I'm arnlan, i have been researching this project the whole summer. I literally know every background research information about this experiment. But! I have 3 questions : What is the dependent variable, independent variable, and constant concerning this project This always stumps me on every single project i have done. Its better to get it out as soon as possible
It's great that you've been working so hard on learning about this project. I'm not going to tell you what the dependent and independent variables, because I think it will help you to really think about it on your own, so let's see if I can give you some hints and get you there. Remember that the independent variable is the thing that YOU are changing/manipulating in your experiment. So look carefully at your procedure for this experiment. You'll have three tubes: −pGLO plasmid (negative control), +1× pGLO plasmid, and +10× pGLO plasmid. What is different between the tubes? What are you changing from the second tube to the third one? For the dependent variable, what outcome are you measuring/counting?
I found the objective statement of your experiment, "The goal of this project is to measure bacterial transformation efficiency as a function of plasmid DNA concentration." to be helpful in thinking about this, as well.
As for your constants, remember that a good experiment only tests one variable (your independent variable). In order to determine the effect of that variable alone, everything else has to remain constant. So basically, once you know your independent and dependent variable, everything else should be your constants.
You're about half way there. The dependent variable is what you are measuring, which is the efficiency of the bacterial transformation, just as you said. Now, with regards to the independent variable, remember that it is what you are changing. You are changing something ABOUT the pGlo plasmid in each sample. Can you read through the protocol again and figure out what that is? Remember that in one tube you are using no plasmid, in the next you are using 1X pGlo plasmid, and in the final tube you are using 10X pGlo plasmid. What did you change?
As for your constants, you are right that ideally everything else would be constant, but can you come up with a few specific examples? These are things that you are keeping the same between each of your tubes.
You're getting there. Keep trying and posting with questions.
JMP
Very good Arnlan. I would absolutely define the independent variable as the amount of the pGlo plasmid that you are using. Remember that you are interested in how the amount of pGlo plasmid you use affects bacterial transformation efficiency, so you are in fact changing the amount of plasmid used, and thus that is your independent variable. What you are measuring is your dependent variable, so technically you are counting the number of colonies on the plate, but you are using that as a measure of transformation efficiency, so I think you are fine calling either the transformation efficiency or the number of colonies your dependent variable, so good job.
As for the control, it's absolutely true that a tube with no plasmid is a control for your experiment. Without plasmid, you shouldn't get any colonies, so if you do, then you know that something has gone wrong with your experiment, and you can't trust the rest of your results. But you were really asking more about your constants, and constants are something that you keep the same between every part of your experiment, so in this case, I would consider things like temperature, the amount of bacteria per tube, and stuff like that your constants.
I hope this conversation has helped and that you feel a little more comfortable thinking about variables, controls, and constants. Please post again if you still have questions, but I think you're really starting to get it.
JMP
It's absolutely okay to mention more than one constant. In fact, I would encourage it. A well designed experiment will probably have many things kept constant, so go ahead and come up with as many as you can.
Great job working on your hypothesis. I only have one comment about it. You have framed your hypothesis that, "if the amount of pGlo plasmid changes, you will get more colonies on the plate" but you didn't specify whether you are increasing or decreasing the amount of pGlo plasmid, so it sounds like you expect either more plasmid or less plasmid to increase the colonies on the plate. Is that what you are hypothesizing and testing? In this procedure, you are only increasing the pGlo plasmid from 1X to 10X, so you won't be testing what happens if you were to decrease the amount of plasmid. Thus, that shouldn't be included in your hypothesis. If you just changed your hypothesis to, "if the amount of pGlo plasmid increases, then there will be more colonies on the petri dish" or even more specifically, "if I increase the amount of pGlo plasmid 10X, the number of bacterial colonies will increase."
JMP wrote:
"if I increase the amount of pGlo plasmid 10X, the number of bacterial colonies will increase."
JMP
I have a question about that. What is the difference of plasmid 1x, plasmid 10x, and plasmid (0)? Does plasmid 1x contain more plasmid than plasmid (0), and does plasmid 10x contain more plasmid than plasmid 1x? If so, do all of the 3 plasmid tubes contain the same amount of liquid (example: are all of them 1ml of plasmid?)
If i do increase the amount of the pglo plasmid, wouldn't that set a disadvantage on the other 2 plasmid groups?
Did you carefully read through the procedures on the Bacterial Transformation Efficiency page? I think it mostly answers your question:
Add 10 μL of the pGLO plasmid solution to the tube labeled +1× pGLO plasmid. (If you don't have access to automatic pipettors, 10 μL is one sterile loop full.)
Add 100 μL of the pGLO plasmid solution to the tube labeled +10× pGLO plasmid.
Mix by covering and flicking the tubes.
Do not add pGLO plasmid DNA to the −pGLO plasmid (negative control) tube.
The plasmid (0) tube does NOT contain any plasmid, and that's actually the point. This tube serves as your negative control. You should not be able to grow any colonies on the plate that didn't have plasmid, because none of the bacteria should have been transformed. If you DO get colonies on that plate, you'll know that something didn't work properly, and you can't trust any of your results and should repeat the experiment. This is the sort of internal control that it is necessary to include in all experiments when possible.
The plasmid (1) tube will contain 10uL of the plasmid solution, which they are considering a "normal" amount of plasmid. The plasmid (10) tube will contain 10 times as much plasmid as you added the plasmid (1) tube. You are right that ideally you would increase the amount of plasmid without increasing the total volume the way you are here (remember in the plasmid(1) tube you add 10uL of plasmid, while in the plasmid(10) tube you add 100uL), because technically you are introducing two variables here: 1) the amount of plasmid and 2) the overall volume of the solution. That said, in this circumstances, the change in volume probably won't be too much of an issue.
As for whether
If i do increase the amount of the pglo plasmid, wouldn't that set a disadvantage on the other 2 plasmid groups?
, isn't that kind of the point? You have hypothesized that increasing the amount of plasmid will increase your transformation efficiency (i.e. the number of colonies on the plate in the end), so you can't test that without increasing the amount of pGLO plasmid. It's not a disadvantage; it's merely testing your hypothesis. And I'll tell you from personal experience, that there is no guarantee that what you hypothesized is correct, either. It is certainly a very valid hypothesis, but bacterial transformation actually requires a careful balancing act, and there is such a thing as adding too much plasmid, so you won't actually know whether your hypothesis is true until you test it (which is kind of the point of science anyway!)
Do you understand the experiment you are going to do better now? I hoped this help.
JMP
I'm not really sure what you mean when you say two different projects. You currently have an experiment with three different conditions: no plasmid, normal amount of plasmid, 10 times the normal amount of plasmid. This tests the hypothesis you formulated that is "if I increase the amount of plasmid, I'll have increased transformation efficiency (i.e. more colonies on the plate). If you break this up into two different projects, by which I assume you mean experiments, then what are you testing? What is your hypothesis for each "project"?
JMP wrote:
"if I increase the amount of pGlo plasmid 10X, the number of bacterial colonies will increase."
I now understand! I will use the hypothesis quoted above.
I still have 1 question. If i do increase the amount of pglo plasmid 10x, will the volume increase? Or will only the amount of plasmid increase. Also how much would i increase it by? (example 10x,20x,30x?)
I'm still kinda shaky on it, but with you're unconditional help, i think i'll understand.
Have you read the procedure for this experiment carefully? I think that it will answer your question.
Day 2: Transforming the Bacteria
Important: do not refrigerate the starter plate prior to transformation.
Label 3 tubes with the following:
−pGLO plasmid (negative control),
+1× pGLO plasmid,
+10× pGLO plasmid.
Using a graduated pipette add 1 mL of the transformation solution to a clean tube.
With a sterile loop, choose 4 well-separated colonies from the starter plate and resuspend in the tube containing 1 mL of transformation solution by flicking the tube or by twirling the loop around in the solution. (Note that each colony contains millions of bacterial cells—do not use too many colonies).
When the colonies are completely resuspended, use a clean graduated pipette to transfer 250 μL of the bacterial solution to each of the 3 tubes labeled −pGLO plasmid, +1× pGLO plasmid, and +10× pGLO plasmid. Add 10 μL of the pGLO plasmid solution to the tube labeled +1× pGLO plasmid. (If you don't have access to automatic pipettors, 10 μL is one sterile loop full.) Add 100 μL of the pGLO plasmid solution to the tube labeled +10× pGLO plasmid.
Mix by covering and flicking the tubes.
Do not add pGLO plasmid DNA to the −pGLO plasmid (negative control) tube.
Incubate the 3 tubes on ice for 15 minutes.
Heat shock the 3 tubes at 42°C for 50 seconds exactly.
Immediately place tubes on ice for 2 minutes.
Add 250 μL of LB broth to each of the three transformation tubes and incubate at room temperature for 10 minutes. Use a clean pipette for each addition.
Spread 100 μL of bacterial suspension on to the appropriate LB:AMP plates, use 2 plates per transformation. Use the average number of colonies from the two plates in subsequent calculation.
Incubate the plates overnight at 37°C (or at room temperature for 2 to 3 days).
Read the section I bolded and see that you will be adding more volume when you increase the amount of pGlo plasmid. In fact, you'll add 10X more volume (100uL into 10X divided by 10uL into 1X = 10).
I do understand that, but my question is how is that going to be related to my hypothesis. I just don't see the comparison. If i do increase the the pglo plasmid 10x, how much do i increase it by. When i think about it, it sets a disadvantage to the other amounts of plasmid. Thats the only part i'm getting stuck on. The procedures are clear as they can be.
I'm having trouble understanding where you are getting stuck. Increasing the pglo plasmid 10x means increasing it 10 times or 10 fold. For example, if you had a starting concentration of 1mg/mL, a 10X increase would give a concentration of 10mg/mL. If I started with 2mg/mL, my 10X concentration is 20mg/mL. You are increasing the plasmid by an order of 10. Is that clear?
In an ideal situation, you would add the same VOLUME of plasmid, but your plasmid would be 10 times more concentrated in the pGlo 10X condition. Since you only have one concentration of plasmid available to you (from the kit), instead you are increasing the VOLUME that you add 10 times. Instead of adding the 10uL (microliters, a measure of volume) that you add to the 1X condition, you add 100uL (10uL x 10). Therefore, your final amount of pGlo plasmid in the 10X condition will be 10 times that in the 1X condition. Does that make sense?
I still don't understand what you mean when you say,
When i think about it, it sets a disadvantage to the other amounts of plasmid
. The entire point of the experiment (i.e. your hypothesis) is to test whether adding more plasmid will lead to more colonies. You can't do that while keeping the amount of plasmid added equal, right? Whether more plasmid turns out to be an advantage or a disadvantage remains to be seen, but that is why you are testing it.
Did that help clarify everything or is there still something I'm not explaining well?
JMP
So my hypothesis is "if I increase the amount of pGlo plasmid 10X, the number of bacterial colonies will increase." So as i'm reading the procedures right now i see that there are three tubes. PGLO plasmid, 1x PGLO Plasmid, 10x PGLO Plasmid. This is where i get confused with the hypothesis. The procedures are telling me to put 10 x PGLO Plasmid, while the hypothesis is telling me to increase the amount of 10x PGLO plasmid. Am i missing anything in between? In the procedures i see that you had 10 ul to the 1x PGLO plasmid and 100 ul to the 10x PGLO plasmid. So if i do increase the amount of PGLO plasmid 10x (100 ul), how will i know the difference between the 10x Pglo Plasmid, and the amount increased. I'm really confusing my self!!
Okay, I think I might see the confusion now. pGlo plasmid is the name of the plasmid. 1X pGlo plasmid and 10X pGlo plasmid are exactly the same plasmid. This is just the way the procedure is marking the increased AMOUNT of plasmid.
Basically:
in the 1X pGlo plasmid you are adding 10uL of the pGlo plasmid
in the 10X pGlo plasmid you are adding 100uL of the pGlo plasmid
the 1X and 10X don't signal a change in the plasmid; they are simply being used to refer to the experimental condition where you add the "normal" amount of plasmid (1X) and the "increased" amount of plasmid (10X). There will only be one tube in the kit labeled pGlo plasmid that you will use for all of your different conditions. From that one tube, you will create two different conditions depending on how much of the plasmid you add (10uL for 1X, 100uL for 10X). The other condition you mention (a tube labeled pGlo plasmid) is actually being labeled –pGlo plasmid. That dash in the front stands for negative. That tube is the condition where you DON'T add pGlo plasmid and will serve as your negative control.
So in the end, you'll have three tubes:
-pGlo plasmid: 0uL of pGLo plasmid
1X pGlo plasmid: 10uL of pGlo plasmid
10X pGlo plasmid: 100uL of pGlo plasmid
The plasmid itself is the same each time (pGlo plasmid); the ONLY thing you are changing is the amount of plasmid that you are adding to your tubes.
You are absolutely correct, that if you were changing both the plasmid and the amount of the plasmid you would have a very bad experiment, so good job recognizing that.
Did that clarify it?
Don't worry, we'll get there.
JMP
In what i 'm seeing, you would have to have 2 experiment. 1 of the with the normal amount and the other is the increased amount of pglo plasmid.
Test 1:
-pGlo plasmid: 0uL of pGLo plasmid
1X pGlo plasmid: 10uL of pGlo plasmid
10X pGlo plasmid: 100uL of pGlo plasmid
Test 2:
-PGlo plasmid: 0ul of pGLo plasmid.
1x PGlo plasmid: 10ul of pGlo plasmid
10x pGlo plasmid: 1000ul of pGlo plasmid.
According to the hypothesis
if I increase the amount of pGlo plasmid 10X, the number of bacterial colonies will increase.
I think that test 1 and test 2 will produce the correct data. Because since you are adding pGlo plasmid 10x, you can see the difference between teh 100ul and 1000ul. Also to make the experiment more complicated i could test the 1x pGlo plasmid too.
I don't understand why you think you need two experiments.
In test 1 you have everything you need to see if increasing the amount of pGlo plasmid 10 times will increase the number of colonies.
Three conditions:
-pGlo plasmid: 0uL of pGlo plasmid
1X pGlo plasmid: 10uL of pGlo plasmid
10X pGlo plasmid: 100uL of pGlo plasmid
These are equivalent to your:
negative control condition (-pGlo plasmid: 0uL of pGLo plasmid)
"normal" condition (1X pGlo plasmid: 10uL of pGlo plasmid)
"increased amount" of pGlo plasmid (10X pGlo plasmid: 100uL of pGlo plasmid).
The 10X pGLo plasmid in test 1 IS your "increased amount" condition.You don't need to increase it even further in a second test in order to test your hypothesis.
Will it be fine if i put this as my problem ? : It can be very expensive to chemically synthesize very short peptides, never mind complex polypeptides and whole proteins which may have post-translational modifications. As the biology of bacteria becomes clearer, coupled with the abundance of bacterial species and strains available and the exciting advances made in molecular biology research and biotechnology, the possibilities and applicability of transformation becomes phenomenal.
Also, will it be ine if i put this as my purpose? :Transformation in and of itself is a very important basic tool in molecular biology. Transformation is used for cloning or to move DNA molecules around between strains. Bacteria are transformed for numerous different reasons. Some of these reasons may include expression of medically useful recombinant proteins such as insulin for treating a disease or vaccines for prevention of disease. Other reasons could be expression of proteins that confer on bacteria the ability to survive in particular environments such as to "clean up" contaminated environments in bioremediation.
IF possible what other things should i add to the problem and purpose?
PS- i have a few more weeks before i head to the lab to experiment.
I think you have a great start in both your problem and your purpose, but that you need to tie both your problem and your purpose back to your specific experiment. You say in your problem that it is "expensive to chemically synthesize very short peptides" but how does your experiment help address that problem? What benefit do you derive by learning how the concentration of a plasmid affects the efficiency of transformation? I'm not saying it doesn't affect your problem, but spell it out for me. Don't make me have to fill in the dots.
The same thing is sort of true for your purpose. I generally think of the purpose of an experiment as the very specific reason that you are doing THIS experiment. So your purpose would be very similar to the objective statement on the experiment's information page. That is not to say that the information you discuss in your purpose isn't true, and highly relevant, and it may show up in your experimental write up (I would tend to think some sort of background section), but most of it doesn't fit in to the purpose to me, or at least, it doesn't fit without a great deal more specifics that relate specifically to YOUR question, "how plasmid concentration affects transformation efficiency"
I'm currently reviewing my pre experimental procedures with the scientist i will be experimenting with, and she said there are a few problems with the control. Currectly my control is :The tube without the plasmid/ temperature/ the amount of bacteria per tube.
This is what she had to say about the control:
Your experiment needs an appropriate control. You want to increase the amount of pGLO transfected, but if your control is bacteria that haven’t been transfected, how will you know if you improve transfection efficiency? Can you think of a more useful control in this instance? Alternatively, you could explore how different conditions affect transfection efficiency (i.e. varying temperature, pH, salt concentration, etc.). I’ll let you think about it and decide what you’d like to investigate.
What steps should i take to make this control more proper?
This always stumps me on every single project i have done. Its better to get it out as soon as possible 
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