Question

does anyone know anything about molecular biology i have an assignment on the Transformation of E.coli with a green fluorescent protein plasmid and have no idea what im doing

Answer 1

I know an insane amount about molecular biology. I am a molecular biologist. I will answer any questions that are not clearly for a test.

Answer 2

@jazzie97 See above post.

Answer 3

it's for an assignment is that okay ?

Answer 4

so i sort of understand but i don't know how to write it and cant find anygood information online but what is transformation and how is it achieved i have to explain it in the intro? @mrdoldum

Answer 5

Transformation is the artificial version of a naturally occurring process called conjugation. First, bacteria have a single chromosome that is a literal loop (circle) of DNA. This loop is kept relatively in the center of the cell. Bacteria can have a plasmid, a very small, <20,000 bp, circular piece of DNA. Some bacteria can pick this up from the environment, or, more commonly, by conjugating with another bacterium that has the plasmid.

Conjugation is not reproduction. What happens is the bacteria become connected via a tube and pass either the plasmid or a duplicate of the plasmid through this tube. This was famously shown with pneumonia. There was a specific bacterium that had two forms: one with a capsule that allowed it to cause pneumonia and one that does not that have a capsule. A study showed that the non-infectious bacteria could become infection when exposed to alive or dead infection bacteria.

When we do this in the lab, we take a man made plasmid (technically called a construct) and try to force it into the bacteria. Usually for learning purposes we use a plasmid that has a gene that provides resistance to an antibiotic and the GFP gene inserted into the operon that provides the ability to digest arabinose. Operons are like cellular assembly lines, multiple genes that are grouped together and work together. The GFP gene inserted in this operon means that if the arabinose sugar is present the operon turns on and starts working, but since we stuck the GFP gene in the middle of the operon, arabinose is not broken down and GFP is made.

Answer 6

We achieve transformation in multiple methods, but the most common way for students and labs is to create what are called "competent" cells. You place the cells in calcium chloride to make the cell wall and membrane "leaky" and then mix these competent cells with a mixture with the construct. You then heat shock the bacteria, rapid transition from near freezing to 65 C or so. This increases plasmid uptake by the bacteria. It also kills a lot of them. This is why from literal billions of cells you may get less than 100 that live and grow into colonies on your plate.

We add antibiotics to the media (what the bacteria are put on to grow) to kill any untransformed bacteria. We also try to use a amount of water with very few bacteria in it and spread it out over the media. The goal is that we get colonies that are from a single founder, important for other reasons not really important for a transformation lab.

Answer 7

thanks @mrdoldum a friend sent me this for what IPTG is is there anything else that should be added and is it right becuase it just seems confusing ??

IPTG (isoprophyl-B-D-thioglacopyraoside) promotes the E.coli to take up the plasmid

Scientists can regulate the experiments recombinant protein using a genetic switch called inducible promotes these sequences allow precise control b/c expression of gene will only ‘turn on’ in the presence of a small material like arabinas tehacyclineer.

Under normal circumstances the bacteria make a protein called lac represser which binds to this protein and blocks expression of the t7 polymerae (i.e. the GFP will not be evident), without the t7 polymerae.

Answer 8

@jazzie97 The description of IPTG is good enough. The "inducible" genes are the same concept of the on/off switch of the arabinose operon. I did not go into it, but the way it works is a protein sits on the DNA at the start of the operon and blocks the operon from being expressed. If the sugar arabinose is present it binds to this regulatory protein, causing a change of shape so that the operon can be expressed. It is self regulating. As the organism uses up the arabinose there is less arabinose to bind to the protein, eventually there will not be enough and it will turn off again.

You have a bit more technical than I bother to go into when I teach this lab. What level of schooling are you in?

Answer 9

@mrdoldum i'm in year 12 but i go to school in Australia so i don't know the american equivalent this is our last big assessment but i've been sick and missed a bit of school and it's due in about 8 hours and i know very little (as you can probably tell)

Answer 10

If I recall, by that point you have a bit more specialized and targeted schooling we do in USA.

Answer 11

@and my teacher won't check drafts so i have absolutely nothing to go off. yeah it' maybe it's very general up until the end of year 10 where you choose what area (if any) you would like to do (chem,physics,Bio) and then you do a different area of that science each term.

Answer 12

@mrdoldum

Answer 13

if you search for "transformation labs" on google, bing, yahoo, whatever you will get lots of results.

Answer 14

@mrdoldum we have to explain what GFP is this is what i have is it ok or just a load of rubbish cause i just got it off a random site??

Green Fluorescent protein (gfp) is found in jelly fish which contain a bioluminescent protein (aequorin) that emits blue light that the GFP then converts into green light. GFP absorbs ultraviolet from sunlight and then emits it as a lower energy green light. GFP is incredibly useful in in scientific research as it allows you to look directly into the inner working if the cell this works due the fact that the GFP glows and can easily be followed throughout a cell this is used in many situations such as you can attach it to a virus. Then, as the virus spreads through the host, you can watch the spread by following the green glow.

also we have to explain How will the GFP gene be transformed into bacteria (e.coli represser) how is this done ??

Answer 15

@jazzie97 I have never heard of it, GFP, being used with viruses. For the bacteria, I think it is probably beyond the class to describe how the gene is inserted into artificial plasmids. This is how it is done. You get your custom made plasmid and transform the bacteria to take up the plasmid. I doubt you need to discuss how the gene is inserted into the plasmid.

Answer 16

Just to clarify, in my last post when I wrote "This is how it is done" I was referring the whole process without the details. The actual cutting and inserting of the genes and the vectors used to get the genes into the plasmid (again, this is artificial and the correct term is actually "construct") and are probably not needed by anyone on here.