The first steps in developing Pseudomonas sp. (CT 364) involve identifying antibiotics active that it is susceptible to in order to select transformants and optimistaion of transformation protocols. Five antibiotics were tested and two were found to be active against Pseudomonas sp. Working concentrations were identified for each antibiotic using lab automation. Additionally, a new streptomycin resistance cassette was constructed to be used in building plasmids for Pseudomonas sp.

Pseudomonas sp. (CT 364) was obtained from DSMZ, Germany (DSM No.: 25356). The strain arrived freeze-dried and was revived according to the protocol recommended by DSMZ.(Figure 1)

Screening on tryptone soy agar (TSA) showed Pseudomonas sp. to be resistant to chloramphenicol, kanamycin and carbenicillin. Antibiotic concentrations of 50 and 100 µg/ml were tested with lawns forming on agar containing 100 µg/ml of each antibiotic. (Figures 2, 3 & 4) after 24 hours incubation at 28 °C.

Figure 2. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing chloramphenicol (100 µg/ml)

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)

Screening on TSA showed that Pseudomonas sp. was susceptible to both streptomycin (Figure 5) and gentamicin (Figure 6) with no colony forming units (CFUs) visible on agar containing 50 µg/ml of either antibiotic after 24 hours incubation at 28 °C.

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)

After identifying which antibiotics were active against Pseudomonas sp. The next step was to identify working concentrations of these antibiotics to be used when selecting transformants. This was done by carrying out minimum inhibitory concentration (MIC) experiments where growth was tested against a range of antibiotic concentrations.

The results of our MIC 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).

After identifying the MIC in liquid culture, the MICs in agar were determined for gentamicin as this was the antibiotic being taken forward for transformation.

Figure 8. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (4 µg/ml)

Figure 9. Pseudomonas sp. DSM 25356 plated on tryptone soy agar containing gentamicin (6 µg/ml)

Agar assays showed that concentrations of 2 µg/ml and 4 µg/ml of antibiotic were insufficient to prevent growth of Pseudomonas sp. on agar with a lawn forming on 2 µg/ml and some colonies forming on 4 µg/ml (Figure 7). A gentamicin concentration of 6 µg/ml was sufficient to prevent growth of Pseudomonas sp. as no colonies formed at this concentration (Figure 8).

Once working antibiotic concentrations had been characterised for both antibiotics, transformations could be carried out. Addgene plasmid number 79813 was selected for transformation protocol optimisation due to its Pseudomonas origin of repolication and gentamicin resistance gene. Two minipreps were carried out on the plasmid containing E. coli strain, one of which was aliquoted and spun down giving three DNA solutions of varying DNA concentration (Table1).

Table 1. Concentration of DNA solutions obtained from miniprep of E. coli containing Addgene plasmid #79813

Four electroporation experiments were carried out, one for each DNA solution and a negative where water was added in place of a DNA solution. Gentamicin resistant colonies formed for each of the DNA solutions with solution 1 giving the highest number of CFUs (Figure 9) and miniprep 3 the lowest (Figure 11). No colonies formed on agar inoculated with the negative control (Figure 12). Colony forming units obtained from each miniprep are displayed in Table 2.

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).

Table 2. Colonies formed from electrocompetent Pseudomonas sp. inoculated with solutions containing Addgene plasmid #79813

An additional MIC experiment was carried out to characterise gentamicin resistance of the transformant (Figure 20). The transformant was resistant to gentamicin at the highest concentration tested (50 µg/ml).

Figure 14. 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 bars are standard error of the mean)

A composite part containing the antibiotic resistance gene aadA, an anderson promoter, a strong RBS and a native terminator was designed. This part conferred resistance to streptomycin, the second antibiotic found to be active against Pseudomonas sp. This part was assembled into pBS1C3 for characterisation and submission to the registry. The Gibson assembly reaction mix is shown in Table 3.

Table 3. Volume used of each reagent in Gibson assembly reaction mix.

Following assembly, chemically competent E. coli cells were transformed using each reaction mix. CFUs were obtained for both the streptomycin resistance Gibson assembly reaction mix (Figure 13) and the positive control (Figure 15). No colonies formed on agar inoculated with the negative control (Figure 16).

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)

Following transformation, colonies were re-streaked on agar containing streptomycin (50 µg/ml) (Figure 17) confirming resistance and on agar containing chloramphenicol (25 µg/ml) as resistance was confirmed by the pBS1C3 backbone (Figure 18).

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.

Streptomycin resistance of the transformed E. coli was compared to the wild type (Figure 19). The transformant was resistant to streptomycin at the highest concentration tested (64 µg/ml).

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).

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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 bars are standard error of the mean)

Pseudomonas sp. was found to be susceptible to two antibiotics, streptomycin and gentamicin. Using a Pseudomonas origin plasmid containing a gentamicin resistance gene electroporation protocols have been optimised for Pseudomonas sp. The new streptomycin resistance part confers strong resistance to streptomycin in E. coli.