User:Derek K.L. Chu/Notebook/Investigation training

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Investigation topic

Transformation: Investigate the best recovery time of ampicilin, kanamycin, chloramphenicol, etc in our lab.

Motivation

Transformation using antibiotics as a selection system requires a recovery time for the cell to express antibiotic resistance gene. This takes from 30 to 60 minutes. Our group is trying to investigate the minimal recovery time needed for a successful transformation, in order to save the time for cloning.

Overview

We define the best recovery time as the shortest time needed to get a usable amount of colony. We assign 6 colony as the amount of colony needed for a convincing transformation result. The project is planned to measure the colony forming unit (CFU) for transformation using different antibiotics. The project is planned to test for the CFU using 5 different recovery time. Transformation using ampicillin (AMP), kanamycin (KAN) or chloramphenicol (CHL) as a selection system for transformant are tested. In our project, we use pCMV/myc/mito/GFP, pEGFP-N1, pSB1C3-BBa_K1119003 for the transformation using AMP, KAN, CHL as the selection system for successful transformant. We use DH10B as the competent cell.

Method

We first search for the amount of DNA needed for transformation which would give a countable CFU. After selecting the amount of DNA for giving CFU within countable range, transformation using different recovery time using the chosen DNA amount was carried to search for the best recovery time.

For the first part of experiment, we transform the cell with different amount of DNA, using 1 hour of recovery time. From previous measurement, the transformation efficiency of the competent cell is 1*10^8 CFU/ug. To generate plate that give CFU within range of 30~300CFU, 1pg, 10pg, 100pg of DNA was used for testing the amount of DNA needed.

In the second part of the experiment, we plan to transform the plate using different recovery time, using 0 min, 15 min, 30 min, 45 min, 60 min in order to search for the best recovery time.

Results

1. Transformation using different amount of DNA
File:Derekgroupgraph1edited.jpeg
Since we have assigned the range for countable colony as 30~300 CFU, we decided that 10pg of DNA would be appropriate to use for our investigation project. According to the table, most of the number of colonies formed by 10pg of DNA lie in between the rage of 30~300 CFU.
However, the results are later identified as inconclusive, since there are replicas eg. 100pg transformation using pSB1C3-BBa_K1119003 show large discrepancy among the replica.

2. Transformation using different recovery time
Derekfigure1.png

Derekgroupgraph2.png

The data for AMP and CHL are not conclusive. As it is shown above, there are transformation with the result of replica with large discrepancy. Since we failed to maintain the confidence of our experimental result, we decided that none of the data can be used to support a conclusive analysis.
The value shown in the red circle indicates the most opposing data towards the overall trend according to the other data plotted on the graph.

For the KAN transformation, cell lawn was obtained for all recovery time. In fact, it was recalled that our KAN plates did not pass the plate testing. For the negative control test, there were bacteria grown on a DH10B plate. Therefore, we were unable to collect any data from the KAN.

Conclusion

Since the result of the replica does not agree with each other, we are unable to draw any conclusion based on the data.

Future Work

Statistical software eg Minitab could be used to calculate the correlation coefficient between the recovery time and the CFU. The p-value will tell the significance of the correlation, and the sign of r will indicate whether it is positively or negatively correlated.
To select best recovery time, we would select the range of recovery time that would give 6 CFU. If recovery time = 0min gave 0 CFU, and recovery time = 15min gave 12 CFU, recovery time of 0~15 min would be suggested.

Reference

http://www.addgene.org/plasmid_protocols/bacterial_transformation/
http://openwetware.org/wiki/IGEM:Paris_Bettencourt_2012/Protocols/Heat_Shock_Transformation_of_E_Coli
http://parts.igem.org/Part:pSB1C3
http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf
https://www4.vanderbilt.edu/vapr/sites/default/files/pEGFP-N1_map.pdf

Appendix: Protocol for transformation

transformation using different amount of DNA
1) Dilute the plasmid into 1pg/ul, 10pg/ul, 100pg/ul using ddH2O.
2) Thaw competent cells on ice.
3) Add 1ul plasmid to tubes containing 50ul DH10B competent cell. Mix gently with the pipette tip.
4) Chill on ice for 10 minutes.
5) Heat shock at 42°С for 90 seconds.
6) Chill on ice for 2 minutes.
7) Add 1ml LB medium, incubate at 37°С for 60 minutes.
8) Centrifuge the cell at 13.2krpm for 30 seconds, remove 900ul supernatant, resuspend the cell pellet by gently pipetting it.
9) Plate the cells on LB with corresponding antibiotics added.
10) Incubate all plate at 37°С for 16 hrs.

transformation using different recovery time
1) Thaw competent cells on ice.
2) Add 1ul plasmid of 10pg/ul to tubes containing 50ul DH10B competent cell. Mix gently with the pipette tip.
3) Chill on ice for 10 minutes.
4) Heat shock at 42°С for 90 seconds.
5) Chill on ice for 2 minutes.
6) For groups using different recovery time, apply different treatment:
0 minutes: directly plate the cell without doing recovery.
15 minutes: incubate at 37°С for 15 minutes.
30 minutes: incubate at 37°С for 30 minutes.
45 minutes: incubate at 37°С for 45 minutes.
60 minutes: incubate at 37°С for 60 minutes.

7) Centrifuge the cell at 13.2krpm for 30 seconds, remove 900ul supernatant, resuspend the cell pellet by gently pipetting it.
8) Plate the cells on LB with corresponding antibiotics added.
9) Incubate all plate at 37°С for 16 hrs.



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