I need help on this thing called SIMULATING INSERTING A HUMAN INSULIN GENE INTO A PLASMID?

Question

I need help answering these 3 questions based on biology and plasmids.

1. Just mixing your insulin gene and plasmid DNA w/ sticky ends may temporarily result in a new plasmid, but it is being held together by weak hydrogen bonds. What enzyme do you need to add in order to make ur new plasmid a solid circle of DNA?

6. What is the purpose of using plasmids with antibiotic resistance in them?

7. Why were no restriction enzyme tested that would produce bunt ends instead of sticky ends?

Answer 1

1. DNA ligase ligates the DNA to a solid plasmid

2. Antiboitic resistance allows you to screen bacteria that contain the new plasmid. Any bacteria without the antibotic resistance gene will die when plated out on a plate containing the proper antibiotic. Without antibiotic on the plate, you would have a plate with a lawn of bacteria.

3 Two reasons. Blunt ends are difficult to ligate and that lowers the cloning efficiency. Also if it does get ligated and some portion will, a second round of analysis will be needed to determine the orientation of the gene. Which goes to the second point

If you use two restriction enzymes with different sticky ends you can clone the insulin gene in to the vector in a directional fashion. ie. Its not cloned in bacwards.

hope this helps

Stooleo

Answer 2

The answer to #1 is my best friend, DNA Ligase! hehe

That one cements the backbone bonds between the insulin gene insert and plasmid.

We use plasmids with antibiotic resistance because we use bacteria to clone (mass-produce) our altered plasmids. When we "transform" the bacteria, shoving plasmids into them, not all bacteria will get a plasmid. So, those non-plasmid bacteria will be killed off by the antibiotic, leaving only plasmid-bearing bacteria.

I am not sure what you are asking on question 7 exactly. Blunt end enzymes in general are fairly useless in cloning individual genes, since after you cut open your plasmid to shove in the insert, your DNA ligase then has to connect both backbones of the DNA strands on both ends. Because the ends are blunt with blunt-cutting enzymes, DNA ligase will not have the assistance of hydrogen bonds to get it all together.

Answer 3

The gene for antibiotic resistance isn't inserted on an identical time; it pre-exists interior the plasmid. You advance th micro organism interior the presence of the antibiotic with the intention to be certain that no different micro organism are starting to be apart from those that have the plasmid, yet additionally to stress the micro organism to maintain the plasmid. interior the absence of the antibiotic the cells won't difficulty to replicate the plasmid (because of the fact it only isn't needed for their survival) and after some generations it's going to be diluted out of the inhabitants.