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 7. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (4 µg/ml)

Figure 8. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (6 µg/ml)

Figure 9. Pseudomonas sp. DSM 25356 transformed with gentamicin resistance plasmid from miniprep 1 plated on TSA containing gentamicin (10 μg/ml).

Figure 10. Pseudomonas sp. DSM 25356 transformed with gentamicin resistance plasmid from miniprep 2 plated on TSA containing gentamicin (10 μg/ml).

Figure 11. Pseudomonas sp. DSM 25356 transformed with gentamicin resistance plasmid from miniprep 3 plated on TSA containing gentamicin (10 μg/ml).

Figure 12. Pseudomonas sp. DSM 25356 transformed with sterile water (Control) plated on TSA containing gentamicin (10 μg/ml).

Bright field microscopy was used to visualise wild type Pseudomonas sp. as a biofilm on root surfaces, as an endophyte in the root and as an endophyte in the hypocotyl of Arabidopsis thaliana seedlings.

Week Beginning 17/09

Gibson assembly contents table?

Figure 13. E. coli strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml).

Figure 15. E. coli strain DH5α transformed with Gibson assembly positive control plated on LB agar containing ampicillin (100 µg/ml)

Figure 16. E. coli strain DH5α transformed with sterile water (negative control) plated on LB agar containing streptomycin (50 µg/ml)

Figure 17. E. coli strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml)

Figure 18. 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 19. 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 20. 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)

Bright field microscopy revealed that the negative control of E. coli DH5α was present on root surfaces but not as an endopyhte in Arabidopsis thaliana seedlings.