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ScienceWeRa student research team trying to synthesize a component of Saffron, the new hope against cancer. AUA!

Jul 24th 2017 by igemuppsala • 10 Questions • 59 Points

Our story:

We are the iGEM team of Uppsala University (Sweden)! iGEM is an international contest for genetically engineered machines. Picking up on the Front Page article about saffron, we thought we'd do a quick AUA about how artificial saffron works! Saffron itself is increcibly expensive, hence we are working on the pathway that enables us to let bacteria produce it for us!

We are planning to synthesize alpha-Crocin in E.coli. It is responsible for the red color of Saffron, has potential medical implications and the famous Crocetin, which had been mentioned in the article, is an intermediate in this reaction! We will go to the USA in November (MIT in Boston! Exciting!) to present our project together with hundreds of other teams from all over the globe!

My Proof:

Group picture in the lab

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Ask Us Anything!

Edit

Thank you for your questions and your interest guys! The working day is coming to an end in Sweden, but we will be back tomorrow to check one more round of questions! Cheers!

Q:

Is the process similar to the way insulin is synthesised using E.coli?

A:

Thank you for your question! The answer is: Yes and No! Also for insulin production, the plasmid (circular DNA) of E. coli is cut in a special way, the gene for insulin inserted, and E. coli proceeds to produce human insulin for us.

The difference is that we are not producing Crocin (or ultimately Saffron) directly, because it is not a protein that can be encoded in the genes. Instead, we are inserting the codes for enzymes (little machines that make A --> B) that are doing this job for us in the cell. The principle of insertion remains the same though.


Q:

How close are you guys to getting it?

A:

We have transformed the enzymes of our interest and the E. coli are carrying the genes! The next step, and our current work, is the isolation of the enzymes for in vitro testing, a process with many challenges as they can be delicate to handle, especially if not well characterized!

In more simple words: The first step was the construction set-up, which we completed. The second step is the re-isolation of the construct, which we are working on. The third step is testing the system and improving it!


Q:

Good luck to you guys

A:

Thank you! And thank you for your interest!


Q:

Hello, which pathway are you using MEP -> b-carotene -> Zeaxanthin -> Crocin? Don't you think you might have problems expressing complex eukaryotic enzymes in E.coli? // Mupp

A:

We are not expressing the whole pathway, as this would be indeed a very complex problem to tackle and because it is possible to engineer strains that produce intermediates of the pathway. We are focussing on crocin and its immediate precursors!


Q:

Witch immediate precursors? And will you add the precursors to the media? :O

A:

Zeaxanthin --> Crocetin dialdehyde --> Crocetin --> Crocin is the pathway we are particularly interested in. We are inserting the enzymes required for these convertions into E. coli! The precursors are firstly added in vitro, so to purified enzymes outside the cell. The ultimate goal, however, is to have an intermediate = Zeaxanthin producing strain that does not require added precursors!


Q:

what other approaches do you see being pursued -- for example are there any attempts to make saffron as a whole easier to grow (rather than one component)?

A:

If I understand correctly, you are wondering why we don't engineer the plants to just grow better? The problem with the plant, Crocus sativa, is that each flower only contains a tiny amount of saffron, which has to be carefully hand-picked and dried. It still remains unknown how exactly the plant accumulates the spice, which makes it hard to engineer it. A study for example found many of the enzymes expressed in the plant, which helps us understand the pathway, but does not tell us where to start improving the plant. Also, not all plants are suitable for genetic engineering and Crocus is not a model plant with decades of accumulated data.

Overall, there are definitely other approaches from molecular plant scientists to improve the growth of the plant, and you will find many projects going on working with Crocus sativa. In terms of cost and also necessary resources, it is well worth trying to implement the system in bacteria in the meantime - like our project.


Q:

what are some of the more exotic approaches you've heard of?

A:

The most common are yeast or E. coli engineering, and molecular plant genetics on Crocus sativa. I have not heard about another method!


Q:

How much money is it costing you guys?

A:

So the lab, the facilities, the instruments and many chemicals are generously sponsored by our university. They also cover our orders, and we get to borrow some equipment from other labs in our faculty. Once you have the basics in the lab, the main costs come from the intermediates (like Zeaxanthin for testing, its very expensive), some of them cost like 400$ for 1 mg.

We will pay ourselves for the trip from Sweden to Boston plus accommodation, though we received some generous donations via our gofundme.


Q:

Synthesized cannabis doesn't seem to match up to the real deal, and may in fact have negative side effects (to be fair, I haven't been reading up on it lately). Do you foresee similar issues with saffron?

I don't mean to discourage, science isn't always easy and scientists are heroes.

A:

Hi! You are absolutely right with being careful of conducting conclusions from in vitro to in vivo. Often enough, things do work surprisingly differently in living organisms, because the level of complexity and influences dramatically increases. Hence the procedure is discovery/testing in isolated systems, then animal testing, then clinical trials in humans if a product is designed for humans. In many cases, it leads to being able to create something that is beneficial for us, so it's worth a try in our opinion!

Edit: This is assuming we want to implement it in medicine. For industrial dye, another potential use for Crocin, we do not have to deal with metabolic issues of course.


Q:

Skimming the previous article I noticed it works by blocking lactate production. Given respiration is a major source of free radicals, isn't it likely inhibiting the lactate production is causing a surge of free radicals and killing the cancer cells with oxidative stress? If that is the case surely this would do the same in normal cells, with the caveat the cell must be using the lactate producing pathways (exercise in muscles etc). In that case it could be reasonably assumed to be carcinogenic too (oxidative stress massively increases cancer risk).

The recommendation in the article "maybe eat a bit more saffron" seemed to be jumping the gun by a few steps. Still we don't know until we try. Thanks for your contributions to science.

A:

It sounds like the cancer in the tube is inhibited in growth by removing not only its extra source for energy, but also it needs the lactate as precursor for growth. The article speaks about "growth inhibition", not killing them off immediatetly, so I am not sure if there are free radicals involved. I have to also make a disclaimer here that I did not study physiology and others can probably give more sophisticated answers regarding this specific crocetin mechanism.

The reason it leaves the normal cells alone is that also muscles cells do not use anaerobic respiration unless they absolutely have to, for example in intensive exercise. Normally, a cells would always chose aerobic respiration with oxygen, because it is far more efficient. So yes, cells can use the lactate producing pathway, but they normally don't. That is why I believe it did not have a carcinogenic effect or growth inhibiting effect on normal cells.

Again, I am not a physiologist, but it seems that the cells would not up-regulate the aerobic respiration, because the oxygen is the limiting factor. The reason the other system is switched on is because of the lack of oxygen. Hence up-regulating would not make sense in a lack of oxygen molecules, and the other system is being used. The radicals can be side-products from aerobic respiration, but only if that oxygen is actually available. Therefore, I believe the radicals won't be a problem.

Good approach with being careful with quick conclusion! But since saffron tastes amazing, I certainly don't mind eating some more lussebullar or saffron risotto! :-)