Thursday, 25 October 2007

Research - Lab Techniques (week 18)

Hi all,

Previously I said I’d cover colony pcr and restriction digestion, but I believe those underlying principles have been covered in my first post, so it’s a bit redundant. Hence, I’ll be covering a bit on the yeast work for today.

First off, my protein interaction study is based on yeast. There are about 3 types of yeast 2 hybid assay. For my project, I’m using yeast 2 hybrid system 2. As I haven’t been exposed to the other 2 systems, I’m afraid I will not be able to cover anything on it.

Basically, the main principle of the yeast 2 hybrid is that the transcription factor in yeast, (GAL4) can be broken up into 2 pieces to form GAL4-BD (binding domain) and GAL4-AD (activation domain).
- GAL4-BD will then be subcloned into a vector for the bait gene, which is the gene of interest for study. The BD is the domain responsible for binding to the UAS (upstream activation site)
- GAL4-AD is subcloned into a vector for the prey cDNA library which is used for testing against the bait gene to see what interactions may take place. AD is the domain responsible for activation of transcription located downstream.

Hence, in event of a protein interaction between the bait and a particular prey, the 2 domains (GAL4-BD and AD) will be brought into close proximity, linking both the GAL4 UAS and transcriptional site in yeast, resulting in transcriptional activation of the beta/alpha-galactosidase, the reporter gene in yeast (MEL1, the reporter for producing alpha-galactosidase is naturally occurring in some strains of yeast, while LacZ is an E.coli enzyme that is cloned into the yeast genome).
Yeast strain that can metabolize alpha-galactosidase such as AH109, Y187 etc from clontech can metabolise x-alpha-gal (a substrate) directly as alpha-galactosidase is secreted (naturally occurring in these yeast strains); colonies can be plated on x-alpha-gal plates. However, for beta-galactosidase, LacZ gene being not naturally occurring means that the beta-galactosidase is not secreted by yeast, but rather produced in the cell upon activation. Hence, to develop blue phenotype, colony lift assay is necessary as x-gal needs to enter cells (have to permeablise yeast cells first by colony lift assay) for metabolism by beta galactosidase.
For more info on colony lift assay, please refer to > hdohlman > lift_protocol.html

For a clearer demonstration of activation by GAL 4 transcription factor, refer to the diagram below:

Taken from:> wiki > Two-hybrid_screening

To recap, AH109 strain of Saccharomyces cerevisiae contains 4 reporters: ADE, HIS (nutritional markers), LacZ (for beta- galactosidase, artifically cloned from E.coli), MEL1 (for alpha- galactosidase, naturally occuring in some yeast strains).

My previous posts have been focusing on obtaining the bait construct. This is done by cloning in a pcr gene of interest into the bait vector that contains
1) gal4-BD
2) a nutritional factor like TRP marker for yeast selection
3) kanamycin for antibiotic selection
4) Multiple cloning site for cloning purposes.

Once bait construct has been obtained, the plasmid is transformed to yeast strain AH109, and plated on SD/-trp plates for selection of transformed yeast (yeast that is not transformed cannot grow on –trp plates) in 3 serial dilutions of 1/10, 1/100, 1/1000 for calculating transformation efficiency. Once yeast has grown, typically 3-4 days initially when incubated at 30oC, it is restreaked onto fresh -trp plate to form the master plate for mating with the cDNA library, which is commercially obtained. Alternatively, it is possible to clone your own library into a vector, though this is much more of a hassle. cDNA prey library is tittered to find amount of viable cells by plating dilutions of 1/10, 1/100, 1/1000 and 1/10,000 on SD-leu plates (Prey vector contains leu nutritional marker) and making suitable calculations - multiply cfu by plating vol and dilutional factor and divide by total volume available in mL.

Eg, 100ul of 1/10,000 dilution of prey library was plated and gave a cfu of 245 from a total cdna vol of 1ml will yield:
100ul x 10,000 df x 245cfu / 1mL = 2.45x10^8 cfu/mL

Typically, at least 2x10^7 cfu/mL is necessary to produce enough clones for screening in mating (at least 1 million diploids).

If viability of prey is high enough, proceed to mating.

*For easier reference purposes, a table below shows the comparison of bait vector and prey vector:


After this, comes the actual mating. Grow out bait colonies in SD/-trp liquid overnight. Next, combine 1mL of bait and prey together in 2x YPDA media (50mL) and incubate at 30oC overnight at 50rpm to ensure cells do not settle down. Check for zygotes after 30hrs under microscope at 40x. If present, pellet at 700g, 5 mins and resuspend into fresh media before plating on 24.5cm x 24.5 cm SD/-trp/-leu/-ade/-his / x-alpha-gal (QDO x-alpha-gal). It is important to note that only mated colonies can grow on QDO (quadruple dropouts as prey provides leu (leucine) marker and bait has trp (tryptophan) marker and interactions between these 2 causes activation of transcriptional factors adenine (ade) and histine (his) as these 2 are under influence of the same promotor.
Within 2-3 days of incubation at 30oC, some colonies may start to turn blue. These colonies are the ones with activation of x-alpha-gal, indicating possible prey protein interaction with bait. These colonies are to be restreaked onto freah QDO x-alpha-gal plates to determine that blue phenotype is not a false positive, which typically turns white after the 2nd streak.

Rescue prey plasmid:

If many colonies are detected, a yeast colony pcr can be used to eliminate same copies of interaction. Upon confirmation, or if there are few colonies for study, yeast plasmid can be extracted by either sonication or using extraction kit (which uses lyticase to break down yeast wall). Plasmid obtain will consist of a mixture of prey, bait and yeast plasmids. To select only prey, the ampicilin selection shall be used – transform to competent cells and plate on amp plates. Bait and yeast plasmids will not be able to grow, hence resultant colonies are all from prey. Miniprep to obtain plasmid from competent cells.

Confirm interaction:

Using the rescued prey plasmid, transform to bait gene and plate to QDO x-alpha-gal plates. If colonies turn blue, use the plasmid obtained from the competent cells for sequencing to determine what protein it is. Blast results using protein query from > BLAST > blastx

That wraps basically everything in a nutshell. Controls were not discussed as they are too bothersome and the principle is the same. Feel free to ask any questions, though I suggest you guys focus on the mp reports/logbook/whatever else instead and leave me to do the same =)

Debra, TG02

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