This was our first important lab of the year. Today we performed a transformation of competent e-coli cells with the IDT plasmid constructs 1-8 our team had designed and stored since 2020. To evaluate the effectiveness of the antibiotic, we placed competent cells in plates with LB+AMP and LB as negative control and LB control, respectively.
*Results from transformation of competent cells with constructs 1-8
There were e coli colonies in the negative control, we noted that some possible conclusions could be that the ampicilin is not working properly, or that there was some contamination to the negative control with one of the transformed cells. We noted a lot of growth on the plates with constructs, but we were expecting only a few colonies.
Based on the results, we decided to make a new AMP/H2O solution. We then poured LB+AMP plates to test the antibiotic with the competent cells we received this year and an LB plate with cells as a control. Similarly, we streaked the plates from the previous experiment to see if the transformation was carried out effectively.
Remaking AMP solution
Testing with Competent Cells (No transformed cells):
We believe that the 6 colonies in the LB + AMP are probably E Coli, so there wasn’t contamination in the previous experiment. However, we can’t certainly determine that the ampicillin is the problem because there is less growth on the LB than the plates with ampicillin.
Replating transformed bacteria on the new LB + AMP:
There was growth on all the plates with transformed cells and smaller colonies that are closer to the result we were expecting (single cell colonies).
We created a new ampicillin sodium solution diluting it in ethanol 1 gram of ampicillin with 5 ml of ethanol and 5 ml of distilled water. We tested the K1724000 tube and there was no growth in LB+AMP or LB.
We concluded that the cells from 2019 are dead, but can’t be sure the AMP is not working.
Finally, we placed some of the colonies of transformed cells from the plates into tubes with LB+AMP to store them.
Final Testing of AMP:
We used the AMP/Etoh solution to make LB+AMP plates with 12.5 LB and 7.5 Agar. The plates were prepared on a 500ml bottle. We have four tubes of LB+AMP to store.
For this experiment we decided to test the AMP/H2O from September 10th, the one from September 15th, and the AMP/Etoh we made on September 16th. In this way we could identify if it was a problem with the mixture or if the entire supply of ampicillin had expired. Finally we used chloramphenicol plates as negative control.
Everything grew on LB+Amp, we concluded that our supply of ampicillin was no longer working.
Testing the transformation (RFP in presence of Cadmium):
We created a new concentration of 0.001M cadmium concentration.
Cadmium concentration:
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Cadmium concentration: |
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0.1 M |
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0.01 M |
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0.001 M → |
100μL in 1 ml + 900μL distilled water |
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0.0001 M → |
100μL in 1 ml + 990μL distilled water |
We added 600 microliters of the bacteria with constructs 4, 7, and the single RFP stored on LB + AMP from the tubes incubated since yesterday. In total we have 16 tubes. Four negative controls (no cells), four of construct 4, four of construct 7, and four of the single RFP.
We added 600 microliters of the bacteria with constructs 4, 7, and the single RFP stored on LB + AMP from the tubes incubated since yesterday. In total we have 16 tubes. Four negative controls (no cells), four of construct 4, four of construct 7, and four of the single RFP.
Expected results:
Results:
There was no shade of red shown in the tubes. Hence, we can conclude that the transformation didn’t work or there was a problem with the plasmids for the RFP expression.
We bought new ampicillin from a local provider in Centro de Lima and used it to pour new LB+AMP plates.
Testing if the transformation worked (ampicillin resistance):
To test if the cells were properly transformed or there was a problem with our RFP expression constructs we needed to see if the cells at least had acquired ampicillin resistance. Therefore, we decided to grab different colonies from the plates of supposedly transformed cells and streak them on the new LB+AMP and LB.
Since there is no growth on the antibiotic, we can conclude that the cells were not transformed correctly.
Reviews from last experiment: The streaks were not transformed since they grew on lB but not LB+AMP.
Our next steps are to streak more plates with more bacteria to check if there are any transformed bacteria on the transformation plates. We will also do another bacterial transformation getting DNA in E Coli Cells and plating them on LB+AMP.
Transforming three constructs only using a negative control:
All constructs are getting 50μl of E coli competent cells.
We are following heat shock transformation in bacterial transformation protocol using a negative control instead of a positive control of iGem 2019.
To test the concentration of the DNA we were working with, we ran a gel electrophoresis of the 8 constructs we were working with alongside the 100bp DNA and lambda hindIII DNA that we have stored in our fridge. Fortunately, the bands show that the DNA is a supercoiled DNA, which is considered the healthiest and thus viable.
After finding the receipt from IDT and reading the description of the product, we realized that we had confused the linear DNA constructs for a plasmid. This explains why the transformation didn’t work, along with the bad ampicillin we used in the process: the linear DNA needs to be cloned before transformation. However, we were able to find the real plasmids at the back of the freezer.
We started a new transformation with the competent cells we had left and the constructs 1, 2, 3, 6, and the single RFP. Then, the negative control was just competent cells.
Since there wasn’t growth on the plates with ampicillin, the transformation didn’t work. The possible reasons for this could be because:
The ampicillin from the local provider was too strong because it was probably pharmaceutical grade. The cells were no longer competent. The plasmid no longer works. There was an error in our protocol