replcatio POLO 371 OFP Multiple Cloning Site AmpR promote 1. Above is an annotat

Question

replcatio POLO 371 OFP Multiple Cloning Site AmpR promote 1. Above is an annotated image of the pGLO plasmid that we used in Labs 1-5 10 Draw a chart similar to the one below, telling me a. What the feature is for/could be for (purpose) b. If the feature was necessary for what we did in lab(i.e., would it have made any difference if that feature was not part of the plasmid?) (necessary yltn) c. If we made use of the feature, how/when/which lab (when/how used) (This chart only indicates organization; the boxes should be larger to accommodate your answers Purpose Necessary (Yes/No) When/How Used Feature araC araC promoter araBAD promoter GFP Multi ni polyA signal AmpR promoter AmpR Origin of replication

Explanation / Answer

This plasmid can have different uses, being primarily as a cloning vector (ie, save genetic information) or even as an expression vector (assuming that the genetic information will code for some specific protein).

Remember that there are other plasmids such as fertility plasmids (F plasmids), resistance plasmids (Plasmids R), virulence plasmids, col plasmids among others.

I can not answer the other questions of your exercise since i did not know what you did in the laboratory, however i can explain to you what each of the elements of the plasmid serves so that you can answer if it is necessary or not some given feature.

The ara gene encodes an enzyme that allows the use of arabinose by the microorganism in question. For the gene to work it is necessary to have the promoter (araC promoter) because if you put this gene without the promoter, the enzyme will never be expressed, so when putting araC you must always go along with its promoter. AraBAD is a regulator of the expression of AraC, which in the presence of arabinose will favor the expression of the AraC gene, however if there is no arabinose then it will repress the expression of the AraC gene with which the bacterium saves energy, it works similar to the Lac operon. Those genes only serves to select bacteria with the use of media containing arabinose. It can be used with a selection method so that when you put a medium containing arabinose as the only carbon source, bacteria that have not internalized the plasmid die and can purify your culture.

GFP has reporter gene function, that is, it informs you whether the bacterium internalized the plasmid or not, independently of the conditions (that is, subjecting it to stress by carbon source or exposure to antibiotics)

The multiple cloning site (or MCS) is a known sequence in which you know that restriction enzymes will act exclusively in that place (that is, they are not going to cut the plasmid in another part whereby the gene or the genetic information of interest it would be inserted in another part, which runs the risk of deactivating the other plasmid genes) (If your plasmid is going to be used as a cloning vector or as an expression vector, you must necessarily have this site)

The polyA signal is a sequence of many adenines stuck one after the other which serve as a sign of transcription completion, in addition to functioning as a protective agent of mRNA degradation, so if you want to express a gene it is necessary to have this signal.

AmpR is a resistance gene to Ampicillin, if you want to select the bacteria that internalized the plasmid by exposure to ampicillin then you must include this gene (with its respective promoter) so that you can select the bacteria that will serve as a genetic library in case be used as a cloning vector.

The origin of replication is the site in which the enzymes (DNA polymerases) replicate the plasmid to keep it inside the cell. This feature is must necessarily regardless of the conditions because if you put the plasmid without the OriC (origin of replitacion) as the generations pass and the bacteria divide, the genetic information will be lost.

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