Janet B. Matsen:Key Ideas
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Notes for my project by date:
- we don't measure [formaldehyde] b/c we don't believe the number will be accurate. When you lyse a cell, there are a lot of things it can react with.
- It is very difficult to engineer Km of enzymes compared to kkat
- Why does formate --> formaldehyde conversion require ATP if it the reaction free energy is downhill?
- Why are we calling ADH AcDH now?
- Ac is for "acylating," the process of adding an acyl group to a compound. An acyl group is
- ATP for our pathway comes from the TCA cycle
- the theoretical maximum of amino acid 13C labeling is 80-90% on our timescales
- used 13C glycerol as a control for our tests
- expect downstream metabolites to have a lag in 13C labeling relative to early metabolites in a pathway
- should we have used His-tags in our pathway constructs?
- tempting because then you can purify the enzymes and run them on SDS gels to make sure they are expressed, OR test them for enzyme activity.
- bad because His-tags chelate metal, and may stick to things in the cell
- Regulation is one of the first targets when you put an organism under selective pressure.
- If you are trying to evolve an enzyme in a strain, you will first get many regulatory changes on the genome of the cell.
- Regulation is combinatorial. You can't assume that knowledge of one regulator will allow you to change a enzyme activity however you want, as that gene may be regulated by several key regulatory elements. You might not even know the others exist. ** Often there are multiple promoters within a gene. RNAseq can be useful for identifying this scenario.
- Synthetic biologists often think of the cell as they do about circuits. One of the key problems with this is that they are neglecting context. Cells are very complicated at every level and there are many interactions one will not know about. Thus it is important to investigate how an added pathway interacts with the rest of the cell. Of course this is difficult and expensive due to the required techniques, and won't necessarily give clear answers as there is a lot of nonlinearity.
- Cells don't usually need regulation at steady state. Regulation is used to move from one steady state to the next.
- Often transcript abundance correlates with metabolic flux, but this isn't always the case. Our lab often finds anticorrelation between the two, likely due to the necessity to keep the concentration of toxic intermediates low.
2012/08: when you have cells under starvation, it can look like they grow for two reasons:
- 1. they start eating the proteins in the ribosomes
- 2. they can divide, which increases OD
- note: neither of these cause increases in dry cell weight, so this is a good way to measure growth.
2012/04: You can drive a few kb with LacI: naturally it drives ~4.9 kb (lacZ = 3075, lacY: 1254 bp, lacA: 612 bp) 2012/05: Do we have an easy (non enzymatic) assay for methanol? Betsy's Answer: Not that I'm aware of but you could probably come up with a bioassay using feeding experiments if it doesn't have to be quantitative. What are you trying to do?