Team:Newcastle/Results/Endophyte
Alternative Roots
Endophytic Chassis Results
RESULTS
Developing Pseudomonas as a new endophytic chassis
Week Beginning 06/08
Pure cultures were obtained following plating out of the sample that arrived from DSMZ.
Week Beginning 13/08
Screening on tryptone soy agar (TSA) showed Pseudomonas sp. to be highly resistant to chloramphenicol with lawns forming on plates containing the highest concentration of chloramphenicol tested (Figure 1).
Figure 2. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing chloramphenicol (100 µg/ml)
Screening on agar also found Pseudomonas sp. to be resistant to kanamycin and carbenicillin with lawns forming on plates containing the highest concentration of chloramphenicol tested (Figure 1)
Figure 3. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing carbenicillin (100 µg/ml)
Figure 4. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing carbenicillin (100 µg/ml)
The results showed that Pseudomonas sp. was susceptible to both streptomycin (Figure 5) and gentamicin (Figure 6) with no colony forming units visible on either the 50 µg/ml or 100 µg/ml plates for both antibiotics
Figure 5. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing streptomycin (100 µg/ml)
Figure 6. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (100 µg/ml)
Week beginning 20/08
Week beginning 27/08
Wild type Pseudomonas sp. Isolated in pure culture from surface-sterilised seedlings inoculated by the seed-coating method. Surface-sterilised non-inoculated seedlings were used as a control from which only wild endophytes were obtained.
Week beginning 03/09
The results of our minimum inhibitory concentration experiments showed a clear dose response between antibiotic concentration and growth of Pseudomonas sp. for both streptomycin and gentamicin (Figures 7 and 8). Gentamicin was found to be the more effective antibiotic with a concentration of 1.5 µg/ml sufficient to prevent growth. A concentration of 6.0 µg/ml of streptomycin was required to prevent growth. A slight increase in absorbance was observed for the positive control for both antibiotics. This is likely due to release of compounds by bacterial cells upon death.
Figure 7. Pseudomonas sp. DSM 25356 grown in tryptone soy broth containing gentamicin at varying concentrations. Cells were grown in 96-well plate format in 200 µl volumes at 37 °C over 24 hours. (n=4 replicates, error bars are standard error of the mean)
Figure 8. Pseudomonas sp. DSM 25356 grown in tryptone soy broth containing streptomycin at varying concentrations. Cells were grown in 96-well plate format in 200 µl volumes at 37 °C over 24 hours. (n=4 replicates, error bars are standard error of the mean).
Week Beginning 10/09
Miniprep DNA concentrations,
Transformation plates
Table of DNA concentrations and CFUS
Prior to tran
Figure 9. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (6 µg/ml)
Figure 10. Pseudomonas sp. DSM 25356 transformed with gentamicin resistance plasmid from miniprep 1 plated on TSA containing gentamicin (10 μg/ml).
Figure 11. Pseudomonas sp. DSM 25356 transformed with gentamicin resistance plasmid from miniprep 2 plated on TSA containing gentamicin (10 μg/ml).
Figure 12. Pseudomonas sp. DSM 25356 transformed with gentamicin resistance plasmid from miniprep 3 plated on TSA containing gentamicin (10 μg/ml).
Figure 13. Pseudomonas sp. DSM 25356 transformed with sterile water (Control) plated on TSA containing gentamicin (10 μg/ml).
Using seedlings inoculated with wild type Pseudomonas sp., DAPI staining and bright field microscopy were used to visualise the relationship between plant and bacteria. This revealed a biofilm was present on the surface of the root however it was unclear if the bacteria was inside the root, so seedlings were washed in distilled water. Examination of the cleaned seedlings revealed clear signs of colonisation and Pseudomonas sp. was present in intercellular spaces along both the root and hypocotyl. The bacteria were still motile and we could see them moving in real time.
Figure 14. Bright field microscopy of a DAPI stained Arabidopsis thaliana root at 40x magnification. Wild type Pseudomonas sp. is visible in the intercellular spaces.
Week Beginning 17/09
Gibson assembly contents table?
Figure 15. E. coli strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml).
Figure 16. E. coli strain DH5α transformed with Gibson assembly positive control plated on LB agar containing ampicillin (100 µg/ml)
Figure 17. E. coli strain DH5α transformed with sterile water (negative control) plated on LB agar containing streptomycin (50 µg/ml)
Figure 18. E. coli strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml)
Figure 19. E. coli strain DH5α carrying the streptomycin resistance part BBa_K2797002 plated on LB agar containing chloramphenicol (25 μg/ml). The part is in the pSB1C3 backbone conferring resistance to chloramphenicol.
Week Beginning 24/09
Figure 20. E. coli DH5α with or without the BBa_K2797002 part in pSB1C3 were grown in LB medium containing streptomycin at varying concentrations. Cells were grown in 96-well plate format in 200 μl volumes at 37 °C over 24 hours. (n=3 replicates, error bars are standard error of the mean).
A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed and DAPI stained.Endophytic bacteria were visible inside the root and hypocotyl of both Arabidopsis thaliana and Eruca sativa seedlings showing that transformation had not altered the bacteria’s ability to colonise.
Figure 21. Bright field microscopy of an Arabidopsis thaliana seedling root at x100 magnification,showing transformed Pseudomonas sp. living as an endophyte in the intercellular spaces.
Week Beginning 01/10
Figure 22. Pseudomonas sp. with or without gentamicin resistance plasmid were grown in tryptone soy broth containing gentamicin at varying concentrations. Cells were grown in a 96-well plate format in 200 μl volumes at 28 °C over 24 hours. (n=5 replicates, error bats are standard error of the mean)
A selection of seedlings were again selected for microscopy, this time negative control E. coli DH5α inoculated seedlings were examined. Microscopy revealed that though E. coli was present on the root surface, there was no sign of endophytic relationship like that of Pseudomonas sp.. Further images were taken of transformant Pseudomonas sp. inoculated seedlings to aid visual demonstration of our transformed endophyte working.
Figure 23.Bright field microscopy showing the root of our negative control Arabidopsis thaliana at100x magnification, where E. coli DH5α was visible on the surface of the root in small numbers but not inside the root as an endophyte.
REFERENCES & Attributions
Attributions: Frank Eardley and Lewis Tomlinson