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Some things to consider. If you spinning too fast and too long, that is going to pack the pellet more. You can spin longer at a slower speed and you will notice your pellet is not as tightly packed. I always do 2000 rpm, 20 minutes, 4C if I'm doing a midi or maxi sized prep and harvesting bacteria in a 50ml conical. If I'm doing a miniprep I will take the ...


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NAD+ is important in this step, since it is co-factor for the glyceraldehyde-3-phosphate dehydrogenase (G3PDH), which acts as a acceptor for the hydrogen atom from the C1 (see below). If you look at the reaction, the aldehyde from the C1 is oxidized to a carboxylic acid which in a second step is turned into a phosphoester. To do so, a cysteine from the ...


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Let me first clarify the difference between anaerobic respiration and fermentation. Literally respiration refers to breathing, but its definition has been extended to include cellular metabolism that leads to ATP generation. Fermentation, as you said depends on substrate level phosphorylation. Anaerobic respiration on the other hand just means ATP ...


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This question got me thinking about what are the metabolic enzymes that take oxygen up in E.coli. I searched the metacyc database for reactions that consume molecular oxygen and there are only 3 that take in oxygen and one that produces oxygen. All three consumers of oxygen in E.coli are the oxidation of ubiquinone by at two sites in cytochrome-bcl or by ...


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The plasma membrane is quite permeable to oxygen and thus oxygen enters the cell simply by diffusion. Reactive oxygen species can be reduced enzymatically in aerobic organisms. Obligate anaerobes lack or don't produce sufficient quantities of these enzymes. An organism that doesn't use oxygen for metabolism but is also not relatively harmed by it can be ...


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There are three variables being shown here: The amount of time samples were stored in the freezer to allow $^{32}$P decay (x-axis), The length of time samples were allowed to grow in a nonradioactive environment before being frozen (points with different symbols), and The rate of $\beta$-galactosidase production when cells were thawed and grown in the ...


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The traditional blue/white screening is set up so that blue colonies are considered positive for the insert, and white colonies are negative. The gene responsible is the lacZ gene, or beta-galactosidase. This enzyme converts a synthetic substrate, X-gal, into an insoluble blue compound. The pCR2.1 TOPO plasmid is a blue/white selection plasmid in which ...


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I found that E. coli exposed to predators such as amoeba changes completely from normal form to filaments (on agar) that look wound up like spaghetti but are not only longer but significantly wider. In this form the predator can't attack E. coli and it lives among the amoeba for extended periods. It does not look like a simple point mutation to me, which ...


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Answer clarified based on comment by @DurgaDatta What would it mean to say the enzyme activity was tested in vitro on the mutant? When we say the response of the a single gene knock out mutant, the cell should be intact except for lacing one gene, which would mean that the response is in vivo. The knockouts were performed inside the E. coli cells and, ...


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It is possible to conduct an in vitro experiment with cell extracts. Meaning that you break up the cells and put their pieces into a reaction glass and do your experiment. It is assumed that the cell molecules (enzyme, sugar or cofactors) will still carry out their functions even though the cell is no more. In other words, you used the mutant to produce the ...


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I did try another transformation: again with our electrocompetent cells Cells form other lab chemically competent cells I made The good thing is that on chemically competent cells there is no weir colonies, however efficiency of transformation was very low, but still, I think it is ok - will confirm it in next day if I really got the right insert. On ...



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