most industrial strains, plus a wide variety of different strain types are
>>>> avaiilable.
>>>>
>>>>
>>>>
>>>> There are a number of repositories that have collections of yeast
>>>> strains-alas I do not know how willing they would be to send something to a
>>>> home biologist. You could search on the internet for biological
>>>> repositories or if you have access the latest issue of journal of
>>>> industrial microbiology and biotechnology has a review on yeast culture
>>>> collections (Boundy-Mills, K. 2012. Yeast culture collections of the world:

>>>> meeting the needs of industrial researchers. JIMB 39:673). ****

>>>>
>>>>
>>>>
>>>> Like I said earlier working with yeast is just as easy as E. coli with a
>>>> few caveats. First of all because most antibiotics don't work the most
>>>> often used selections are nutritional selections. What this means is that
>>>> the starting strain is unable to make a particular nutrient (usually an
>>>> amino acid or a nucleotide precursor) because of a mutation in a single
>>>> gene. The selectable marker carried on the plasmid has an unmutated copy of
>>>> the same gene. Therefore cells carrying the plasmid can grow in the absence
>>>> of the nutrient but cells that do not have the plasmid cannot-just like E.
>>>> coli cells with a plasmid can grow in the presence of ampicillin but cells
>>>> that don't have the plasmid cannot. From a practical standpoint it means
>>>> that the media used is a bit more complex so instead of growing cells in
>>>> rich media (which is relatively simple) you need to select for recombinant
>>>> cells on media that lacks only that single nutrient. You can but the media
>>>> known as drop-out media or you can make it yourself but it is more complex
>>>> to make from scratch. Cells are transformed similar to E. coli cells
>>>> although the method used to make the cells competent to take up DNA is
>>>> different. With yeast Li salts (Li acetate for cerevisiae and LiCl for
>>>> Pichia ) are used. You can also electroporate yeast like you can E. coli,

>>>> and there is a method based on digesting away the outer cell wall. ****

>>>>
>>>> A number of different plasmid vectors exist for use in yeast and a
>>>> number of different inducible promoters are available as well. So you can
>>>> make use of high oor low copy episomal vectors or you can use integrating
>>>> vectors. Promoters, such as the GAL promoter, can be turned on just by
>>>> shifting the carbon source from glucose to galactose (making sure the
>>>> glucose is used up). One really cool thing about yeast is that it is
>>>> relatively easy to make genome modifications either by knocking out certain
>>>> genes or adding new ones. Plus you can do some nice genetic experiments

>>>> through simple mating and sporulation. ****

>>>>
>>>> Working with yeast is fun, pretty easy to do, and they smell a lot

>>>> better than E. coli! ****

>>>>
>>>> On Saturday, June 23, 2012 4:15:23 AM UTC-4, Brian Soe wrote:
>>>>
>>>>> Hi friends
>>>>>
>>>>> I want to learn some biology experiment at home for fun. I wonder
>>>>> is there any yeast culturing protocol that can be done without a
>>>>> research grade laboratory but a simple bench, a light microscope,
>>>>> micro pipette and syringe, etc. May I know where are the potential
>>>>> sources where can I get them? Thanks.
>>>>>
>>>>> Sincerely
>>>>>
>>>>> Aung
>>>>>
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>>>
>>>
>>>
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>>> PGP Public Key: http://bit.ly/CathalGKey<http://letters.cunningprojects.com>

[Andreas Sturm]

Ok, thank you!!

So probably it's not the best idea to try it with a rare/expensive plasmid :D

2012/6/26 Cathal Garvey <cathal...@gmail.com>

[Cathal Garvey]

Most plasmids for Agrobacterium are E.coli-compatible, they have origin
sites for both. E.coli is used when assembling the new construct, and
Agrobacterium is used to transfer the DNA to plants.

If that's so, start out by hacking it into E.coli with an appropriate
antibiotic, and then you can make as much as you need for working with
Agrobacterium.

Run the plasmid sequence through BLASTn, and you should find a region
that's homologous to the origin on lots of E.coli plasmids.. or post the
plasmid map, and perhaps it's labelled?

[Avery louie]

If you have the sequence, try plasmapper.  It does a pretty sweet visual and textual annotation.

--A

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[Andreas Sturm]

Clearly, it has e.coli origin.
I was thinking of pGreenII 0049 , which I'd like to insert the modified lux genes (once I got funding)  ;)  Or better spoken, the gene synthesizing company shall clone it into the vector. 

2012/6/27 Avery louie <inact...@gmail.com>

[Jonathan Cline]

On Sunday, June 24, 2012 11:58:10 AM UTC-7, shamrock wrote:

...

 Added to the FAQ !

http://openwetware.org/wiki/DIYbio/FAQ/Projects

## Jonathan Cline
## jcl...@ieee.org
## Mobile: +1-805-617-0223
########################

[Nathan McCorkle]

AKS, this is my favorite yeast kit:
http://www.neb.com/nebecomm/products/producte1000.asp

On Sat, Jun 23, 2012 at 4:15 AM, AKS <aung...@gmail.com> wrote:

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College of Science, Biotechnology/Bioinformatics