User:Thewinster/Sandbox/B8/1W2

Lecture 1

Rough and Trivia

To Do

• Highlight Doubts
• Try and guess the learning objective of each section
• Ask what is the SPECIFIC experiment that is being discussed!
• Update the syllabus.

Section 1

• Plasmids can be used for cloning or expression. Cloning Cannot be used for expression but expression can be used to cloning. (this might be a good place to start for explanations.)
• we generally use phage promoters for the Vector (?). T3, T7, M13 are some examples of her face promoters that I used for the same. These are used both upstream and downstream of the gene to be cloned or expressed. (?)
• There was also discussion where the plasmid, a circular collection of genes, was discussed. There are three different parts that were discussed here. The origin site, the antibody marker site and the multiple cloning sequencing. It is the multiple cloning sequence item when you put your DNA segment.

the following concepts are discussed in this particular segment of The lecture:

• the need for primer was discussed. The need for primer is that if you do not know what the DNA sequences, to start cloning you need a primer. However I do not understand where this discussion came in from a discussion of plasmids Vectors and promoters. this was actually a part of the methods of cloning where if you do not know what sequence you are cloning, you can use this concept.
• the binding site has to be known because you need To design the prima for initiation of the cloning. However the following details are hazy: how are the discussing the necessity off unknown binding site and primer in the case of cloning, What is it that The are discussing in terms of cloning and what is the Objective here?
• the need for a unique sequence was also brought up when it came to the point of having a primer for cloning. Perhaps this is because the one the primer to stick to the site where the cloning has to start and we do not want the particular prima to bind somewhere else. This might actually be one of the few things that I understand from this section of the lecture.

Section 2

The cell lysate and the p53 were the major keywords in of this particular section. Somehow, the activity of cell lysate (in vitro?) Was limited by the short half-life of ATP. It was also discussed that small amounts of Coomassie blue dye was used to stain cells.

Questions are: what is being stained and what is the purpose of staining?

Also, the ability to replace sulfur containing amino acids namely the Cysteine and methionine with the S35 isotope labeled the particular product was brought up.

The questions you are, what is the cell lysate being used for? Also, if the amino acids are being labeled radioactively, then for sure we want the protein. What is the common ground on which staining the Coomassie blue and radioactively billing achieve the same particular purpose?

The problem here is that, it is still not clear as to what is the context of discussion. The general context is cloning and expression using plasmid vectors. However, what is the procedure that is being discussed and what is the context in which cell lysate has been introduced is not clear.

This is the section in which in vitro processes have been emphasized. However, what is the stage in which we start the in vitro part of the experiment? What goes into the test tube from the bacteria and what is added as a secondary reagent to the test tube? What we want to get out of the test tube?

Now, we are also discussing RNA sequences. There are two key word here, first is sequencing and the second is in vitro RNA. Both of these are related to a promoter. However again, it is not clear what we are discussing. We started of with cloning and discussed expression as well. When it comes to expression we are sure to discuss RNA. However, is that the RNA from our cloned sequence that we are talking about? Also, the purification of RNA sequences is difficult, because of the presence of RNA binding sequences. (?)

Also, how does RNA polymerase come into the picture question mark RNA polymerase is used to generate RNA sequences. What are they generating the RNA polymerase from? Again, this has been discussed with relation to plasmids.

'Question'

We have also talked about codon optimization. The fund I here is that different codons code for the same amino acid. In case a particular tRNA does not serve the function (again, the question is, why would tRNA not be useful? Is it related to the vastness of a particular tRNA? Which also forces us to ask a particular question, if tRNAs are rare, why are they rare?), We can make use of this. (Also of note is the fact that the sequence will change. So, what is the sequence that we are changing? The sequence of the original DNA?). One example of a scenario in which such: optimization would be required to be done is nothing but viral proteins.

Suggestion: it will be good if you could make a sequential, step-by-step diagram of this process and mark out the concerns.

Also, a question regarding Splicing was asked. In response, the importance of Nucleus (and NOT Cytoplasm) was put forward. Perhaps this was regarding the availibility of Proteins. It's also a grey area :-(