Bacteria That Glow #GlowNation
Devansh Taori (Period Two)
Biology Advanced Placement
pGLO Laboratory Report
Introduction
The ultimate objective of this lab was to perform a genetic transformation. It is well established that gene expression is controlled by DNA. Genes are usually expressed through protein synthesis, a process where mRNA reads a sequence of DNA (transcription), processes it (usually only in eukaryotic cells), and proceeds to be transformed into a polypeptide when tRNA matches its anticodon with the codons on the mRNA (translation). Thus, if DNA can be altered in some way, then the gene expression can be altered as well. In this particular lab, we wanted to see if we could inject a sequence of DNA that codes for Green Fluorescent Protein into E. Coli bacteria. Since the specific pGLO plasmid codes for resistance to ampicillin, while the green fluorescent protein is only expressed in the presence of the sugar arabinose (a molecule which prompts transcription of the gene), the presence of the plasmid, ampicillin, and arabinose in a petri dish should prompt the expression of glowing bacteria colonies. The absence of any of those traits will yield different results and is explored within this experiment.
Hypothesis
I hypothesize that the injection of the pGLO plasmid into the E. Coli bacteria in the presence of arabinose and ampicillin will yield the expression of the Green Fluorescent Protein in sparse bacteria colonies. In addition, the absence of arabinose or the pGLO plasmid will keep the gene for GFP turned off. Furthermore, the absence of ampicillin will cause unrestricted bacteria growth as natural selection is eliminated as a factor.
Materials
- pGLO plasmid
- Transformation solution (Calcium Chloride)
- Mini test tubes
- Petri dishes (x4) with the following in each:
- LB (x1)
- LB/Ampicillin (x2)
- LB/Ampicillin/Arabinose (x1)
- E. Coli Bacteria
- Inoculation loops
Procedure
First, we wanted to inject the pGLO plasmid into the E. Coli bacteria. We put 250 micro liters of the transformation solution into two test tubes, one labeled pGLO+ and the other labeled pGLO-. This will facilitate the entry of the pGLO plasmid into the cell membrane. We then placed both tubes on ice.
Next, we placed 2-3 colonies of bacteria into the 2 mini test tubes. One tube will have the plasmid in it and the other one will not. This establishes the independent variable for the experiment.
Then, we added the pGLO plasmid into the pGLO+ tube. Using an inoculation loop, we “grabbed” some of the plasmid and mixed it into the preexisting solution. Both tubes were then incubated in the ice for 10 minutes.
Next, we performed the heat shock procedure. The purpose of this is to “soften” the cell membrane so that it is easier for the plasmid to enter the cell. We placed both tubes on a floating styrofoam holder and placed it in a 42-degree water bath for 50 seconds. Immediately after, we transferred both tubes back onto the ice for 2 minutes.
Afterwards, we placed 250 micro liters of LB nutrient into each tube. This will help the bacteria grow.
Next, using a pipette, we transferred the solutions onto the petri dishes. We placed 100 micro liters of the pGLO+ solution onto the LB/Ampicillin/Arabinose plate and 100 micro liters onto one of the LB/Ampicillin plates. We placed 100 micro liters of the pGLO- solution onto the LB plate and 100 micro liters onto the other LB/Ampicillin plate. The purpose of the four different petri dishes is to analyze the effect of each factor on the transformation process.
Results
Questions
I completed the questions in the pGLO manual directly on the worksheets. Here is a link to a PDF of it: https://drive.google.com/open?id=15RP2Ow_RDzTqmDBzZFfwM2lnoa9rze8K
Conclusion
Overall, the hypothesis was supported. As evident in the results of the experiment, the green fluorescent protein was only expressed when the plasmid, ampicillin. arabinose were all present within the petri dish. Without arabinose, the bacteria did not glow because arabinose is a necessary transcription factor to begin the expression of the green fluorescent protein. And without the plasmid, the antibiotic resistant gene is also not expressed. Therefore, the plate without the plasmid but with arabinose had no bacteria in it. Furthermore, the bacteria plate with only LB in it resulted in unrestricted bacteria growth as evident by the bacteria lawn.
Furthermore, the transformation efficiency in the LB/Amp/Ara plate was approximately 758 transformants/microgram. Though this number was on the low end of the normal transformation efficiency (in comparison to the norms stated in the worksheet), genetic transformation undoubtedly occurred.
Overall, I learned a lot about the various factors that affect gene expression. Not only does the DNA sequence need to be present, but environmental conditions can introduce additional factors that affect gene expression. For instance, a lack of a specific transcription factor can lead to the failure to express a certain gene because transcription of that gene sequence doesn’t even occur. Furthermore, new genes can be affected once a specific gene is turned on or off. In this instance, the presence of the antibiotic resistance trait is also expressed when the plasmid was introduced.
For future experiments, I would like to see the results of this experiment without ampicillin. Without ampicillin, a lawn of bacteria showed up. Perhaps introducing the plasmid without ampicillin can lead to a growing lawn of bacteria. However, without ampicillin, resistant and nonresistant cells will be competing for the same resources. This way, we can see whether the pGLO plasmid is genetically viable or offers other naturally advantageous traits besides antibiotic resistance.
Just wanted to give a special shout-out to Mr. Wong and – of course – Manas. Thank you for your support, dude. :) <3
#WongLifeIsGoodLife #HappyWongEqualsHappyDevansh
Biology Advanced Placement
pGLO Laboratory Report
Introduction
The ultimate objective of this lab was to perform a genetic transformation. It is well established that gene expression is controlled by DNA. Genes are usually expressed through protein synthesis, a process where mRNA reads a sequence of DNA (transcription), processes it (usually only in eukaryotic cells), and proceeds to be transformed into a polypeptide when tRNA matches its anticodon with the codons on the mRNA (translation). Thus, if DNA can be altered in some way, then the gene expression can be altered as well. In this particular lab, we wanted to see if we could inject a sequence of DNA that codes for Green Fluorescent Protein into E. Coli bacteria. Since the specific pGLO plasmid codes for resistance to ampicillin, while the green fluorescent protein is only expressed in the presence of the sugar arabinose (a molecule which prompts transcription of the gene), the presence of the plasmid, ampicillin, and arabinose in a petri dish should prompt the expression of glowing bacteria colonies. The absence of any of those traits will yield different results and is explored within this experiment.
Hypothesis
I hypothesize that the injection of the pGLO plasmid into the E. Coli bacteria in the presence of arabinose and ampicillin will yield the expression of the Green Fluorescent Protein in sparse bacteria colonies. In addition, the absence of arabinose or the pGLO plasmid will keep the gene for GFP turned off. Furthermore, the absence of ampicillin will cause unrestricted bacteria growth as natural selection is eliminated as a factor.
Materials
- pGLO plasmid
- Transformation solution (Calcium Chloride)
- Mini test tubes
- Petri dishes (x4) with the following in each:
- LB (x1)
- LB/Ampicillin (x2)
- LB/Ampicillin/Arabinose (x1)
- E. Coli Bacteria
- Inoculation loops
Procedure
First, we wanted to inject the pGLO plasmid into the E. Coli bacteria. We put 250 micro liters of the transformation solution into two test tubes, one labeled pGLO+ and the other labeled pGLO-. This will facilitate the entry of the pGLO plasmid into the cell membrane. We then placed both tubes on ice.
Next, we placed 2-3 colonies of bacteria into the 2 mini test tubes. One tube will have the plasmid in it and the other one will not. This establishes the independent variable for the experiment.
Then, we added the pGLO plasmid into the pGLO+ tube. Using an inoculation loop, we “grabbed” some of the plasmid and mixed it into the preexisting solution. Both tubes were then incubated in the ice for 10 minutes.
Next, we performed the heat shock procedure. The purpose of this is to “soften” the cell membrane so that it is easier for the plasmid to enter the cell. We placed both tubes on a floating styrofoam holder and placed it in a 42-degree water bath for 50 seconds. Immediately after, we transferred both tubes back onto the ice for 2 minutes.
Afterwards, we placed 250 micro liters of LB nutrient into each tube. This will help the bacteria grow.
Next, using a pipette, we transferred the solutions onto the petri dishes. We placed 100 micro liters of the pGLO+ solution onto the LB/Ampicillin/Arabinose plate and 100 micro liters onto one of the LB/Ampicillin plates. We placed 100 micro liters of the pGLO- solution onto the LB plate and 100 micro liters onto the other LB/Ampicillin plate. The purpose of the four different petri dishes is to analyze the effect of each factor on the transformation process.
Results
I completed the questions in the pGLO manual directly on the worksheets. Here is a link to a PDF of it: https://drive.google.com/open?id=15RP2Ow_RDzTqmDBzZFfwM2lnoa9rze8K
Conclusion
Overall, the hypothesis was supported. As evident in the results of the experiment, the green fluorescent protein was only expressed when the plasmid, ampicillin. arabinose were all present within the petri dish. Without arabinose, the bacteria did not glow because arabinose is a necessary transcription factor to begin the expression of the green fluorescent protein. And without the plasmid, the antibiotic resistant gene is also not expressed. Therefore, the plate without the plasmid but with arabinose had no bacteria in it. Furthermore, the bacteria plate with only LB in it resulted in unrestricted bacteria growth as evident by the bacteria lawn.
Furthermore, the transformation efficiency in the LB/Amp/Ara plate was approximately 758 transformants/microgram. Though this number was on the low end of the normal transformation efficiency (in comparison to the norms stated in the worksheet), genetic transformation undoubtedly occurred.
Overall, I learned a lot about the various factors that affect gene expression. Not only does the DNA sequence need to be present, but environmental conditions can introduce additional factors that affect gene expression. For instance, a lack of a specific transcription factor can lead to the failure to express a certain gene because transcription of that gene sequence doesn’t even occur. Furthermore, new genes can be affected once a specific gene is turned on or off. In this instance, the presence of the antibiotic resistance trait is also expressed when the plasmid was introduced.
For future experiments, I would like to see the results of this experiment without ampicillin. Without ampicillin, a lawn of bacteria showed up. Perhaps introducing the plasmid without ampicillin can lead to a growing lawn of bacteria. However, without ampicillin, resistant and nonresistant cells will be competing for the same resources. This way, we can see whether the pGLO plasmid is genetically viable or offers other naturally advantageous traits besides antibiotic resistance.
Just wanted to give a special shout-out to Mr. Wong and – of course – Manas. Thank you for your support, dude. :) <3
#WongLifeIsGoodLife #HappyWongEqualsHappyDevansh
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