pGLO lab quiz
Compare and contrast the number of colonies ( or other type of growth) on each of the following pairs of plates. Explain why you obtained these results.
-LB/amp and -LB there was nothing in this one because the ecoli did not have the resistance to the amp so ti could not repoduce +LB/amp and -LB/amp there are 19 here which means 19 ecoli were transformed they were restintant if they transformed +LB/amp and +LB/amp/ara there was 26 in this one and they glow the resistance was all good and it cound produce GFP because there was ara to induce the operon
why is it good to use ecoli
. coli reproduce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight. Like all bacteria, E. coli has no nuclear envelope surrounding the bacterial chromosome and thus no true nucleus. All of the genes required for basic survival and reproduction are found in the single chromosome.
Why do the E.coli cells produce the Green Fluorescent Protein if it is of no use to them at all? Describe the detailed sequence of events that makes this possible.
1. arabinose binds to araC regulatory protein→changes its shape 2. ara C no longer hold the DNA in a loop →DNA loop breaks open 3. promoter is now accessible to RNA polymerase 4. Meanwhile, ara structural genes(BAD) were replaced by GFP genes 5. RNA polymerase now transcribes GFP gene instead, then translates it into GFP
what was the function of adding calcluim chloride
Addition of calcium chloride solution- the calcium has a positive charger so it shields the negative phosphate in the nucliotide. You nutrelize it so it can go through the cell membrane because it is non polar. Adding the calcium chloride serves to shield the negative charge of DNA phosphate groups, neutralizing the DNA which makes it easier to go through the cell membrane which is nonpolar
What do you think each of the two environmental factors you listed above is doing to cause the genetically transformed bacteria to glow green?
Arabinose- serves as switch, explained in #8 UV- jellyfish are bioluminescent so can energize the GFP. Without that ability we need UV to shine on GFP in bacteria, in turn it emits a lower energy green fluorescence.
when are ecoli most likely to take in foreign DNA
E. coli cells are more likely to incorporate foreign DNA if their cell walls are altered so that DNA can pass through more easily. Such cells are said to be "competent." Cells are made competent by a process that uses calcium chloride and heat shock. Cells that are undergoing very rapid growth are made competent more easily than cells in other stages of growth.
What type of operon is the arabinose operon, inducible or repressible? Inducible Name another specific operon system studied in class that is most similar to this one. the Lac operon What is the advantage of having the structural genes ( like the ones for the arabinose enzymes) arranged as part of an operon system?
For efficiency, all of the proteins/enzymes needed are transcribed and translated at the same time, when you need them. And they are turned off when you don't need them. The operon is similar to an inducible operon, like the lac operon we studied in class. The advantage is that multiple genes can be expressed at once. Like say the enzyme need to parts to function the gene for one part and the gene for the second part can be expressed simultaneously Inducible operon lac operon one master switches switches all the genes on and off at once efficent
What are two possible sources of fluorescence within the bacterial cell when exposed to UV light?
GFP Gene (in the plasmid) or GFP (protein)
why is GFP used
GFP can be used to see if you successfully made a GMO by tagging the gene
How is genetic engineering used commercially? HGH
Hypopituitary dwarfism is currently treated by administering hGH extracted from the pituitaries of human cadavers. ... The plasmid with the hGH gene was inserted into a special bacterial strain. Once inside the bacteria, the hGH DNA is used a a template to produce the hGH protein.
What type of bacterial growth (Lawn, colonies, nothing) do you expect for the - pGLO tube added to the LB plate? Why?
I think that a lawn to grow on the LB plate, i think this because there isnt any ampicillin to kill the bacteria if it is no resistance plasmid in ecoli. Each colonies is 1 million bacteria but in each colony they are all cloned, it can not reproduce but it is still there. The lawn will grow because it has nutriets, optimal position for hte bacteria
What type of bacterial growth do you expect for the + pGLO tube added to the LB/amp plate? Why?
I think that colonies in this plate since only some transformed the pGLO and others did not which would mean they would be the only one to survive making a colony. Colonies because so will have plasmid some will not, there are only a few survivors because only some of the bacteria will grow, because it is hard to do transformation with bacteria
What type of bacterial growth do you expect for the - pGLO tube that is added to the LB/amp plate? Why?
I think there will not be any growth, it is not resistant to ampicillin so none it will survive. No growth because they dont have resistance to ampicillin don die but no
In bacteria, translation of the mRNA molecule immediately follows transcription. How can this be a problem when trying to get a eukaryotic gene (like the GFP gene) expressed by E.coli?
INTRONS in mRNA are not spliced out by bacteria because they don't have a system to edit mRNA (no spliceosomes, etc)
what was function of incubation on ice
Incubation slows down the movement of the bilayer in cell. slows fluid cell membrane- fluid mosatic model
Which of the two possible sources of the fluorescence can now be eliminated? What does this observation indicate about the source of the fluorescence?
It can't be the plasmid, so the protein must be what is glowing
How do you suppose this problem was overcome by the scientists who engineered our pGLO plasmid?
Scientists needed to give the bacteria "clean" versions of the eukaryotic gene. This was accomplished by reverse transcribing already-edited mRNA into cDNA, then using the cDNA for the transformation process.
Bacterial Colonies
The bacterium you use in your laboratory activity is Escherichia coli, which has been grown in a petri dish on Luria Broth (LB) agar. Each colony in the petri dish is made up of millions of individual cells.
What is meant by a control plate? How many control plates are used in this lab, and what is the purpose of each?
The control group are the plates with no plasmid and no amicillin, with the other one being a non transformed bacteria on the ampicillin plate. The purpose of them is to show how non transformed bacteria grows and how the normal ampicillin plates to compare to the other plates They are all controls with out expression except the LB/Amp/ara. There will only be glowing in the LB/ amp/ ara. To prove what is rewuired to get a glowing green colony
How is genetic engineering used commercially? Insulin
The gene for human insulin is inserted into the gap in the plasmid. This plasmid is now genetically modified. The genetically modified plasmid is introduced into a new bacteria or yeast cell. This cell then divides rapidly and starts making insulin
Which plate(s) do you expect the bacterial cells to glow under UV light? Why?
The plasmid is expressing the jelly fish gene LB amp arrow has to have the sugar so that it can turn on the operon arrow= sugar
Sometimes satellite colonies form in the areas (halos) directly surrounding the transformed colonies growing on the ampicillin plates. These cells are not transformed. Why are they able to survive in the ampicillin plates? How could you prove that they are not transformed?
The transformed colonies have an amp -resistance gene that allows them to make an enzyme that digests (neutralizes) ampicillin, thereby removing it from their local environment. Non-transformed cells in this zone are suddenly able to divide, and create satellite colonies. They are smaller than the transformants because there was a lag time for when they could start dividing. You can prove they are not transformed by shining a UV light on them: they will not glow.
how did they get the ecoli to express the gene
What the company did they cut in the middle of the operon and but in the jelly gene so the cell thinks it is making sugar and it is making glowing stuff.
bacterial lawn
a very dense layer of bacterial growth looks like a whitish film this indicates ton of bacterial growth/ reproduction
what is the ARAc
activator and respressor
what was the function of the nutrient bath 10 min recovery
allows gene expression of the plasmid- recovery area you can not make a bacteria that just got a plasmid, it needs time to express the gene. The more time to express the more time you have more enzymes.
What two factors must be present in the transformed bacteria's environment for you to see the glowing green color?
arabinose and UV
how do you coax the ecoli to transform
at cacl to nuetrilze the DNA so ti will base throught the bilayer. then you ice it so it slows down the movement of the mosiaic bilayer then you heat shock it to increase the permeabiliy of the membrane allowing the plasmid to slip in
why do not all cells undergo transformation
because the dont want to they need coazing
a kid messes up and does not transfer the ecoli in the ice bath to the heat bath what effect could it have
decrease because the heat shock was not complete so it did not open the phosopholipid bilayer correctly
whether your organism is unicellular or multicellular, has a long generational time or a short generational time, and whether your organism is pathogenic or nonpathogenic. Given these standards, why are bacteria such as E.coli good candidates for transformation? Explain, in detail.
easier to get DNA into one cell vs. millions short generational time- can show you right away if the organism passes on the new DNA to offspring and future generations because E.coli reproduce every 30 minutes in ideal environment. nonpathogenic for safety: E.coli HB 101-k-12 (the strain that we used) cannot survive outside of lab
How do you get bacteria to transform to force them because ecoli doesnt want to, electrically shock the cell to allow gaps to form and let it slip in
electrically shock the cell to allow gaps to form and let it slip in Or create a chemical substance to do the same thing which will cause the cell to open up temporarially to let the plasmid in
what is the LB bath for
food for bacteria, everything they need
what is the procedure for the heat shock
ice hot ice
what was function of the heat shock
increases permeability of membranes- time is important exposing it to heat give it a small window of time The heat shock increases the permeability of the membrane and 37 degrees is the temp for max cell growth
how is the ara operon different
it has two control regions the initiator and the operator and it is both positively and negatively controled by the produce of arac
what is the function of LB
it is a nutrient filled broth making a perfect envirnment for the ecoli to grow
what gene was the amp restintant gene transfromed into
it was put in the gene that produce the enzymes that digest arabinose sugar the plasmid have been genetically engineered by cutting out structural genes that code for arabinzoes enzyme and replace it with GFP gene
the to find what you divide the colonies by you
multiple total number of DNA time sfraction of DNA used
Recall what you observed when you shined the UV-light source onto a sample of original plasmid DNA and describe your observations
no glow
how does the ara operon work
normal structure pretty much promoter region where RNA poly lands all structural genes in a row upstream of the promoter is the operator where the gene is when it is on the rna oply can land and get to structural genes, when it is off it can not land and express dna
colonies
one ecoli that divide lots of time
what does the operon look like when it is off
operator is folded over on the iniator and it can be expressed araC is holding it together
activiation of the operon
operons ara operon, like lac operon, they only want it on when ara is there. The way it is shut if is not normal there is a protein which twist the DNA into a loop which makes it hard for RNA poly to land and when it is needed a sugar lands breaks the loop and RNA poly can land
Transformation efficiency" is a measure of how successful your lab group was in producing transformed bacteria. It is roughly estimated as the number of bacteria(colonies) that successfully absorbed the plasmid. What are TWO factors that might influence transformation efficiency? Explain your answer
procedual errors like leaving the heat bath for to long or not centrifuge of vortexing for long enough
Plasmids
re circular pieces of DNA that exist outside the main bacterial chromosome and carry their own genes for specialized functions. In genetic engineering, plasmids are one means used to introduce foreign genes into a bacterial cell. To understand how this might work, consider the plasmid below.
what is ARa c
regulatory gene two conformations one in the presence of arabinose and the other in the absence when it is absent the iniator binds to the operator and folds itself over
How does ampicillin work?
stops bacteria ecoli from reproducing
what happens when arabinose is present
the araC leaves and breaks the loop allowing expression
steps in experiment basics
the ecoli is transfored to the CaCl solution then the amp+ is added to half then the cells are heat shocked to transform the cell the cells are spread on agar with amp on it and incubated
how is the thing resistant to the amp
the plasmid brings in the GFP gene because it is the vector and then it cuts out isloate plasmid from bacteria with RAmp on it then put in the GFP where the ara gene was the insert plasmid into bacteria
how did they get the transformed cell to express the GFP gene
they had to make sure arabinose was present in the environment
how does the heat shock work
total numver of colonies growing on agar/ amount of plasmid DNA spread on agar plate
how to find the fraction of DNA used
volume spread/ totl colume of that tube
what are satalite cells
when the amp restint cell kill all the amp around them the nonactive cells see the gap in amp and begin to reproduce around the amp resistant cell