Team:Newcastle/Results/Endophyte

Alternative Roots/Results

Alternative Roots

Endophytic Chassis

Introduction Antibiotic Testing Transformation New Part Conclusion
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Introduction

Root colonisation experiments indicated that Pseudomonas sp. strain CT 364 was successfully able to colonise the roots of Arabidopsis. The next step was to develop Pseudomonas sp. as an endophytic chassis, so that roots could be colonised by a Pseudomonas sp. strain that had been genetically engineered to express gene(s) of interest. This involved three main objectives: identifying suitable selectable markers, establishing a transformation protocol for Pseudomonas sp., and the design, assembly and characterisation of a new genetic part.

Antibiotic Testing

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.As Pseudomonas sp. is a soil bacterium it has an optimum growth temperature of 28 °C. Pure cultures were obtained following revival after overnight incubation (Figure 1).

Figure 1. Pseudomonas sp. strain CT 364 plated on tryptone soy agar.

The first step in the chassis development was to identify antibiotics that Pseudomonas sp. was susceptible to. This would indicate which selectable markers were suitable for use in transformation procedures. Five antibiotics were tested, chloramphenicol, kanamycin, gentamicin, carbenicillin and streptomycin. Kanamycin, gentamicin and streptomycin are all aminoglycosides with similar but independent modes of action interfering with protein synthesis via ribosomal binding. Chloramphenicol prevents protein chain elongation and carbenicillin interferes with cell wall synthesis. Many Pseudomonas species are known to be resistant to chloramphenicol but this antibiotic was tested to determine compatibility with pSB1C3 (1). Carbenicillin and the aminoglycosides have been identified as active against many Pseudomonas species (2 and 3).

Antibiotic 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 two replicates for each concentration. Lawns formed on agar containing 100 µg/ml of each antibiotic (Figures 2, 3 & 4) after 24 hours incubation at 28 °C.

Figure 2. Pseudomonas sp. strain CT 364 plated on tryptone soy agar containing chloramphenicol (100 µg/ml).

Figure 3. Pseudomonas sp. strain CT 364 plated on tryptone soy agar containing carbenicillin (100 µg/ml).

Figure 4. Pseudomonas sp. strain CT 364 plated on tryptone soy agar containing kanamycin (100 µg/ml).

Screening found Pseudomonas sp. to be susceptible to both streptomycin (Figure 5) and gentamicin (Figure 6). Antibiotic concentrations of 50 and 100 µg/ml were again tested with two replicates for each concentration. No colony forming units (CFUs) were visible on tryptone soy agar containing 50 µg/ml of either antibiotic inoculated with Pseudomonas sp. after 24 hours incubation at 28 °C.

Figure 5. Pseudomonas sp. strain CT 364 plated on tryptone soy agar containing streptomycin (100 µg/ml).

Figure 6. Pseudomonas sp. strain CT 364 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. To minimise variation between replicates MIC experiments were automated using an Opentrons OT-2 pipetting robot.

The results of 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 minimum concentration was clear for gentamicin with a significant drop in growth rate between concentrations of 1.0 µg/ml and 1.5 µg/ml. The minimum inhibitory concentration for streptomycin was less clear, with a small drop in growth rate for each increase in concentration. A working concentration of 6.0 µg/ml was determined for streptomycin. A slight increase in absorbance was observed in the positive control for both antibiotics. This is likely due to release of compounds by bacterial cells upon death.

Figure 7. Pseudomonas sp. strain CT 364 grown in tryptone soy broth containing gentamicin at varying concentrations. Cells were grown in 96-well plate format in 200 µl volumes at 28 °C over 24 hours. (n=4 replicates, error bars are standard error of the mean).

Figure 8. Pseudomonas sp. strain CT 364 grown in tryptone soy broth containing streptomycin at varying concentrations. Cells were grown in 96-well plate format in 200 µl volumes at 28 °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 use in transformation. Concentrations of 2.0 µg/ml, 4.0 µg/ml (Figure 9) and 6.0 µg/ml (Figure 10) were tested as the working concentration was expected to be slightly higher than the 1.5 µg/ml MIC in liquid culture due to lower availability of antibiotic in agar. It was necessary to establish a working concentration in agar as cells would be plated onto agar following transformation. Gentamicin was selected as the working antibiotic as it is known to stunt Arabidopsis growth (4). Treatment of Arabidopsis with a gentamicin resistant transformant could prevent stunting of growth by breaking down gentamicin in the rhizosphere (5). Demonstrating that a plant colonising microbe can be used to change the physiology of the plant.

Figure 9. Pseudomonas sp. strain CT 364 plated on tryptone soy agar containing gentamicin (4 µg/ml).

Figure 10. Pseudomonas sp. strain CT 364 plated on tryptone soy agar containing gentamicin (6 µg/ml).

Agar assays showed that concentrations of 2.0 µg/ml and 4.0 µg/ml of antibiotic were insufficient to prevent growth of Pseudomonas sp. on agar. A lawn formed on agar containing 2.0 µg/ml and some colonies formed on agar containing 4.0 µg/ml (Figure 9). A gentamicin concentration of 6.0 µg/ml was sufficient to prevent growth of Pseudomonas sp. as no colonies formed at this concentration (Figure 10).

Transformations

Once working concentrations had been determined for both antibiotics, transformation of Pseudomonas sp. could be attempted. Addgene plasmid number 79813 was selected for transformation protocol optimisation due to its Pseudomonas origin of replication and gentamicin resistance gene. The function of this plasmid was as a helper plasmid for conjugation. This plasmid could therefore also be used to optimise conjugation protocols for Pseudomonas sp. if transformation of larger plasmids is required. The plasmid was supplied by Addgene in E. coli and a Qiagen QIAprep Spin Miniprep Kit was used to extract the plasmid from overnight liquid cultures. Plasmid extractions were divided into multiple aliquots and a vacuum concentrator was used to remove water from some samples resulting in three solutions with varying DNA concentrations (Table1).

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



Electroporation was chosen as the first transformation method to be tested on Pseudomonas sp. primarily because it was the most utilised transformation method for Pseudomonas in available literature. It was also advantageous due to the quick procedure for preparing electrocompetent cells allowing high throughput experimentation. Heat-shock and conjugation were identified as potential other transformations methods should electroporation fail.

Four electroporation experiments were carried out, one for each DNA solution and a negative control where water was added in place of a DNA solution. Electrocompetent cells were prepared by carrying out five washes with 300 mM sucrose solution. Electroporation reaction mixes consisted of 100 µl of competent cells with 2 µl of plasmid DNA solution.

Gentamicin resistant colonies were obtained for each of the DNA solutions with solution 1 containing the highest DNA concentration yielding the highest number of CFUs (Figure 11) and solution 3 containing the lowest DNA concentration yielding the fewest colonies (Figure 13). Testing with higher DNA concentrations would be required to determine if more CFUs can be obtained. No colonies formed where the negative control had been plated onto selective media (Figure 14). Colony forming units obtained from each DNA solution are displayed in Table 2.

Figure 11. Pseudomonas sp. strain CT 364 transformed with gentamicin resistance plasmid from DNA solution 1 plated on TSA containing gentamicin (10 μg/ml).

Figure 12. Pseudomonas sp. strain CT 364 transformed with gentamicin resistance plasmid from DNA solution 2 plated on TSA containing gentamicin (10 μg/ml).

Figure 13. Pseudomonas sp. strain CT 364 transformed with gentamicin resistance plasmid from DNA solution 3 plated on TSA containing gentamicin (10 μg/ml).

Figure 14. Pseudomonas sp. strain CT 364 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 conducted to characterise gentamicin resistance of the transformant (Figure 15). This experiment was carried out on a 96-well plate using the OT-2 robot. More replicates were used than previously as fewer concentrations were being tested leaving more space on the plate, as a result standard error was reduced compared to previous MICs. A low concentration of 5.0 µg/ml was tested to determine if the transformant was significantly more resistant than the wild-type that is eliminated by a working concentration of 1.5 µg/ml. A 10 times concentration of 50 µg/ml was also tested to determine if there was an upper limit to resistance. The transformant was resistant to gentamicin at the highest concentration tested (50 µg/ml) whereas the wild type was unable to grow at the lowest concentration tested (5.0 µg/ml).

Figure 15. 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).

New Part

A composite part containing the antibiotic resistance gene aadA (BBa_K125520), an Anderson promoter (BBa_J23119), a strong RBS (BBa_B0034) and a native terminator (BBa_K2797001) new to the registry was designed (Figure 16). This part conferred resistance to streptomycin, the second antibiotic found to be active against Pseudomonas sp. The part was synthesised as a gBlock by IDT with compatible ends for insertion into the pSB1C3 backbone using Gibson assembly. This part was assembled into pSB1C3 for characterisation and submission to the registry. The composite part was inserted into the backbone using a NEBuilder Hi-Fi Gibson assembly kit. A vector:insert molar ratio of 1:2 was used; the reaction mix is shown in Table 3. A positive control assembly reaction was also carried out using a standard NEBuilder control DNA mix containing a backbone and two gBlocks. This was mixed with NEBuilder Hi-Fi assembly mix in a 1:1 ratio.

Figure 16. Composite part BBa_K2797002 conferring resistance to streptomycin.



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



Multiple E. coli strains have been found to be resistant to streptomycin; therefore before transformations were carried out, DH5α the strain of E. coli being used for transformations was tested for resistance to streptomycin. Concentrations of 5.0, 50 and 100 µg/ml were tested in LB agar, with 50 µg/ml being the concentration recommended by Addgene. A concentration of 5.0 µg/ml was insufficient to prevent growth (Figure 17) with colony forming units visible after 24 hours incubation at 37 °C. No colonies formed on agar containing 50 µg/ml of streptomycin (Figure 18), this concentration was therefore used when plating transformants.

Figure 17. E. coli strain DH5α plated on LB agar containing streptomycin (5.0 μg/ml).

Figure 18. E. coli strain DH5α plated on LB agar containing streptomycin (50 μg/ml).

Following assembly, chemically competent E. coli cells were transformed by heat shock using each reaction mix. CFUs were obtained for both the streptomycin resistance Gibson assembly reaction mix (Figure 17) and the positive control (Figure 18). No colonies formed on agar inoculated with the negative control (Figure 19).

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 transformed with Gibson assembly positive control plated on LB agar containing ampicillin (100 µg/ml).

Figure 19. 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 onto agar containing either streptomycin (50 µg/ml) (Figure 20) or chloramphenicol (25 µg/ml) (Figure 21). This indicated that transformants containing the correctly assembled plasmid (BBa_K2797002 inserted into the pSB1C3 backbone) were resistant to both antibiotics. As the BBa_K2797002 and the pSB1C3 backbone confer resistance to streptomycin and chloramphenicol respectively.

Figure 20. E. coli strain DH5α transformed with streptomycin resistance part BBa_K2797002 plated on LB agar containing streptomycin (50 μg/ml).

Figure 21. 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 22). The transformant was resistant to streptomycin at the highest concentration tested (64 µg/ml). The wild-type E. coli showed greater resistance to streptomycin than Pseudomonas sp. with residual growth in wells containing both 16 µg/ml and 32 µg/ml of streptomyin.

Figure 22. E. coli DH5α with or without the BBa_K2797002 (SmRp) 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).

Conclusions

Pseudomonas sp. was found to be susceptible to two antibiotics, streptomycin and gentamicin. Working concentrations were determined for both of these antibiotics using solid and liquid media. These data firstly aided the selection of a suitable plasmid for use in transformation protocols, and also helped to inform the design of a new part. The selected plasmid (Addgene plasmid 79813) was used in successful transformations of Pseudomonas sp. using electroporation, demonstrating that Pseudomonas sp. is genetically tractable and offers potential as an endophytic chassis. Finally, a new streptomycin resistance part, conferring strong resistance to streptomycin in E. coli, was designed, built and characterised.

The Pseudomonas sp. transformation protocol could be optimised by implementing the automated transformation optimisation methods established in our Measurement work. These methods have proved effective for the optimisation of E. coli transformation protocols, so we anticipate that the same methodologies could be applied to improving transformation efficiency in Pseudomonas sp. These optimised protocols could also be used to examine promoter strength in Pseudomonas sp. using the improved measurement devices constructed by the team. The new streptomycin resistance part (BBa_K2797002) could also be introduced into Pseudomonas sp. using optimised transformation procedures and used for selection of Pseudomonas sp. transformants.

Refrences & Attributions

1.Fernandez M, et al. (2012) Mechanisms of Resistance to Chloramphenicol in Pseudomonas putida KT2440. Antimicrobial Agents and Chemotherapy 56(2):1001.

2.Grollier G, Agius G, & Borion N (1991) Determination of Pseudomonas aeruginosa susceptibility to aminoglycosides using API and Autobac semi-automated systems. Journal of Antimicrobial Chemotherapy 27(6):868-870.

3.Hill SF, Haldane DJ, Ngui-Yen JH, & Smith JA (1985) In vitro susceptibility of Pseudomonas species to carbenicillin and trimethoprim-sulfamethoxazole. Journal of Clinical Microbiology 22(3):465.

4.Hayford MB, Medford JI, Hoffman NL, Rogers SG, & Klee HJ (1988) Development of a plant transformation selection system based on expression of genes encoding gentamicin acetyltransferases. Plant physiology 86(4):1216.

5.Gopalan S & Ausubel FM (2011) A High Throughput Amenable Arabidopsis- P. aeruginosa System Reveals a Rewired Regulatory Module and the Utility to Identify Potent Anti-Infectives (Host-Microbe Adaptations and Anti-Infectives). PLoS ONE 6(1):e16381.

Attributions: Frank Eardley and Lewis Tomlinson