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[Challenge Topic] MANAGING STRESS FROM ENGINEERED METABOLIC PATHWAYS

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IP  162.105.160.10

Posted at 2012-4-30 12:08:24 | All floors |Read mode
original link: http://www.ucsf-pku.org/index.ph ... _METABOLIC_PATHWAYS
(Ignacio Zuleta, David Pincus)

Challenge statement:
Identify, quantify and combat the stressful costs of metabolic engineering.A major effort of synthetic biology is to engineer cellular metabolic pathways to produce desired chemicals.A side-effect of such metabolic engineering is that cellular fitness is decreased.What are the sources of the fitness costs? Can we mitigate the deleterious effects of metabolic engineering?

Anticipated outcomes:A strategy and action plan for how to solve the challenge. Deliverables will be a list of reporter constructs, yeast strains, and a list of experiments organized into phases (including a decision tree).

Background:
Basic microbiology of yeast;
stress pathways in S. cerevisiae;
fluorescent reporters;
industrial synthetic biology applications involving yeast;
temporal encoding of transcriptional output over fermentation/growth (e.g. diauxic shift);
metabolic pathways;
quantitative cell physiology

Outline:
Intro lecture to frame the challenge.
Go over homework and establish conceptual inventory and review technique details.

Basic techniques summary:
how to do genetic manipulations and build reporters.
Idealization I: How to identify and quantify sources of stress.
Group refinement of idealization I.Action plan/decision tree for identification/quantification of stress.
Idealization II: Strategies for managing/mitigating stress.Group refinement II.Final action plan/decision tree.

Homework:Review articles (6 papers?) that we will put on the wiki.Learn to define S. cerevisiae promoters: pick 2 genes from Derisi et al. and find their promoter sequences.Papers: Joe's microarray paper, reingeneered wine strain, yeast stress review, reporter construction example, stress homeostasis, msn2 deletion partners paper, industrial pathway reingeneering example, close control loop of culture example.
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IP  162.105.248.55

Posted at 2012-5-9 21:11:28 | All floors
interesting

but why only concern yeasts

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IP  162.105.21.109

Posted at 2012-5-9 21:42:38 | All floors
Reply zhiyuanli Add Thread


    Maybe the side effect of some metabolic stress is coming from the accumulation of the product, and microfluid equipments can be employed  to dilute the product. Another reason is perhaps the product compete with bacteria growth for some nutrition, for example, arginine. Thus we can strengthen the arginie producing pathway by promoting some crucial enzymes' activity.

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IP  124.128.37.18

Posted at 2012-5-15 21:20:31 | All floors
Reply yscao Add Thread


      yes, and the accumulation of the product inside the cell can be solved by adding a signal to the end of the product to transport it out of the cell, which is already used in bioengineering.
however, aside from the resource and product problem, what if the cell is just gets "tired" due to too much work? i mean, to make excess product, machines (such as ribosomes, ER...) & time are also needed. just like that u cannot work all day and night without rest, and u dont have more than two hands, so u cannot finish infinite work even thongh u've got infinite material & a huge depot for ur products...
stress comes from many aspects, can we find a reporter for all kinds of stress (more reporters means more stress & complexity)? can we define something like "general stress"? is there an exchange rate between different kinds of stress, or a "common currency" for stress, like ATP for energy?
王紫薇

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IP  169.230.87.173

Posted at 2012-5-23 05:13:29 | All floors
Reply Fleo Add Thread

Well it actually concerns any situation in which you repurpose a pathway - be it to secrete a recombinant protein or by adding an entire biosynthetic pathway for producing a fine chemical or fuel. In practice this is a challenge when working in industrial E.Coli strains as well as fungi like Picchia pastoris... soon there might be examples using human cell lines too.
   

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IP  162.105.23.18

 Author| Posted at 2012-5-23 09:06:25 | All floors
Reply izuleta Add Thread

Welcome! and .. are you Ignacio?


   
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IP  162.105.248.40

Posted at 2012-5-23 10:04:57 | All floors
oh...the initiator

welcome

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IP  162.105.23.162

 Author| Posted at 2012-5-23 21:52:58 | All floors
I remember that yeast is quite "inflexible" in sensing the glucose and make corresponding response (tell me if I remember it wrongly). It mainly relies on the receptors that sensing the glucose concentration outside, but not the real situation inside the cell.  Therefore, you can trick the yeast by overexpression/knockdown some of its receptors, and make it think it's "full" or "hungry".
I'm wondering how many cells use this "sensing outside" strategy, and it may provides us a easy way to manipulating the metabolic pathway from upstream.
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IP  162.105.116.89

Posted at 2012-5-29 11:59:35 | All floors
I'm quiet interested in this topic,  especially in strategies for managing/mitigating stress, which reminds me of data mining for network, although i'm not familiar with the biological background...
btw, how does cellular fitness decreased? Is it related with analysis method? such as FBA, MOMA, ROOM or something else...
It takes two of us to discover truth: one to utter it and one to understand it.

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IP  222.29.74.11

Posted at 2012-5-30 15:11:33 | All floors
I am interested in this topic too. However, I have little knowledge about the engineered metabolic pathways. Are there any papers that could lead me quickly into this topic? Thanks a lot.
潘兴杰

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IP  162.105.100.165

Posted at 2012-5-30 23:22:51 | All floors
Can cells feel tired? If the rate of metabolism is too fast by some reason, do they die early?

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IP  123.120.154.132

Posted at 2012-6-1 23:55:42 | All floors
In my opinion, like chemical equilibrium, with some protein in the cell being more active, some protein become less active, and I strongly believe this is the source of stress. To link the microcosmic world and macroscopic world is not easy, maybe my could design an experiment: place normal cell and “stressful” sparately in two same culture dish(with all ingredients and chemical elements known), several days later, we would detect the difference between the concentration of all ingredients which reflects difference in metabolic path. Then we would have a rough idea about the change in the metabolic path. Then we would be able to design more accurate experiment to detect changes within certain metabolic pathway.

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IP  123.120.154.132

Posted at 2012-6-2 00:03:04 | All floors
Answer: lyxiong
   If the metabolic rate is too fast, the cell is supposed to die early. (I think there is something to do with oxygen radical which formed in metabolic process and plays an important role in cell aging)

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IP  123.120.154.132

Posted at 2012-6-2 00:05:10 | All floors
Reply lyxiong Add Thread


   
Answer: lyxiong

Ifthe metabolic rate is too fast, the cell is supposed to die early. (I thinkthere is something to do with oxygen radical which formed in metabolic processand plays an important role in cell aging)

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IP  123.120.154.132

Posted at 2012-6-2 00:14:51 | All floors
Reply Amber.Zw.W Add Thread
Answer:

    As for a "commoncurrency" for stress, from my perspective, we could measure the livenessof some certain protein like respiratory enzyme. Since the liveness ofrespiratory enzyme changes with the age of the cell, we could create a matchupbetween the liveness of respiratory enzyme and the liveness of telomerase whichindicates cell’s age. The ration of them would indicate if the cell is in anactive or stressful state. Through the comparison we could know the differencewith normal cell and “stress” cell(and the stress level as well ).

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IP  123.120.154.132

Posted at 2012-6-2 00:58:32 | All floors
As far as I know, many gene within a yeast have been decoded, if we could use a DNA sequencer, would it be an easy job for us to detect genetic change between normal cell and stressful cell?(if there is a difference then it would probably be a source of stress, if there is no difference we could move our focus to the metabolic process within cell, like the accumulation of certain thing in the cell. Then the problem is easier but could we use such a equipment like DNA sequencer?)

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IP  115.25.240.160

Posted at 2012-6-2 21:42:41 | All floors
Hi, I have an idea: would it be possible if we use chemical reagent polyethyleneglycol to fuse a cell with normal metabolic pathway and a cell with a stress pathway? Will we gain a super cell with both great production and relatively “healthy” state? If we could separate syncretic cells and compare them with normal cells we would be able to see if this way could help us alleviate the “pain” of cells with stress pathway. Do you have any comments or other ideas about this topic?

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IP  218.199.171.180

Posted at 2012-6-5 16:48:47 | All floors
Reply zhiyuanli Add Thread
We know that cell can adapt different stresses largely depends on the robustness of molecular networks in protein or metabolites level and so on, to mitigate the deleterious effects when engineering metabolic pathways, we can try to design the network topolopy that is tolerant to stress or pertubations, that is, we design a robust engineered metabolic pathway to well adapt for the alteration we act on the pathway.


   

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IP  162.105.38.137

Posted at 2012-6-8 15:49:13 | All floors
I am interested in this topic. Perhaps we can combine this problem with the topic "How to grow cells as large as grapes" together.  

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IP  115.27.93.126

Posted at 2012-6-12 23:03:41 | All floors
Reply Anakinphile Add Thread


    This book in the attachment--Yeast Stress Responds (Topics in Current Genetics)  published in 2003 provides some reviews on the stress responses using S. cerevisiae as model organism.  Maybe it would be of some use to help get into the topic quickly.

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