This is one of those results that was so minor I’d never be able to “publish” it, and so interesting and useful that I can’t help but share it somewhere. Consider this a “mini-report.” (Featured image, above, is “Yeast Membrane Proteins” by Wikimedia user masur.)

Introduction

My student researchers and I are trying to overexpress a protein of interest in S. cerevisiae – a 6xHis-tagged copy of the metacaspase MCA1 (also known as YCA1 and YOR197W). MCA1 is interesting because it seems to be the master controller of apoptosis (programmed cell death) in yeast. Which naturally leads to the question — why the heck would a single-celled organism need to be able to commit suicide? Being able to over-express and purify out this protein might help us answer this question.

We attempted to over-express the protein by putting it under the control of the GAL1 promoter and inducing expression with galactose. The GAL1 promoter is repressed in the presence of glucose and strongly expressed in the presence of galactose, with an induction of up to 1000x. However, we had difficulty expressing our positive control, a green fluorescent protein (Venus) using this system. An internet search, and conversations with colleagues, indicated that others often have the same issue. Here, we test several different induction conditions to determine which one gives us a strong induction.

Results

Galactose induction in YP+sucrose

We used a yeast GoldenGate toolkit to create a transcription unit of MCA1-6xHis under the control of the GAL1 promoter, then integrated it into the HO locus. After verifying the integration, we followed an induction protocol from a JoVE article on over-expressing and affinity-purifying proteins in yeast. The protocol was explicitly written to express proteins under the GAL1 promoter from plasmids (2µ, one assumes). Briefly, the protocol said to:

• Pick a colony from a selective plate and grow a 5 ml culture overnight in selective media (ie, SD-Uracil) with 2% sucrose.
• The next morning, subculture into 33 ml of selective media with 2% sucrose at an OD600 of 0.3. Grow until the culture reaches an OD600 of 0.8-1.5 (mid-log)
• Supplement with concentrated rich media to a final concentration of 1% yeast extract, 2% peptone, and 2% galactose.
• Grow for another 5-6 hours and harvest.

However, because our construct was integrated into the genome, we did not need to use selective media to maintain the plasmid. So, we modified the protocol as follows:

• Pick a colony from a YPD plate. Grow in 5 ml YP (1% yeast extract, 2% peptone) + 2% sucrose overnight.
• The next morning, subculture into 50 ml of YP + 2% sucrose at an OD600 of 0.3. Grow until the culture reaches an OD600 of 0.8-1.5 (mid-log).
• Supplement with 2% galactose.
• Grow for 6 hours.

Unfortunately, when we checked the fluorescence of the GAL1:Venus strain using a flow cytometer, we didn’t see much induction even after 6 hours:

Well poop. That’s not going to give us much protein! After doing some reading, we found that the “usual” protocol for inducing the GAL1 promoter is to use raffinose, a disaccharide of glucose and galactose. The reason is that the GAL1 promoter is strongly repressed by glucose. Sucrose shouldn’t repress the GAL1 promoter like glucose does (it’s a disaccharide of glucose and fructose), but often there is significant glucose contamination in sucrose.

Galactose induction in YP + Raffinose

Our next experiment compared two induction protocols: initial growth in YP + glucose, then replacing the gluose with galactose; and initial growth in YP + raffinose, then adding galactose (the universally suggested solution.) The experiment looked like this:

• Grow a colony overnight in 5 ml of YP + 2% glucose or YP + 2% raffinose
• Subculture into 20 ml of the same media at OD=0.3. Grow 3 hours, until the OD600 is between 0.8 and 1.2.
• For the YP + 2% glucose culture: centrifuge, rinse in 20 ml PBS, resuspend in YP + 2% galactose
• For the YP + 2% raffinose culture: add galactose to a final concentration of 2%
• After induction, take OD600 readings and measure on the flow cytometer every 6 hours.

Confusinger and confusinger. We saw basically no induction — a teeny little bit in the raffinose + galactose condition starting at 180 minutes, but it remains low and fades off by 360 minutes. My rule-of-thumb for changes in gene expression is 1-2 cell divisions — so we should be seeing SOMETHING after 6 hours. Maybe relieving the glucose repression is slow? That would explain the negative glucose –> galactose results. Maybe there’s some chromatin repression or something?

Carbohydrate history affects GAL1 expression

We did some reading and found a couple of interesting papers. The first is a review article — the takeaway is that galactose induction is well-studied, but there are interlocking feedback loops that make things complicated. In particular, there is a “history” effect — the media that the cells were growing in affects how fast the galactose-responsive system comes online. The second study measured and modeled these feedback loops using a system very similar to ours (integraged pGAL1:GFP, but at the ADE2 locus intead of HO). Interestingly, their “uninduced” setup wasn’t just raffinose — it was raffinose AND galactose!

I also wanted to see if there was perhaps some chromatin silencing going on. This is often an issue for genetic circuits that are integrated into the genome. The usual way around that is to maintain selective pressure on the selection marker that you used for the integration — that way you select for cells that have not silenced the locus.

We decided to take a deeper dive into this idea. We grew eight cultures overnight (5 ml), varying BOTH the carbohydrates AND the base media.

• YP + 2% raffinose
• YP + 2% sucrose
• YP + 2% raffinose + 2% galactose
• YP + 2% sucrose + 2% galactose
• Synthetic dropout media without uracil (SD-Ura) + 2% raffinose
• SD-Ura + 2% sucrose
• SD-Ura + 2% raffinose + 2% galactose
• SD-Ura + 2% sucrose + 2% galactose

The next morning, we centrifuged all the cells, rinsed in PBS and resuspended in their original base media plus ONLY 2% galactose. We measured green fluorescence on the flow cytometer just before induction, and after 6 hours.

Here are the results:

There is a lot here that I don’t know what to make of, but a few things were clear:

• We were unable to induce cells that had been growing in just raffinose or just sucrose. The sucrose-in-YP media saw a several-fold increase in fluorescence, but not a “strong” induction like we were expecting.
• Cells that we started on media plus galactose all saw some induction, though the amount of induction varied. In both base medias (SDC-Ura and YP), there was essentially complete, 100% induction in the sucrose + galactose condition.
• The YP + sucrose + galactose condition had very little induction after the overnight growth step — maybe 10-20% — but induced very strongly.

So we definitely have our winner — pre-growth in sucrose and galactose in YP, followed by an induction in just galactose. Most of the cells are “off” pre-induction, then basically all of them turn “on”. But there are still some things to think about here.

Discussion

The GAL1 promoter is an attractive system for over-expressing proteins in yeast. The promoter is strongly repressed in the presence of glucose, allowing for cloning and expression of potentially toxic proteins. The off-to-on ratio is between 100x and 1000x. It uses fairly accessible carbohydrates instead of specialty small-molecular chemicals to do the induction.

However, as we and others have found, things are not necessarily as easy as they could be. The “memory” of the interlocking feedback loops means it’s not necessarily as easy as “replace glucose with galactose and watch your genes turn on.” We did end up finding a condition that could turn our genes on — but it required a pre-incubation in media that “should” have been “inducing”, at least according to much of the existing literature.

It also leaves unresolved the question of why the usual advice, “grow in raffinose then add galactose” didn’t work for us at all. My hypothesis is that this advice comes from folks who use an RS-based shuttle plasmid system — where you’ve got hundreds of copies in the cell instead of just one. It would be easy enough to test this using the GoldenGate cloning system — just drop the same GAL1:Venus transcriptional unit into a plasmid with a 2µ or CEN/ARS origin of replication and repeat the experiment.

Thanks to Brandon and Spencer for all your hard work on this over the last week.

Postscript

We tried the induction “for real” today — 5 ml cultures in YP + 2% sucrose + 2% galactose. I had a quick look on the cytometer as we set up big 50 ml cultures — and a substantial fraction of the culture, at least 50%, is “on” already. I don’t think this will mess us up too badly, but clearly the “mostly off” to “100% on” behavior we saw with these culture condition conditions isn’t 100% repeatable.