DNA Technology Lab
Data and Analysis for pGREEN
LB with -plasmid
Prediction: yes Reason: The bacteria will grow because nothing in the solution will kill the cells since there is only nutrients. LB and Ampicillin with -plasmid Prediction: no Reason: Since the solution contains ampicillin which can kill bacteria and since the plasmid with resistance gene to ampicillin was not taken into this group of bacteria, it will be killed by the medium and not grow. |
LB with +plasmid
Prediction: yes Reason: The bacteria will grow because nothing in the solution will kill the cells since there is only nutrients. Also the additional plasmids the cells will take in will not effect their normal replication functions. LB and Ampicillin with +plasmid Prediction: yes Reason: The bacteria will grow because even though the solution has ampicillin, most of the bacteria will have gained a resistance gene through plasmids they would have absorbed. It is noted though that because not all of the bacteria will successfully absorb the plasmids they will not all have the gene to resist ampicillin, thus there will be less growth than the samples in only LB. |
Prediction Analysis
All of the observed results were accurate with the predictions above, thus showing how awesome the group was and how well the experimented was executed.
Colony Counts
LB +Plasmid = lawn (covers entire surface)
LB -Plasmid = lawn (covers entire surface)
LB/Amp +Plasmid = 9
LB/Amp -Plasmid = 0
Compare and Contrast Colony Counts
a. LB +PLasmid and LB -PLasmid
- Both samples were placed in the same LB solution, though one had an ampicillin resistance gene it will not effect how it reploicates allowing them to be the same
b. LB/Amp -Plasmid and LB -Plasmid
- While both have no additional plasmids added into the bacteria cells, one of the samples has a medium of ampicillin which will kill bacteria without resistance to it. Thus, the sample in only LB will grow normally while the sample with ampicillin and LB will die.
c. LB/Amp +Plasmid and LB/Amp -Plasmid
- Both of these solutions create the same medium for both samples of bacteria, but only one has an added plasmid with resistance to the mediumin which it can still grow. The other sample without the obtained gene will not thrive.
d. LB/Amp +PLasmid and LB +Plasmid
- The two samples of bacteria both had a plasmid added, but had two seperate mediums. Since the bacteria in the LB/Amp medium also had the plasmid with the resistance gene, it will be able to survive, but not all of the bacteria will take in the plasmid so some of the bacteria will die still. This means that the bacteria sample in the LB/Amp medium will grow less than the bacteria sample in the LB medium.
All of the observed results were accurate with the predictions above, thus showing how awesome the group was and how well the experimented was executed.
Colony Counts
LB +Plasmid = lawn (covers entire surface)
LB -Plasmid = lawn (covers entire surface)
LB/Amp +Plasmid = 9
LB/Amp -Plasmid = 0
Compare and Contrast Colony Counts
a. LB +PLasmid and LB -PLasmid
- Both samples were placed in the same LB solution, though one had an ampicillin resistance gene it will not effect how it reploicates allowing them to be the same
b. LB/Amp -Plasmid and LB -Plasmid
- While both have no additional plasmids added into the bacteria cells, one of the samples has a medium of ampicillin which will kill bacteria without resistance to it. Thus, the sample in only LB will grow normally while the sample with ampicillin and LB will die.
c. LB/Amp +Plasmid and LB/Amp -Plasmid
- Both of these solutions create the same medium for both samples of bacteria, but only one has an added plasmid with resistance to the mediumin which it can still grow. The other sample without the obtained gene will not thrive.
d. LB/Amp +PLasmid and LB +Plasmid
- The two samples of bacteria both had a plasmid added, but had two seperate mediums. Since the bacteria in the LB/Amp medium also had the plasmid with the resistance gene, it will be able to survive, but not all of the bacteria will take in the plasmid so some of the bacteria will die still. This means that the bacteria sample in the LB/Amp medium will grow less than the bacteria sample in the LB medium.
Experiment Questions
For this experiment, we will be looking for the samples of bacteria that have taken up the plasmids they were exposed to. This can be done by looking for samples of bacteria that have survived in a medium that would normally be harmful, but because of a specific resistance gene taken in through a plasmid they are able to thrive and grow. In this case, the selected group for the experiment will be the bacteria living in the ampicillin and LB medium.
The phenotype for the transformed solutions will be their blue color compared to the normal milky white coloration. Basically, this means that the sample of bacteria has taken in the plasmids because they had a blue protein producing protein added in so it will carry over to the bacteria it is absorbed by.
To conclude that the transformation of plasmids into the bacteria cells was successful, the first dish that should be checked is the LB/Amp containing dish. If successful, the bacteria in this cell will have survived in this solution medium forming small colonies.
Transformation Calculations
a. Total mass = volume x concentration
.05 μg/μL = 10 μL x .005 μg/μL
b. Total Volume of cell suspended prepared.
510 μL
c. fraction spread = volume suspension spread/ total volume suspension
0.1818 = 100/510
d. Mass plasmid DNA spread = total mass plasmid (a) x fraction spread (c) = mass plasmid DNA spread
.00909 μg = .05 x .1818
e. transformation efficiency = colonies/ mass plasmid spread (d)
.08181 = 9 x .00909
Factors can greatly effect how a plasmid is transformed and its efficiency, here are some examples. One of the main issues is the amount of cells are originally suspended in the solutions. This along with the volume suspension spread can effect how well the cells can work individually by receiving enough nutrients to function and to have enough space to grow. Another factor is how much plasmid is present in the suspended fluid. This can determine if their is even enough for all the bacteria and the ability for the bacteria to take in some. All of these factors lead up to the amount of colonies present. This is so because the bacteria that takes in the plasmids will have resistance to the ampicillin in the medium and not die. These that do not die will begin to grow in colonies. The more colonies there are in the end, the more efficiently the plasmids were transferred into the cells.
a. Total mass = volume x concentration
.05 μg/μL = 10 μL x .005 μg/μL
b. Total Volume of cell suspended prepared.
510 μL
c. fraction spread = volume suspension spread/ total volume suspension
0.1818 = 100/510
d. Mass plasmid DNA spread = total mass plasmid (a) x fraction spread (c) = mass plasmid DNA spread
.00909 μg = .05 x .1818
e. transformation efficiency = colonies/ mass plasmid spread (d)
.08181 = 9 x .00909
Factors can greatly effect how a plasmid is transformed and its efficiency, here are some examples. One of the main issues is the amount of cells are originally suspended in the solutions. This along with the volume suspension spread can effect how well the cells can work individually by receiving enough nutrients to function and to have enough space to grow. Another factor is how much plasmid is present in the suspended fluid. This can determine if their is even enough for all the bacteria and the ability for the bacteria to take in some. All of these factors lead up to the amount of colonies present. This is so because the bacteria that takes in the plasmids will have resistance to the ampicillin in the medium and not die. These that do not die will begin to grow in colonies. The more colonies there are in the end, the more efficiently the plasmids were transferred into the cells.