Experiment - E.Coli Flashcards
Overview of the experiment
ThepGREEN plasmid contains the GFP gene. It also contains antibiotic resistant gene.
- Bacteria only express GFP when an inducer (like IPTG) is present. Antibiotic resistant gene is expressed in recovery broth and does not need an inducer.
- If IPTG is missing, the transformed bacteria may still grow (if they have ampicillin resistance), butthey will not glow under UV light.
- This methoddoes not integrate the GFP gene into the bacterial chromosome. Instead, thepGREEN plasmid remains as an independent circular piece of DNA, which the bacteria can replicate and express under the right conditions. In this experiment, theGFP gene was introduced into the bacteria through a process called transformation, rather than cutting and inserting DNA with restriction enzymes.
Transformation
We did not cut to create the recombinant plasmid - only the method to integrate the plasmid into the bacteria
Calcium chloride
Makes the theE. coli competent by neutralising the negative charges on both the bacterial cell membrane and the plasmid, making it easier for the plasmid to enter. So they do not reject each other.
Heat shock
Cold shock:
- Placed on ice for 10 minutes
- Cold shockslows down molecular movement and stabilizes the membrane.
Heat shock:
- 42°Cforheat shock, for 45 seconds
- Heat shock (42°C)creates a thermal imbalance, forming temporary pores in the membrane, allowing plasmid DNA to enter.
- Increases the permeability of the membrane
Recovery period:
- Recovery broth and the maintenance of the test tubes at 37Callow the bacteria to repair their membranes, enclosing the plasmid and start expressing the new plasmid genes.
- It gives the bacteria time toexpress the antibiotic resistance gene.
-DNA plate
Control plate:
This plate contains E. coli without the pGREEN plasmid. It helps show that normal (non-transformed) bacteria can grow on nutrient agar.
Growth
–DNA / +Amp
This control plate contains E. coli without the plasmid, but with ampicillin in the agar:
- If there is no growth, it confirms thatuntransformed bacteria are susceptible to ampicillinand cannot survive –>ensure validityby confirming that normal bacteria cannot survive ampicillin.
- It also helps to show that any growth on the +DNA/+Amp plate isdue to successful transformation, not random contamination.
No growth
+DNA / +Amp
Experimental plate: tests whether bacteriasuccessfully
transformedwith the plasmid.
- Only transformants grow because they have successfully taken up the pGREEN plasmid, which contains the ampicillin resistance gene.
- Non-transformed bacteria die due to the antibiotic.
- Without IPTG, the GFP gene wasnot induced, so the bacteria did not produce the fluorescent protein (non-coding DNA impact on polypeptide synthesis)
Growth
+DNA / +Amp / +IPTG
Experimental:
Tests if IPTGinduces GFP expression, making bacteria fluoresce. There was growth of transformed bacteria, but now they glow greenunder UV light due to GFP expression.
- Fluorescence only occurs when the GFP gene isexpressed inside a living bacterial cell, which requires transcription and translation.
- This is to show gene regulation - promoters are necessary to code proteins.
No growth
Transformation results
+DNA/+Ampplate:colonies, not a complete lawn of bacteria → suggests that only asmall fraction of bacteriasuccessfully took up the plasmid and survived the ampicillin selection.
-DNA / -Ampplate hasa lawn of bacteria, showing that most of the original bacteria were alive. This difference confirms thatonly a small percentage of the original bacteria successfully underwent transformation.
Recovery broth
Added to the test tubes and let it incubate for 10 minutes.
Note that the broth does not contain ampicilin because transformed bacteria have not yet begun to produce the protein
b-lactamase that gives them ampicillin resistance.
Few/no colonies
Possible sources of error
- Too few or too many plates were collected from source plates
- Plasmid DNA was not added
- The transformation process had not been followed correctly at the right temeperatures and timing → plasmid DNA did not get properly integrated into the bacteria
Colonies don’t glow
Possible sources of error
- IPTG was omitted from the plates. As seen by the +DNA/Amp plate, transformed E. coli will not express green fluorescence if IPTG was not present to activate the inducible promoters in the plasmid
- IPTG may have degraded rapidly because it was added when the agar solution was too hot
Only some of the colonies glow
Possible sources of error
- IPTG was not mixed properly
- IPTG was added when the agar was too cold, causing it to not distribute evenly when swirled → not every bacteria came into contact with the IPTG, so the promoter for GFP was only activated in some bacteria
- Note: can’t be that transformation did not occur properly, as the colonies would be killed by the ampicilin
Glowing colonies are surrounded by small background colonies that don’t glow
Possible sources of error
- The small background colonies are ‘satellite’ colonies that grow as the ampicillin is broken down around each transformed colony
- This is caused by insufficient ampicillin in the plates, or ampicillin breaking down because it was added when the agar was too hot
- Also possible that the ampicillin was added when the agar was too cool, then not mixed properly
Plate is covered by non-glowing colonies
- Can occur if ampicillin has been omitted from the agar or destroyed by adding it to the agar when it was still too hot → in the absence of ampicillin, non-transformants can survive and grow
Validity of E. coli experiment
Validity is determined by whether or not an experiment achieves its intended aim
- Aim of this experiment was to introduce recombinant DNA into E. coli
- Aim was achieved. Controls were used in the experiment: the -DNA plate and the -DNA/Amp plate to prove that untransformed bacteria could not survive in a plate containing ampicillin. This proves that bacteria that survived were transformants, and the introduction of recombinant DNA had been successful
- Variables were controlled: both all the petri plates were incubated at the same temperature for the same durations of time, and equal volumes of CaCl2 and heat shock procedures were applied to the -DNA and +DNA. Also equal volumes of -DNA and +DNA were used, and all equipment had been properly sterilised before use
Accuracy of E. coli experiment
Accuracy is a measure of how close to expected values the experimental results are.
- The experiment was accurate as the control plates and experimental plates expressed their expected result.
Reliability of E. coli experiment
Reliable to a certain exent:
* As the experiment was complex, in which the transformation cycle involved a very precise heat shock, there was some variance in results.
* These variance were contamination though, indicating mistakes carried out in execution and not a fault of the experimental procedure.
* And then talk about the different errors and explain them??
Ampicillin resistance enzyme
- The ampicillin resistance gene, ampR, codes the enzyme beta-lactamase
- Beta-lactamase breaks down the beta-lactam ring structure found in ampicillin and other pencillin-type antibiotics
