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6

I've found a nice review that has many details on plasmid replication in general, and several papers about pSC101 in detail, and I'll try to extract the key information from these papers. First of all your plasmid as an ori region that contains so called iteron: In many cases, the origin of replication contains directly repeated sequences, termed ...


5

There are some databases in which you can search for E.coli gene expression data: GenExpDB: E. coli Gene Expression Database Many Microbe Microarrays Database (M3D): A resource of microbial gene expression data Stanford MicroArray Database (use the search tool to find relevant organisms) Colombos (COLlection Of Microarrays for Bacterial OrganismS) ...


4

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 ...


4

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 ...


4

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 ...


3

It is not uncommon for cells to have parallel pathways for same outcome. This ensures foolproof response and makes the system robust. E.coli also has another sensor for aerotaxis (Aer and Tsr proteins). See my answer on your previous post and the linked paper. Also look for coherent feed forward network motifs.


3

One is not normally required to serially dilute E. coli cultures for spectrophotometric measurements, at least in the experiments where the OD value is important. For most protein expression work, the consensus is to start the induction at OD 0.6, and for most chemically competent cells, the optimal OD ranges from 0.3 to 0.8 depending on the competency ...


3

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 ...


2

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 ...


2

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, ...


1

In general, there is no need for a spacing between stop codon and terminator. They can be right next to each other. When transcribed, transcription terminator forms a hairpin-like structure which interacts with the RNA polymerase to stop the transcription. Usually, the upstream sequence will not interact with the terminator and it will be transcribed ...


1

You should dilute (not necessarily serially dilute) if: You are exceeding the concentration range detectable by the machine Scattering starts overwhelming absorption (at high cell densities scattering will overwhelm absorbance). In such cases you can use a turbidimeter. As such there is no issue of the law not holding true at certain limits of ...


1

Reading the optical density of a log phase microbial culture at a fixed wavelength approximates the Beer-Lambert law where A = ecl. This relationship is not linear as the Absorbance approaches 1.0 so at higher cell densities you will eventually underestimate the cell density of the culture. Likewise the law is not accurate below an Absorbance below ~ 0.05, ...


1

The basic problem is one of prior knowledge. The information you have about E. coli metabolism is in the form of a simple network, i.e. a list of nodes and the edges (along with their weights) that connect them. From this information alone it's impossible to know, for example, that the reactions in the TCA cycle should be plotted as a perfect circle (that's ...


1

I'm not sure if they will be useful for your application, but you should look into software used to visualize ecological networks (and maybe also software used for drawing electrical charts). The type of data used there is very similar to what you want to plot, with nodes and links along with metadata for links on e.g. rates or connection strenghts. I can ...


1

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 ...


1

Non-coding transcripts can be as small as 10-15 nucleotides. Once the RNA polymerase initiates, the DNA is melted and the transcription bubble forms. This region is about 10 nucleotides long. But if you are asking the limit of mRNA length, then the answer is different. First of all, to get a protein, the ribosomes have to translate the transcript. To do ...


1

E.coli strains that have dam or dcm methylases can methylate plasmid at adenines or cytosines respectively. See here — It is a NEB web page but has links to the cited references. DH5α has both the methylases- dam+ dcm+ , BL21 is dam+ dcm− and ET12567 is dam− dcm−



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