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MI pGLO flowchart
6
STEP ONE:
PLTW MI pGLO Lab Flow
Riley Hooks- A3
BACTERIAL TRANSFORMATION
Bacterial Transformation
Start Here:
The main purpose of this step as a whole is to insert the pGLO plasmid into the bacteria so it produces GFP protein
A professional that may be involved with plating bacteria and studying it would be a:
E. COLI is divided into two tubes:
E. COLI
In one tube you will add pGLO plasmid:
In another tube, you will leave the bacteria as is with no plasmid
+pGLO tube
+pGLO
he pGLO plasmid is present in this tube, as this is the experimental group in this part of the lab. It contains a gene commonly found in jellyfish that will cause the bacteria to glow in UV light. It also contains an ampicillin-resistant gene.
- The plasmid is transformed into the bacteria through a heat shock: by sitting in ice, followed by immediate exposure to hot water, then back in the ice water.
After transformation, a sterile loop is used to divide bacteria from this tube onto two plates.
LB/AMP +pGLO plate
LB/AMP
+pGLO
This plate contains LB (essential to bacterial growth) and ampicillin (the antibiotic the plasmid causes the bacteria to be resistant to). There is NO arabinose (described in LB/AMP/ARA +pGLO tab) in the tube, however, so the gene that causes the bacteria to glow is not activated.
Bacterial growth is present due to ampicillin resistance, but it does NOT glow under UV light due to the lack of arabinose.
Note: This plate will go on to be used in step 2 of the lab- linked in the LB/AMP/ARA plate tab.
LB/AMP/ARA +pGLO plate
LB/AMP/ARA
+pGLO
This plate contains LB (basically bacteria food), ampicillin (the antibiotic that the pGLO plasmid is resistant to), and arabinose (a substance that allows the glowing gene on the plasmid to activate, causing the bacteria to glow.
PRESENT: Growth of E. coli bacteria that contains the pGLO plasmid- and it glows due to the presence of arabinose.
Note: This plate will go on to be used in step 2 of the lab, shown here:
STEP TWO:
INOCULATION
**Note that the same process is done for bacteria from both the LB/AMP plate and the LB/AMP/ARA plates**
Inoculation is done in order to grow the E. coli some more and see if the LB/AMP bacteria can re-activate the GFP gene.
- Sterile loops are used to inoculate E. coli bacteria into liquid medium:
INOCULATION
LB/AMP/ARA
LB/AMP
- Tube
-
Contains bacteria from the LB/AMP plate
The purpose of this portion of the lab is to see if the gene from the bacteria that grew on the plate without the arabinose can be reactivated.
After being incubated, the gene is re-activated and the bacteria glows, but not as much as those in the + tube. Due to this, the bacteria from the - tube will be discarded.
Note that the plasmid and the E. coli bacteria are still present.
+ Tube
+
- Contains bacteria from the LB/AMP/ARA plate
After being incubated, the liquid that contains the bacteria will glow more than that in the - tube. This is because the GFP gene is already activated and enhanced. The tube serves as a POSITIVE control to see if the gene in the - tube can be re-activated.
The bacteria from this tube will go on to be used in Step 3.
STEP THREE:
Bacterial Concentration
The main purpose of this step is to separate te bacteria from the liquid medium it was growing in.
Bacterial Concentration
In order to concentrate the bacteria into one place, some of the bacteria culture from the + tube is transferred into another tube and centrifuged.
When the culture is centrifuged, it is separated into a pellet at the bottom with a supernatant at the top.
PELLET
Pellet
STEP FOUR
The pellet produced from the centrifuge is composed of the bacteria itself that had been grown in the liquid medium.
TE solution is then added and the pellet is resuspended. Note that the E.Coli with the plasmid are still present.
Lysozyme is then added to the resuspended solution, which causes the bacteria to lyse- releasing GFP protein.
Bacterial Lysis
The main purpose of this step is to release the GFP protein that had been produced by the bacteria earlier.
The bacteria are then frozen, therefore the cell walls can lyse thanks to the lysozyme, releasing GFP PROTEIN.
**Note that the bacteria and plasmid are no longer present.
During this step- after the lysis:
Bacterial Lysis
Note that the pGLO plasmid and the E. coli bacteria are no longer present- however the GFP protein has been released from the bacteria.
Protein Chromatography Prep
One who may manufacture purified proteins using protein chromatography such as insulin is known as a:
Protein Chromatography Prep
- Equilibration buffer is added to the newly acquired protein column to get it set up for the purification.
- On the other hand, the tube with the GFP protein is centrifuged again, forming another pellet and supernatant.
Protein Purification Scientist
Protein Purification Scientist
- Protein purification scientists are in charge of manufacturing and purifying proteins and protein solutions- mainly for medical use.
For example, insulin injection production for those with diabetes is the job of a protein purification scientist- as their pancreas cannot produce insulin on their own.
Pellet
The pellet produced from this centrifuge will be discarded, as it contains dead bacteria remnants.
The GFP protein was released from this bacteria, and it is now present in the supernatant.
Supernatant
Supernatant
The supernatant from the centrifuge contains the GFP protein that was released from the bacteria upon lysis, as well as the TE solution the pellet of bacteria was resuspended in during the Bacterial Concentration step.
This product will go on to be used during protein chromatography.
STEP FIVE
Protein Chromatography Process
The main purpose of the protein chromatography process is to purify the GFP protein and filter out any other unwanted substances.
Protein Chromatography
STEP SIX
The remaining buffer is dripped out of the tube, and then the GFP protein supernatant is filtered through the column. A wash buffer is then added to filter out any unwanted proteins.
A TE buffer is then added to the GFP will stick to the beads present.
Now that the protein has been purified, it will need to be analyzed through protein electrophoresis.
The main purpose of this step is to discover the purity and the size of the GFP protein molecules through running it through an agarose gel.
Protein Electrophoresis
Protein Electrophoresis
A loading buffer is added to the protein sample while the agarose gel is prepared by being placed in a chamber.
The protein sample is then loaded into the gel, and the chamber is turned on to send an electric current through the water, causing the protein to travel through the gel.
As soon as the protein is done, they are stained to make it easier to observe, and GFP should be at a certain spot on the gel.
Gel electrophoresis is a common process used in many labs. A professional that may be able to assist with this is a:
Lab Technician
Lab Technicians typically assist with processes that occur in scientfic laboratories. Gel electrophoresis of any kind is a common one.
Lab Technician
Sources Cited
Sources
USBLS. (2022, April 18). Clinical Laboratory Technologists and technicians : Occupational outlook handbook. U.S. Bureau of Labor Statistics. Retrieved May 12, 2022, from https://www.bls.gov/ooh/healthcare/clinical-laboratory-technologists-and-technicians.htm
ZIppia. (2021). What does a protein purification scientist do? - zippia. Zippia. Retrieved May 12, 2022, from https://www.zippia.com/protein-purification-scientist-jobs/what-does-a-protein-purification-scientist-do/
Betterteam. (2022, March 15). Microbiologist job description. Betterteam. Retrieved May 12, 2022, from https://www.betterteam.com/microbiologist-job-description
Ata, S. (2019, October 10). Protein analysis techniques explained. ATA Scientific. Retrieved May 12, 2022, from https://www.atascientific.com.au/3-protein-analysis-techniques/
Sino. (2021, July 15). Chromatography for protein purification - Sino biological. SinoBiological. Retrieved May 12, 2022, from https://www.sinobiological.com/resource/protein-review/chromatography-purification
Worthington. (2022). Lysozyme. Lysozyme - Worthington Enzyme Manual. Retrieved May 12, 2022, from https://www.worthington-biochem.com/LY/
SUPERNATANT
Supernatant
The supernatant is composed of the liquid medium that the bacteria was grown in. This is going to be disposed of- as it is not needed for the remainder of the lab.
-pGLO tube
-pGLO
The pGLO plasmid (described in the +pGLO tab) is NOT present in this tube. This will serve as the negative control group of the lab, as the plasmid will not be transformed into the bacteria.
Bacteria from this tube will be divided onto two plates using a sterile loop:
LB, -pGLO plate
LB, -pGLO
This plate contains LB Agar, which is essential to bacterial living. There are no antibiotics to kill the bacteria, nor are there any extra proteins to enhance the bacteria in any way.
The growth of a bacterial lawn is present. This is meant to serve as a control- showing growth of bacteria with no plasmid or antibiotic.
Note that this plate is just observed as a control and will not be used for the remainder of the lab.
LB/AMP -pGLO plate
LB/AMP
-pGLO
This plate contains LB, which is essential to bacterial living, and ampicillin, a beta-lactam antibiotic that kills bacteria by destroying the cell wall. This is meant to set a control for when wild-type (no plasmid) bacteria are plated onto ampicillin.
There is no growth at all present on the plate due to the ampicillin present and lack of the resistance gene from the plasmid.
(Note that this plate is observed for a control and is not used for the remainder of the lab)
Microbiologist
A microbiologist conducts research and studies microbiological factors such as bacteria, viruses, algae, and fungi. They also study how they interact with other living things.