Pglo Transformation Lab Report
Genetic transformation happens when an organism is altered by the introduction of new genetic information which is merged into the organism’s genome. Bacterial transformation is a type of genetic transformation that was used in lab and mainly used due to the single celled nature of bacteria. In this lab, the engineered pGLO plasmid is integrated into E. Coli bacteria, and adds the genes which code for the proteins GFP in the modified bacteria’s genome (Hanahan, Studies on transformation of Escherichia coli with plasmids, 1983). To see the reaction of this plasmid on the cells, bacteria treated with the plasmid were grown on two separate agar plates containing LB nutrient broth and ampicillin, and another containing LB nutrient broth, ampicillin
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and arabinose. To compare the difference of these plates two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that didn’t have the plasmid.
Introduction:
What is bacteria transformation?
It is when a bacterial cell takes up DNA that is foreign to it and integrates it into its own DNA. What is genetic transformation? That is change that is caused by genes involving the insertion of a gene into an organism to change the organism trait. This transformation typically occurs in plasmids. So, what are plasmids? Plasmids are small circular DNA molecules that are isolated from its chromosomes. So, what are genes and plasmids and how are they related to bacteria and genetic transformation. (Hanahan, Jessee, & Bloom, Plasmid transformation of Escherichia coli and other bacteria, 1991)Well genes are part of DNA which basically give instructions to make molecules which are called proteins. In this bacteria transformation procedure bacteria are transformed with a gene that has codes for GFP (Green Fluorescent Protein). The bacteria with this gene will cause them to glow a brilliant green color under UV light. So, plasmids carry a gene that contains one or more traits that may be beneficial to survival. In this scenario, the pGLO has a resistance to antibiotics such as ampicillins, which is the one used in this method along with the bacteria E coli because it can easily be grown gene can be turned on in transformed cells by adding arabinose to the cells. SO therefore, the transformed cells will grow on plates with LB/amp and appear white on plates not contain
arabinose.
Hypothesis:
The hypothesis is if bacteria with pGLO plasmids are resistant to the ampicillin and have that gene for GFP, the colonies will survive and grow on the plates that have LB/amp. The pGLO bacteria on a plate with LB/amp/ara should grow and glow green under UV light because of the addition of arabinose. The control plates the --pGLO bacteria should be amp sensitive and will not grow on the LB/amp plates. The second control plate with -pGLO bacteria and no ampicillin will host a lawn of colonies.
Method & Materials:
In this experiment two micro test tube were labeled +pGLO and -pGLO and were placed in tube rack. A pipet, was used to transfer 250 µl of transformation solution into each tube. Both tubes were placed on ice. Using a sterile loop, a single colony of bacteria was selected up from the starter plate. Individual colonies are taken because the bacteria must be actively growing to attain high transformation effectiveness. Only bacterial colonies that are uniformly circular and with smooth edges were chosen. The +pGLO tube was then immersed with the loop into the transformation solution at the bottom of the tube. The loop was spun to make sure solution was dispersed in the transformation solution. The tube was placed back in the tube rack in the ice and another sterile loop is used and repeated for the -pGLO tube. The pGLO DNA solution is examined with a UV light. A new sterile loop is immersed into the pGLO plasmid DNA stock tube. The loopful was mixed into the cell suspension of the +pGLO tube. The tubes were then incubated on ice for 10 minutes. While tubes are sitting on the ice, there are four tubes labeled one LB/amp plate: + pGLO • Second tube is labeled LB/amp/ara plate: + pGLO • Third is labeled LB/amp plate: - pGLO • And last Labeled the LB plate: - pGLO. The racks are removed from ice. Using a sterile pipet 250 ul og LB broth was added to each tube. They tubes are left at room temperature for 10 minutes. A sterile loop is used for each tube, 100ul were pipetted into the appropriate pales. The suspensions were spread evenly around the surface of the agar. Plates are stacked up taped and placed upside down and left in an incubator.
Results:
Transformation plates: +pGLO LB/amp did not glow because it contained no ribosomes contained 24 colonies. +pGLO LB/amp/ara had 13 colonies and glowed green under UV light with many small colonies with a greenish tint meaning that the DNA became resistant.
Control plates: -pGLO LB/amp had no bacteria growth and -pGLO LB had a lawn of colonies
So, the Transformation efficiency can be calculated. It is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the capability of the cells. It can be calculated by using formula down below. The most direct way to determine the total number of bacteria that were transformed with the pGLO plasmid is to count the colonies on the plate.
Total number of colonies=37
The total amount of DNA we began with is equal to the product of the concentration and the total volume used, or (DNA in pg.) = (concentration of DNA in ug/pl) x (volume of DNA in PI)
Total amount of pGLO DNA (pg) used in this experiment =
In this experiment, we used 10 pl of pGLO at concentration of 0.08 pg/pl. This means that each microliter of solution contained 0.08 pg of pGLO DNA.
0.8ug
Transformation efficiency=Total number of colonies growing on the agar plate Amount of DNA growing on the agar plate(up)
Number of colonies on 37
Micrograms of pGLO DNA spread on the plates 0.16
Now using the data in the table, we calculate the efficiency of the pGLO transformation
231.25
Conclusion:
The purpose in this lab was to understand how transformation transpires. The results lead to the hypothesis being a success. Our hypothesis stated that if bacteria with pGLO plasmids are resistant to the ampicillin and have that gene for GFP, the colonies will survive and grow on the plates that have LB/amp. The pGLO bacteria on a plate with LB/amp/ara should grow and glow green under UV light because of the addition of arabinose. The control plates the --pGLO bacteria should be amp sensitive and will not grow on the LB/amp plates. The second control plate with -pGLO bacteria and no ampicillin will host a lawn of colonies. The +pGLO LB/amp/ara bacteria glowed. This plate has resistance to the ampicillin. The +pGLO LB/amp was also resistance to ampicillin. This was because of the plasmids present. When plasmids are present it helps transforms the bacteria to become resistant. In -pGLO LB/amp no bacteria were present this is because there are no plasmids present.
and arabinose. To compare the difference of these plates two more plates were grown, one with LB nutrient broth and ampicillin and the other with only the LB broth, using cells that didn’t have the plasmid.
Introduction:
What is bacteria transformation?
It is when a bacterial cell takes up DNA that is foreign to it and integrates it into its own DNA. What is genetic transformation? That is change that is caused by genes involving the insertion of a gene into an organism to change the organism trait. This transformation typically occurs in plasmids. So, what are plasmids? Plasmids are small circular DNA molecules that are isolated from its chromosomes. So, what are genes and plasmids and how are they related to bacteria and genetic transformation. (Hanahan, Jessee, & Bloom, Plasmid transformation of Escherichia coli and other bacteria, 1991)Well genes are part of DNA which basically give instructions to make molecules which are called proteins. In this bacteria transformation procedure bacteria are transformed with a gene that has codes for GFP (Green Fluorescent Protein). The bacteria with this gene will cause them to glow a brilliant green color under UV light. So, plasmids carry a gene that contains one or more traits that may be beneficial to survival. In this scenario, the pGLO has a resistance to antibiotics such as ampicillins, which is the one used in this method along with the bacteria E coli because it can easily be grown gene can be turned on in transformed cells by adding arabinose to the cells. SO therefore, the transformed cells will grow on plates with LB/amp and appear white on plates not contain
arabinose.
Hypothesis:
The hypothesis is if bacteria with pGLO plasmids are resistant to the ampicillin and have that gene for GFP, the colonies will survive and grow on the plates that have LB/amp. The pGLO bacteria on a plate with LB/amp/ara should grow and glow green under UV light because of the addition of arabinose. The control plates the --pGLO bacteria should be amp sensitive and will not grow on the LB/amp plates. The second control plate with -pGLO bacteria and no ampicillin will host a lawn of colonies.
Method & Materials:
In this experiment two micro test tube were labeled +pGLO and -pGLO and were placed in tube rack. A pipet, was used to transfer 250 µl of transformation solution into each tube. Both tubes were placed on ice. Using a sterile loop, a single colony of bacteria was selected up from the starter plate. Individual colonies are taken because the bacteria must be actively growing to attain high transformation effectiveness. Only bacterial colonies that are uniformly circular and with smooth edges were chosen. The +pGLO tube was then immersed with the loop into the transformation solution at the bottom of the tube. The loop was spun to make sure solution was dispersed in the transformation solution. The tube was placed back in the tube rack in the ice and another sterile loop is used and repeated for the -pGLO tube. The pGLO DNA solution is examined with a UV light. A new sterile loop is immersed into the pGLO plasmid DNA stock tube. The loopful was mixed into the cell suspension of the +pGLO tube. The tubes were then incubated on ice for 10 minutes. While tubes are sitting on the ice, there are four tubes labeled one LB/amp plate: + pGLO • Second tube is labeled LB/amp/ara plate: + pGLO • Third is labeled LB/amp plate: - pGLO • And last Labeled the LB plate: - pGLO. The racks are removed from ice. Using a sterile pipet 250 ul og LB broth was added to each tube. They tubes are left at room temperature for 10 minutes. A sterile loop is used for each tube, 100ul were pipetted into the appropriate pales. The suspensions were spread evenly around the surface of the agar. Plates are stacked up taped and placed upside down and left in an incubator.
Results:
Transformation plates: +pGLO LB/amp did not glow because it contained no ribosomes contained 24 colonies. +pGLO LB/amp/ara had 13 colonies and glowed green under UV light with many small colonies with a greenish tint meaning that the DNA became resistant.
Control plates: -pGLO LB/amp had no bacteria growth and -pGLO LB had a lawn of colonies
So, the Transformation efficiency can be calculated. It is the efficiency by which cells can take up extracellular DNA and express genes encoded by it. This is based on the capability of the cells. It can be calculated by using formula down below. The most direct way to determine the total number of bacteria that were transformed with the pGLO plasmid is to count the colonies on the plate.
Total number of colonies=37
The total amount of DNA we began with is equal to the product of the concentration and the total volume used, or (DNA in pg.) = (concentration of DNA in ug/pl) x (volume of DNA in PI)
Total amount of pGLO DNA (pg) used in this experiment =
In this experiment, we used 10 pl of pGLO at concentration of 0.08 pg/pl. This means that each microliter of solution contained 0.08 pg of pGLO DNA.
0.8ug
Transformation efficiency=Total number of colonies growing on the agar plate Amount of DNA growing on the agar plate(up)
Number of colonies on 37
Micrograms of pGLO DNA spread on the plates 0.16
Now using the data in the table, we calculate the efficiency of the pGLO transformation
231.25
Conclusion:
The purpose in this lab was to understand how transformation transpires. The results lead to the hypothesis being a success. Our hypothesis stated that if bacteria with pGLO plasmids are resistant to the ampicillin and have that gene for GFP, the colonies will survive and grow on the plates that have LB/amp. The pGLO bacteria on a plate with LB/amp/ara should grow and glow green under UV light because of the addition of arabinose. The control plates the --pGLO bacteria should be amp sensitive and will not grow on the LB/amp plates. The second control plate with -pGLO bacteria and no ampicillin will host a lawn of colonies. The +pGLO LB/amp/ara bacteria glowed. This plate has resistance to the ampicillin. The +pGLO LB/amp was also resistance to ampicillin. This was because of the plasmids present. When plasmids are present it helps transforms the bacteria to become resistant. In -pGLO LB/amp no bacteria were present this is because there are no plasmids present.