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Biology Help - May 24th 2010, 10:56 PM

Well I really need help on a lab in Biology. I don't really know what we are trying to find or do. Basically we are supposed to put "a single colony of bacteria" into two micro test tube (one labeled +DNA and one labeled -DNA) and then put in some plasmid "solution" into the test tube labeled +DNA only.And then we put it in ice, then in a water bath, then back to ice, etc (i think this part isn't that important). We then put it in room temperature for 10 minutes and then we use a pipet and put the solution into the "plates" labeled LB (thats the food for the bacteria to grow in he said), amp (don't know what this is, and ara (he said it was used to "turn on" something I forgot what it was).

I don't know if any of this makes sense, I have the procedure right next to me and we are going to do the lab and he told use to KNOW what we were doing and to not just follow the procedures. If you want to know one of the steps or if something is not clarified just tell me, I really want to know what we are doing!

Last edited by Alvin719; May 24th 2010 at 11:06 PM. Reason: spelling mistake
   
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Re: Biology Help - May 24th 2010, 11:14 PM

We did this lab!! I can't find MY lab report, but I have my friends on my computer... which if I send it to you, it would help a ton. But I'll copy some of it to help?
Quote:
Abstract:
This lab focused on implementing a pre-constructed plasmid containing beta lactamase, GFP, and an arabinose operon into an E.Coli cell. Furthermore, it was observed how this plasmid contributed to the growth and proliferation of bacteria in different environments. For two of the scenarios, the bacteria were cultured without the plasmid, and were either placed in a dish with normal growing conditions or one laden with Ampicillin—an antibiotic. The other two conditions included the integration of the plasmid in the bacteria cells, and consisted of a dish with Ampicillin and one with Ampicillin and arabinose sugar. It was seen that the bacteria without the plasmid produced a lawn in normal conditions, but grew only in colonies in the Ampicillin condition. As for the plasmid-induced bacteria, there were colonies that grew on the Ampicillin plate, and green florescent glowing colonies in the Arabinose/Ampicillin plate.

Introduction:
Escherichia coli (E.Coli) is a bacteria that primarily lives in the lower intestine of human beings, but is also one of the most commonly found strains of bacteria in the world. It is used as a model in many experiments due to its very rapid reproduction rate and its abundance in nature. Most strains of E.Coli are harmless, but there are a few exceptions (such as the strain O157:H7) that cause food poisoning in humans beings. Bacteria in general reproduce asexually by undergoing mitosis. Mitosis is the process in which a single-celled organism replicates its DNA and splits into two identical cells (called daughter cells) with equal number of chromosomes. The process of mitosis allows for bacteria to grow at rapid rates.
Scientists have found that it is possible to integrate foreign DNA into bacteria by inserting a pre-constructed, circular, double-stranded DNA called a plasmid. Plasmids replicate independently from the bacterial chromosomes, and are especially important when bacteria have to evolve to specific environmental hindrances. By creating a plasmid with modified genes and inserting it into the bacteria cell allows the bacteria to code for specifically- targeted proteins. The bacteria then reproduce and the plasmid is replicated in the process as well. The plasmid that was used in this experiment was called “pGLO” and it includes beta lactamase, an enzyme that causes ampicillin resistance, GFP (Green Fluorescent Protein), which allows for the bacteria to glow green under ultraviolet light, and an arabinose operon, which codes for the expression of GFP in the presence of the sugar, arabinose. Operons are made up of nucleotides, and are parts of the DNA that code for specific proteins. In the pGLO plasmid, the arabinose operon codes for GFP when in the presence of arabinose. It is important to note, however, that the operon does not function in the absence of arabinose. This is important for gene expression because it be wasteful for an organism to express a gene when it isn’t needed all the time.
The process of integrating the plasmid into the bacteria cell involves a process called heat shocking. When the bacteria are introduced to a rapid change from cold to hot temperatures, they react by taking in plasmids from their external environment. By placing them in an environment laden with pGLO plasmids, they would most likely integrate the pGLO plasmid into their DNA. This would mean that they would also start expressing the proteins regulated by the genes inserted in the pGLO plasmid.

Materials/Procedures:
First, four Petri dishes were handed out, each representing a different condition used to test the growth of the bacteria. The first plate had growing broth which would allow bacteria to grow normally (this would be used as the control). The second was laced with ampicillin, and would be used with bacteria that did not receive the plasmid. The third plate was the same as the second, except only bacteria with the plasmid would be grown on it. The final plate had ampicillin and arabinose, and plasmid-induced bacteria would grow on it as well. Pre-grown bacteria were extracted by using sterile extraction loops and were placed in two different types of 2 mL microtubes: One with the plasmid in it and the other without the plasmid. The bacteria in the tube with the plasmids were heat shocked for 50 seconds, and were then spread along the Petri dishes that corresponded to their desired condition. The bacteria were then given five days to grow and proliferate, and after that duration, the bacteria were examined and their growth development was recorded.

Sorry I couldn't copy the diagrams... but here is the results section:

According to the diagram above, it can be seen that the bacteria grew normally under optimal conditions (as illustrated in the LB plate). However, when given ampicillin, the bacteria only grew in colonies in the LB/Amp- plate. The bacteria that took in the plasmid in the LB/Amp + plate grew in colonies as well. It is interesting to note that there should have been no bacterial growth in the LB/Amp plate which had bacteria with no plasmid. If ampicillin is an antibiotic, it should have killed all the bacteria on the LB/Amp - plate. Therefore, there must have been a source of error in spreading out the ampicillin in the LB/Amp - plate. Finally, in the LB/Amp/Ara + plate, it was observed that the bacterial colonies that grew with the integrated plasmid glowed green. The arabinose must have contributed to the activation of the expression of the GFP protein due to the lack of glow in the LB/Amp + plate.
Discussion/Conclusion:
In this lab, it was hypothesized that the bacteria that took in the pGLO plasmid would be resistant to ampicillin, and with the presence of arabinose, would glow green as well. This hypothesis was supported in this lab. The control that was used in the experiment was the plate that consisted of only the growing broth. The bacteria grew into a lawn because there was no ampicillin that would hinder its reproduction. However, there was still colonial growth in the plate that had ampicillin with bacteria that did not have the plasmid. This went against the hypothesis, but the only reason for this result was the fact that the ampicillin wasn’t well distributed around the plate. If the ampicillin reacted with the bacteria, the bacteria would have not been able to reproduce, and there wouldn’t have been colonies seen on the plate. However, since there were colonies, there must have been a lack of interaction between the ampicillin and the bacteria. As for the same dish, instead with the pGLO-induced bacteria, the bacteria grew in colonies because not all of the bacteria were able to take in the plasmid. This is simply because the process of heat shocking bacteria doesn’t always work perfectly. The few bacteria the successfully took in the pGLO plasmid were able to reproduce, this producing small visible colonies on the LB/Amp + plate. Finally, the last plate was observed to have similar results as LB/Amp + plate, except for the fact that the bacteria colonies glowed green. The plasmid had an arabinose operon, which would express GFP only in the presence of arabinose. There was no arabinose in the LB/Amp + plate-- only in the LB/Amp/Ara + plate. It can be concluded that the GFP could only be expressed when arabinose was present in the environment. This is because the arabinose operon can only express GFP if arabinose binds to its receptors. Without arabinose, the GFP would not be expressed, and the bacteria would not glow, as seen in the LB/Amp + plate.
There were many potential sources of error in this lab. Firstly, the labels of the Petri dishes could have been incorrect, which could have easily misled the results. Also, when the bacteria was extracted from the pre-grown dish in the beginning of the experiment, they may have not been transferred completely into the microtubes. Since there may have not been any bacteria present in the tubes, this would account for the lack of growth that may have been observed in some of the dishes. The ampicillin on the dishes may have not been distributed very well, which accounts for the fact that there was colonies on the LB/Amp – dish. Finally, when the time in which the bacteria were being heat shocked was not exact, and could have affected the number of bacteria cells that took in the plasmid.
All in all, it can be concluded that bacterial transformation can affect the fitness of bacteria in different situations. In this case, bacteria became resistant to an antibiotic—a chemical that is supposed to induce reproduction. This lab also shows how a genetically engineered plasmid can successfully be implemented in a natural organism’s DNA. This is seen in today’s world when certain strains of bacteria are bred to produce targeted proteins such as those used in pharmaceuticals.



   
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Re: Biology Help - May 24th 2010, 11:37 PM

Thanks Amy for the help I appreciate it
   
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Re: Biology Help - May 24th 2010, 11:39 PM

Before explaining it, I'll say what the things are that you're using are for so you can better understand the lab. First, the bacteria you're using is E.coli (I know this based on the things you're using and it being commonly used in labs). I'll just give really simple definitions so not to confuse.

LB is a lysogeny broth and it's used for the lsyogenic cycle as a way for viral reproduction.
AMP is for adenosine monophosphate, which will become cyclic AMP (cAMP). cAMP is used for tons of things, such as forming ATP, which can later be broken into ADP + Pi (inorganic phosphate) to release energy. If you've learned of the mitochondria, then you'll know what ATP is for.
ARA is the L-arabinose operon for E.coli and you must know this to understand your lab. I'm not sure how much you've learned already but the idea is certain enzymes will convert arabinose into xyulose (not important to know this name probably) and you will then encounter NADP and NADPH via the phosphogluconate pathway. ARA "turns on" because it has a promoter region as well as being an operon, and as you know the promoter region (called araI for ARA) is where transcription will occur.

The plasmid solution that you're putting into the vile of +DNA serves as a means for cell replication by replicating parts of DNA strands.

So, the goal I think is to replicate the E.coli DNA and examine the effects of putting it into a "food" (LB), AMP probably for cAMP and thus cellular respiration, and ARA for transcription. The -DNA is a "control" in the sense that it's without plasmids and you can compare how the +DNA in each of the three indepedent variables affects the E.coli (dependent variables).

I believe that varying the temperature is probably to restrict some of the growth of the E.coli colony and may degenerate some of the proteins so as to make the effects occur slower so they're more visible for observation. It's an error you must include in each dependent variable.

I would also look up "super-bugs" if you haven't already learned of them because that will be the other goal of this lab.
   
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Re: Biology Help - May 24th 2010, 11:46 PM

Quote:
Originally Posted by WOW!USaidSomethingSmart! View Post
Before explaining it, I'll say what the things are that you're using are for so you can better understand the lab. First, the bacteria you're using is E.coli (I know this based on the things you're using and it being commonly used in labs). I'll just give really simple definitions so not to confuse.

LB is a lysogeny broth and it's used for the lsyogenic cycle as a way for viral reproduction.
AMP is for adenosine monophosphate, which will become cyclic AMP (cAMP). cAMP is used for tons of things, such as forming ATP, which can later be broken into ADP + Pi (inorganic phosphate) to release energy. If you've learned of the mitochondria, then you'll know what ATP is for.
ARA is the L-arabinose operon for E.coli and you must know this to understand your lab. I'm not sure how much you've learned already but the idea is certain enzymes will convert arabinose into xyulose (not important to know this name probably) and you will then encounter NADP and NADPH via the phosphogluconate pathway. ARA "turns on" because it has a promoter region as well as being an operon, and as you know the promoter region (called araI for ARA) is where transcription will occur.

The plasmid solution that you're putting into the vile of +DNA serves as a means for cell replication by replicating parts of DNA strands.

So, the goal I think is to replicate the E.coli DNA and examine the effects of putting it into a "food" (LB), AMP probably for cAMP and thus cellular respiration, and ARA for transcription. The -DNA is a "control" in the sense that it's without plasmids and you can compare how the +DNA in each of the three indepedent variables affects the E.coli (dependent variables).

I believe that varying the temperature is probably to restrict some of the growth of the E.coli colony and may degenerate some of the proteins so as to make the effects occur slower so they're more visible for observation. It's an error you must include in each dependent variable.

I would also look up "super-bugs" if you haven't already learned of them because that will be the other goal of this lab.
Thanks, but I have a question. Why do I have to put it in ice? I understood that he said that putting it in the water bath with make the cell membrane permeable, but what is the point of putting it in ice, water bath, back to ice, and then let it stand in room temperature for 10 minutes? Thanks
   
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Re: Biology Help - May 25th 2010, 06:10 AM

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Originally Posted by Alvin719 View Post
Thanks, but I have a question. Why do I have to put it in ice? I understood that he said that putting it in the water bath with make the cell membrane permeable, but what is the point of putting it in ice, water bath, back to ice, and then let it stand in room temperature for 10 minutes? Thanks
Keeping the E.coli in the ice bath decreases the membrane channel activity. For your experiment, you have to get each of the variables (LB, AMP and ARA) inside the cell, so one way is to create small holes in the membrane using chemicals. I'm sure you're going to do this or your teacher will explain it but one way is to use CaCl2. When you rupture the membrane, the contents inside the cell may leak so the ice bath prevents this.

When you put it into the water bath, the membrane undergoes heat shock so the previously made holes will seal.

When you return the cells to the ice bath, you decrease the activity of the channels and prevent the heat shock from causing too much damage. Remeber that the membrane is 3-D so the water will affect all of the membrane, meaning some parts can get denatured so the ice bath serves as a buffer in this sense.

Returning the E.coli to room temperature allows for it to be at around 37C, which is somewhere around 100F. E.coli lives inside you and this temperature is the optimal temperature for it to grow, so when you introduce each sample to each platelet of LB, AMP and ARA, you want the E.coli to be at maximal effeciency for the effects to take place.

I guess an analogy of it would be someone whose heart has stopped beating (the ice bath). When you try to use the paddles, you attempt to bring the person back (the water bath) but don't want to keep using the paddles when the person's heart is beating (the ice bath acting as a buffer). Then, you bring them to normal body temperature, stabalize them and give any necessary medications (room temperature). That's the way I think of it and at each step of the way, I think of how the microbiology will lead to the analogical goal.
   
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Re: Biology Help - May 25th 2010, 11:06 PM

Quote:
Originally Posted by WOW!USaidSomethingSmart! View Post
Keeping the E.coli in the ice bath decreases the membrane channel activity. For your experiment, you have to get each of the variables (LB, AMP and ARA) inside the cell, so one way is to create small holes in the membrane using chemicals. I'm sure you're going to do this or your teacher will explain it but one way is to use CaCl2. When you rupture the membrane, the contents inside the cell may leak so the ice bath prevents this.

When you put it into the water bath, the membrane undergoes heat shock so the previously made holes will seal.

When you return the cells to the ice bath, you decrease the activity of the channels and prevent the heat shock from causing too much damage. Remeber that the membrane is 3-D so the water will affect all of the membrane, meaning some parts can get denatured so the ice bath serves as a buffer in this sense.

Returning the E.coli to room temperature allows for it to be at around 37C, which is somewhere around 100F. E.coli lives inside you and this temperature is the optimal temperature for it to grow, so when you introduce each sample to each platelet of LB, AMP and ARA, you want the E.coli to be at maximal effeciency for the effects to take place.

I guess an analogy of it would be someone whose heart has stopped beating (the ice bath). When you try to use the paddles, you attempt to bring the person back (the water bath) but don't want to keep using the paddles when the person's heart is beating (the ice bath acting as a buffer). Then, you bring them to normal body temperature, stabalize them and give any necessary medications (room temperature). That's the way I think of it and at each step of the way, I think of how the microbiology will lead to the analogical goal.
thank you very much you explain in a lot of detail.
   
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