hanzuniga
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Bacteria Transformation Efficiency

Postby hanzuniga » Wed Oct 22, 2014 11:42 am

I have a question on the purpose of the Bacterial Transformation Efficiency, what exactly is the experiment trying to figure out???? Is there a hypothesis to test for?

MadelineB
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Re: Bacteria Transformation Efficiency

Postby MadelineB » Wed Nov 05, 2014 9:44 pm

Hello Hanzuniga,
Bacterial transformation is an exciting process. The background section of this project idea
http://www.sciencebuddies.org/science-f ... background should help you understand the purpose of bacterial transformation efficiency. You can find additional information by following the links shown in the Bibliography.

The Science Buddies Project Guide is a good place to look for help in figuring out what hypotheses can be tested.
http://www.sciencebuddies.org/science-f ... ndex.shtml. In particular, look at this link:
http://www.sciencebuddies.org/science-f ... esis.shtml

Be sure to let us know if you have additional questions!
Madeline B

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Re: Bacteria Transformation Efficiency

Postby connief » Sat Nov 08, 2014 2:47 pm

Hello Hanzuniga,

Looking at the project procedures, it seems like this project is trying to figure out what factors can affect bacteria transformation efficiency, and they are specifically focusing on how the amount of DNA used affects the efficiency. Hence, the hypothesis would be how you predict this factor would affect transformation efficiency. For example, if you use higher amounts of DNA, do you think the transformation would work better or worse than when you use lower amounts? The previous expert sent you some very helpful links on the scientific method, so you should definitely take a look at those.

In addition, as the previous expert mentioned, the background section of the project does a pretty good job describing why it's important to study bacterial transformation efficiency. We can use bacteria to produce medically important proteins or engineer bacteria to make proteins that can help us clean up environmental spills, for example. Improving bacterial transformation efficiency is also very important for microbiologists. For example, when we study our bacteria of interest, sometimes we want to express tags in the bacteria in order to track them in our experiments. In order to express these tags in the bacteria, we need to be able to transform them. There are many other reasons why bacteria transformation efficiency is important to study, and you should definitely do some more research on that if you are interested in learning more!

Let us know if you have any other questions.

Connie

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Re: Bacterial Transformation Efficiency - Arnlan

Postby arnlan » Sun Nov 16, 2014 11:33 am

Hey,

My hypothesis is : If I increase the amount of pGlo plasmid 10X, then the number of bacterial colonies will increase.
I have read the procedures and i can't determine how my results is going to answer my hypothesis.

I need help with my problem and purpose. Currently this is what i have.

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.

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.

Are these good pertaing to the focus of this project?

Thanks :D
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Best,
-Arnlan

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Re: Bacteria Transformation Efficiency

Postby connief » Sun Nov 16, 2014 12:41 pm

Hi there,

I think the purpose and problem that you have written up sounds really great! Good job! That is also a reasonable hypothesis for this project. As far as I see for the procedures, it should answer your hypothesis. You're going to start off with two bacterial cultures that should be around the same numbers, and for one, you're going to be transforming with 1x plasmid, whereas the other culture, you'll transform with 10x plasmid. Afterwards, you'll plate out your transformants and count the number of colonies. If your hypothesis were true, there should be more colonies on the plates that were plated with bacteria that were transformed with the 10x plasmid compared to the plates that were plated with bacteria that were transformed with the 1x plasmid. In other words, the more efficient the transformation is, the more colonies should grow.

Let me know if you have more questions!

Connie

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Re: Bacteria Transformation Efficiency

Postby arnlan » Sun Nov 16, 2014 12:55 pm

connief wrote:Hi there,

You're going to start off with two bacterial cultures that should be around the same numbers, and for one, you're going to be transforming with 1x plasmid, whereas the other culture, you'll transform with 10x plasmid. Afterwards, you'll plate out your transformants and count the number of colonies. If your hypothesis were true, there should be more colonies on the plates that were plated with bacteria that were transformed with the 10x plasmid compared to the plates that were plated with bacteria that were transformed with the 1x plasmid. In other words, the more efficient the transformation is, the more colonies should grow.



What do you mean by bacterial cultures? Is it as in the E. coli? When i plate out the transformants, how many different plates do i need for the 1x and the 10x?
I have been watching videos on youtube pertaining to this project. Is this what i will be doing in the experiment : https://www.youtube.com/watch?v=c40UudFIlGw?

I will be doing my experiment in a university starting this thursday. So i'm trying to get all the concepts i have not gotten yet.

Thanks for your help,
-Arnlan
-Arnlan
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Best,
-Arnlan

connief
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Re: Bacteria Transformation Efficiency

Postby connief » Sun Nov 16, 2014 1:14 pm

Hi there,

Yes, by the bacteria I mean E. coli. All you need to know is written in the procedures. The procedures mention that you will plate 100 microliters of each transformation reaction on each LB:AMP plate, and you're going to have 2 plates for each transformation reaction (no plasmid, 1x plasmid, 10x plasmid), meaning there will be 6 total. You will count the colonies on each plate, and for each transformation, average the number of colonies on the two plates to get your final counts. That video does a pretty good job showing you how to go through with the procedure.

Let us know if you have more questions.

Connie

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Re: Bacteria Transformation Efficiency

Postby arnlan » Sun Nov 16, 2014 1:21 pm

Ohhh that helps alot.

In my project i will also be testing the part for the visualization of the transformation kit. So will i need to add one more plate for each? To be 3 plates for each? For LB:AMP:ARA?

You mentioned that i will need 2 plates for each, can you explain to me the difference between the 2 plates?
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-Arnlan

connief
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Re: Bacteria Transformation Efficiency

Postby connief » Sun Nov 16, 2014 1:37 pm

Hi,

The procedures mention 2 plates. It's just 2 LB:AMP plates. The reason why you have 2 plates is because they want you to average the colony counts you get on those two plates to get your final numbers to account for any variability. The counts on each of the 2 plates for a specific transformation should have very similar numbers. Does that make sense? The LB:AMP:ARA plates are not necessary for you to determine efficiency in this project. It's just a nice way of confirming that your transformation worked and the bacteria took in the plasmid, since the bacteria can be induced to express a gene encoded on the plasmid if you plate them on LB:AMP:ARA. So 1 plate for each reaction sounds good. You would expect the colonies from the bacteria transformed with 1x or 10x plasmid to turn green when you visualize them under the UV light they provided, whereas the bacteria transformed with "no plasmid" shouldn't be green at all, since they didn't receive any plasmid in the first place. Does all of that make sense?

Connie

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Re: Bacteria Transformation Efficiency

Postby arnlan » Sun Nov 16, 2014 1:52 pm

connief wrote:Hi,

The procedures mention 2 plates. It's just 2 LB:AMP plates. The reason why you have 2 plates is because they want you to average the colony counts you get on those two plates to get your final numbers to account for any variability. The counts on each of the 2 plates for a specific transformation should have very similar numbers. Does that make sense?
Connie

But the procedures mentions 2 different types of plates. The LB and LB : AMP.


connief wrote:
The LB:AMP:ARA plates are not necessary for you to determine efficiency in this project. It's just a nice way of confirming that your transformation worked and the bacteria took in the plasmid, since the bacteria can be induced to express a gene encoded on the plasmid if you plate them on LB:AMP:ARA. So 1 plate for each reaction sounds good. You would expect the colonies from the bacteria transformed with 1x or 10x plasmid to turn green when you visualize them under the UV light they provided, whereas the bacteria transformed with "no plasmid" shouldn't be green at all, since they didn't receive any plasmid in the first place. Does all of that make sense?

Connie


This part does not really make sense. So should i have a 3rd plate for LB:AMP:ARA?
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-Arnlan

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Re: Bacteria Transformation Efficiency

Postby connief » Sun Nov 16, 2014 2:16 pm

The LB plates are just made from nutrient agar. You use these plates to streak out the bacteria and allow them to grow up before you pick single colonies from them to do the transformation. The LB:AMP plates are made from nutrient agar with the addition of an antibiotic called ampicillin. These plates are used to plate the bacteria after they are transformed to select for the bacteria that got the plasmid because the plasmid encodes a gene that allows the bacteria to acquire ampicillin resistance. Hence, after the transformation, bacteria that didn't take up the plasmid will never grow on the LB:AMP plates, and only bacteria that took up the plasmid will grow on the LB:AMP plate. AGAIN, they ask you to plate on 2 LB:AMP plates because they want you to take the average of the colony counts on both plates to give you the final number to account for any variability. Is this still not clear or do I need to explain more?

The reason why the LB:AMP:ARA plates were mentioned is because the plasmid that you're using to transform encodes for a green fluorescent protein, which when expressed by the bacteria, allows them to glow green. This gene is engineered to only be expressed when the bacteria are exposed to a sugar called arabinose. Hence, the LB:AMP:ARA plates (ARA stands for arabinose). Plating your transformants on these plates will only help you further confirm that the bacteria took up the plasmid because in the presence of arabinose, if the bacteria GOT the plasmid, then they should be expressing green fluorescent protein and should glow green. Honestly, this part is unnecessary for the purpose of testing your hypothesis, but it's just another cool technique to confirm that your transformation worked. Does that make sense or is it still confusing?

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Re: Bacteria Transformation Efficiency

Postby arnlan » Sun Nov 16, 2014 2:37 pm

connief wrote:The LB plates are just made from nutrient agar. You use these plates to streak out the bacteria and allow them to grow up before you pick single colonies from them to do the transformation. The LB:AMP plates are made from nutrient agar with the addition of an antibiotic called ampicillin. These plates are used to plate the bacteria after they are transformed to select for the bacteria that got the plasmid because the plasmid encodes a gene that allows the bacteria to acquire ampicillin resistance. Hence, after the transformation, bacteria that didn't take up the plasmid will never grow on the LB:AMP plates, and only bacteria that took up the plasmid will grow on the LB:AMP plate. AGAIN, they ask you to plate on 2 LB:AMP plates because they want you to take the average of the colony counts on both plates to give you the final number to account for any variability. Is this still not clear or do I need to explain more?


This is clear now. I just got confused on that. So in my experiment i will need 6 plates.

connief wrote:The reason why the LB:AMP:ARA plates were mentioned is because the plasmid that you're using to transform encodes for a green fluorescent protein, which when expressed by the bacteria, allows them to glow green. This gene is engineered to only be expressed when the bacteria are exposed to a sugar called arabinose. Hence, the LB:AMP:ARA plates (ARA stands for arabinose). Plating your transformants on these plates will only help you further confirm that the bacteria took up the plasmid because in the presence of arabinose, if the bacteria GOT the plasmid, then they should be expressing green fluorescent protein and should glow green. Honestly, this part is unnecessary for the purpose of testing your hypothesis, but it's just another cool technique to confirm that your transformation worked. Does that make sense or is it still confusing?


I'm still kinda confused. So do i still have 2 plates per plasmid or do i add 1 more to each for the AMP?
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Best,
-Arnlan

connief
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Re: Bacteria Transformation Efficiency

Postby connief » Sun Nov 16, 2014 2:43 pm

What exactly are you confused about? The number of plates to use? You just need 1 LB:AMP:ARA plate for each transformation reaction. The point of this is to see if you seen green colonies. There is no counting involved whatsoever like with the LB:AMP plates. If you are still confused, then just don't use the LB:AMP:ARA plates. That will make things easier.

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Re: Bacteria Transformation Efficiency

Postby arnlan » Sun Nov 16, 2014 3:06 pm

OHH that makes alot of sense. So in the end 9 plates will be used right?
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-Arnlan

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Re: Bacteria Transformation Efficiency

Postby connief » Sun Nov 16, 2014 3:13 pm

Yup, exactly! 2 LB:AMP and 1 LB:AMP:ARA for each transformation reaction!

Let me know if anything else is confusing.


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