1. Student: Lauren Crampton, A00200702, Supervisor: Donal Eardly Department of Life Sciences, Athlone Institute of Technology.
Antibiotics such as penicillin have transformed the way people are treated for a multitude of diseases. The potency of these drugs has reduced dramatically due to
bacteria developing resistance against them. Ling et al. (2015), explains that the majority of antibiotics were found and formulated from isolating easily cultivated soil
micro-organisms, such as Prokaryotic micro-organisms. Antibiotics have been derived from prokaryotic microbes and are produced using a Polyketide Synthase
Pathway. There are two main classes of PKS genes called PKS Type I and PKS Type II.
• Obtain good quality soil samples
• Sequence & Amplify those genes
in PCR machine
• Locate & Identify Type II PKS gene
• Clone these PCR products using
TOPO – TA vectors
• Collected a range of soil samples
• Extracted Total DNA from soil using MO
BIO PowerSoil™ DNA Isolation Kit
• Tested DegKs Forward and Reverse
Primers
• Troubleshooted PCR reactions and DNA
Isolation Techniques
• Collected and Re-Isolated soil samples
No amplification; table below is examples
of troubleshooting efforts
Were aims and objectives met?
Yes, some were met, however the sequencing
and amplification of Type I & II PKS genes in a
PCR machine was unsuccessful.
Recommendations for Future Work
• Fresh Soil Sample Collection
• Re-Isolation (Practise & Improve
Technique)
• Check Purity and Concentration of DNA
Isolated
• Purify and alter DNA Concentration where
required
• Test Master Mix and Primers on other DNA
samples from other research projects
• MO BIO and Further PCR Troubleshooting
• Cloning Vectors
Ling, L.L. et al. (2015). ‘A new antibiotic kills pathogens without detectable resistance.’ Nature. 517 (2015) pp. 455 – 459.
Sriprapun, M. (2014). ‘Principles of PCR.’ [Online Image] Available at: http://www.slideshare.net/MetheeSri/principle-of-pcr. [Accessed 25th November 2015]
Label Primers Dilution
Denaturation
Temperature
(°C)
Annealing
Temperature
(°C)
Extension
Temperature
(°C)
A
DegkKS Forward &
Reverse
- 95 55 72
B
DegkKS Forward &
Reverse
- 95 50 72
C
DegkKS Forward &
Reverse
1:2 95 50 72
D
27 Forward &
1492 Reverse
1:4 95 55 72
![Student: Lauren Crampton, A00200702, Supervisor: Donal Eardly Department of Life Sciences, Athlone Institute of Technology.
Antibiotics such as penicillin have transformed the way people are treated for a multitude of diseases. The potency of these drugs has reduced dramatically due to
bacteria developing resistance against them. Ling et al. (2015), explains that the majority of antibiotics were found and formulated from isolating easily cultivated soil
micro-organisms, such as Prokaryotic micro-organisms. Antibiotics have been derived from prokaryotic microbes and are produced using a Polyketide Synthase
Pathway. There are two main classes of PKS genes called PKS Type I and PKS Type II.
• Obtain good quality soil samples
• Sequence & Amplify those genes
in PCR machine
• Locate & Identify Type II PKS gene
• Clone these PCR products using
TOPO – TA vectors
• Collected a range of soil samples
• Extracted Total DNA from soil using MO
BIO PowerSoil™ DNA Isolation Kit
• Tested DegKs Forward and Reverse
Primers
• Troubleshooted PCR reactions and DNA
Isolation Techniques
• Collected and Re-Isolated soil samples
No amplification; table below is examples
of troubleshooting efforts
Were aims and objectives met?
Yes, some were met, however the sequencing
and amplification of Type I & II PKS genes in a
PCR machine was unsuccessful.
Recommendations for Future Work
• Fresh Soil Sample Collection
• Re-Isolation (Practise & Improve
Technique)
• Check Purity and Concentration of DNA
Isolated
• Purify and alter DNA Concentration where
required
• Test Master Mix and Primers on other DNA
samples from other research projects
• MO BIO and Further PCR Troubleshooting
• Cloning Vectors
Ling, L.L. et al. (2015). ‘A new antibiotic kills pathogens without detectable resistance.’ Nature. 517 (2015) pp. 455 – 459.
Sriprapun, M. (2014). ‘Principles of PCR.’ [Online Image] Available at: http://www.slideshare.net/MetheeSri/principle-of-pcr. [Accessed 25th November 2015]
Label Primers Dilution
Denaturation
Temperature
(°C)
Annealing
Temperature
(°C)
Extension
Temperature
(°C)
A
DegkKS Forward &
Reverse
- 95 55 72
B
DegkKS Forward &
Reverse
- 95 50 72
C
DegkKS Forward &
Reverse
1:2 95 50 72
D
27 Forward &
1492 Reverse
1:4 95 55 72](https://image.slidesharecdn.com/8df4c436-1d15-47ca-978d-835a17b1591b-160720183936/85/FINAL-POSTER-1-320.jpg)
