Team:Newcastle/Notebook/Endophyte
Alternative Roots
Endophytic Chassis Notebook
NOTEBOOK
Developing Pseudomonas as a new endophytic chassis
Week Beginning 20/07
Preliminary work began with the team developing agar-based germination methods, this was mainly due to the lack of plant research experience in the team. The team conceptualised growing Arabidopsis in microcentrifuge tubes within pipette-tip boxes.
The team planted their first set of 16 Arabidopsis seeds in 1% agar in a pipette-tip box placed on the lab windowsill. This would give an indication as to if these conditions were suitable for growth. After 7 days 12/16 seeds had germinated showing this method was appropriate.
Week Beginning 30/07
Development of our new endophytic chassis began on the 1st of August with the arrival of root colonising Pseudomonas sp. DSM25356 from DSMZ in Germany. The strain arrived in a glass ampoule and was inoculated onto tryptone soy agar (TSA) plates using methods outlined by DSMZ. The plates were incubated at 28 ℃ for 24 hours after which they were used to inoculate tryptone soy broth (TSB) for initial growth characterisation and antibiotic testing.
Week Beginning 06/08
In order to select transformants when introducing foreign DNA, we first needed to determine antibiotics active against Pseudomonas sp. We initially planned to transform Pseudomonas sp. with the one of the interlab test devices we modified to contain mNeonGreen instead of GFP. Creating a mutant Pseudomonas sp. with strong fluorescence would be useful for identification when carrying out microscopy on roots inoculated with Pseudomonas sp. As the test devices are in a pSB1C3 backbone, activity of chloramphenicol against Pseudomonas sp. had to be characterised. To test activity 200 µl of Pseudomonas sp. TSB overnight culture was spread onto TSA containing 50 and 100 µg/ml of chloramphenicol.
Week Beginning 13/08
As Pseudomonas sp.. DSM 25356 was found to be resistant to chloramphenicol, a selection of four further antibiotics (kanamycin, gentamycin, streptomycin and carbenicillin) were test for activity against Pseudomonas sp. 20 ml of stock solution was prepared for each of the antibiotics at concentrations show in Table 1 and water or an ethanol solution was used as the solvent. Stock were distributed into 1 ml aliquots and frozen at -20 °C.
TSA plates were prepared containing 50 µg/ml and 100 µg/ml of each antibiotic for screening of activity against Pseudomonas sp. Two replicates were prepared for both concentrations for each of the four antibiotics. 200 µl of overnight culture was spread onto each plate which were subsequently incubated overnight at 28 °C
Week Beginning 20/08
Week Beginning 27/08
After identifying streptomycin and gentamycin as active against Pseudomonas sp. the next step was to characterise the susceptibility to each antibiotic by identifying the minimum inhibitory concentration for each antibiotic. To do this a range of concentrations needed to be tested on a 96-well plate format to produce a ‘kill curve’. To improve the accuracy of these experiments were carried out using the Opentrons OT-2 robot won by the team. For this python code for the robot had to be designed specific to these experiments. Much of this week was spent outside of the lab working on the code. The code was designed in order to distribute liquid from 4 different sources onto a 96-well plate. These four sources were sterile tryptone soy broth, Pseudomonas sp. liquid culture, antibiotic solution and sterile distilled water. The code used a list function to define the volume of each of the four solutions distributed into each well. These experiments also utilised the universal racks designed and assembled by the team originally for carrying out heat-shock transformations using the robot.
Following engagement with GrowUp Urban Farms, it was suggested that we use a seed coating inoculation method, rather than wounding as we intended, this makes our engineered microbe more accessible for commercial use. As a preliminary experiment to test this, Arabidopsis seeds were sterilised before being coated in Pseudomonas sp. liquid culture. Seeds were then planted in 1% agar and allowed to germinate, after 1 week 7 of these seedlings were surface sterilised, cut and plated on nutrient agar plates. On all 7 of these plates Pseudomonas sp. was re-isolated in pure culture.
Week Beginning 03/09
This week started with the first testing of our python code for the minimum inhibitory concentration experiments. The first problem encountered with the code was that when using the 10 µl tip the robot was not completely dispensing all the liquid out of the tip and subsequently dripping into other wells causing contamination. This problem was fixed by adding a blow-out function to the code, this pushed air through the tip after the initial distribution to remove any remaining fluid. The second problem encountered was that the working volume of the 20 ml universals used to hold each of the four solutions was not sufficient for the volume of sterile broth required. As the OT-2 pipette returns to the same position for each aspiration rather aspirating from a lower position as the solution is used up the working volume of the universal is limited by the height of the pipette tip. This equated to a working volume of approximately 8 ml, we therefore added two more universal containers to the labware to hold sterile broth. The perimeter wells were not used for the assay and were each filled with 200 µl of sterile broth due to reduced accuracy when measuring absorbance in these wells. Three plates were run for each antibiotic using three different stock concentrations. The assays used an antibiotic stock concentration of 1 mg/ ml, 100 µg/ml and 10 µg/ml.
The team decided that the most valuable way to assess endophytic relationship would be to use microscopy to visualise Pseudomonas sp. inside the plant. As a positive control a set of 96 Arabidopsis seeds were surface sterilised and coated in wild type Pseudomonas sp. liquid culture. These seeds were planted in 1% agar and allowed to germinate on the laboratory windowsill (PICTURE?).
Week Beginning 10/09
Following on from successful characterisation of the minimum inhibitory concentration in both liquid culture and solid agar for each antibiotic transformations were attempted. Addgene plasmid number 79813 was selected for introduction into Pseudomonas sp. This plasmid was selected as it was the only plasmid found that was isolated from a Pseudomonas species and contained either a gentamycin or a streptomycin resistance gene. The plasmid arrived from Addgene in E. coli and was streaked onto LB agar containing streptomycin (50 µg/ml). After incubation overnight at 37 °C two samples of the plasmid were isolated from E. coli using a Qiagen QIAprep spin miniprep kit. The DNA concentration of each sample was then quantified using a Qubit fluorometer. Each sample was then split into two aliquots and one aliquot from each sample was spun down using a DNA Speedvac resulting in four samples with a range of DNA concentrations.
Electroporation was chosen as the first method of transformation for testing as it was the most documented method for Pseudomonas. An overnight culture of Pseudomonas sp. were made electrocompetent by washing with 300mM sucrose solution. Electroporation was performed with three different DNA concentrations along with a control containing only sterile water. Electroporated cells were resuspended in TSB and incubated at 28 °C for 3 hours before spreading onto TSA containing gentamycin (10 µg/ml).
During this week the team also designed a streptomycin resistance gene that could be used in Pseudomonas sp. The design consisted of an aadA gene under the control of a strong Anderson promoter and strong RBS, a native terminator new to the iGEM registry was also included to prevent any readthrough.
Using the seedlings inoculated with wild type Pseudomonas sp. last week, bright field microscopy was 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 were 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. (PICTURE)
Week Beginning 17/09
The streptomycin resistance G block was assembled into a pSB1C3 backbone by Gibson assembly. The G block was diluted to a concentration 10 ng/µl as advised by IDT. The backbone solution used was a concentration of 26.2 ng/µl. 50 femtomoles of backbone and 100 femtomoles of insert was required for the reaction mix. The backbone solution contained 21.28 femtomoles in 1 µl. 2.35 µl of backbone solution was therefore added to the reaction mix. 1 µl of insert solution contained 17.42 femtomoles, 5.74 µl of insert was therefore added to the reaction mix. This gave a total of 8.09 µl of DNA solution, an equal volume of Gibson assembly master mix was added to the DNA solution. A positive control reaction mix was also prepared containing 10 µl of DNA and 10 µl of Gibson assembly master mix. Each sample was incubated at 50 °C for 60 minutes on a heat block and stored at room temperature until transformation.
Prior to transformation E. coli DH5α cells were tested for susceptibility to streptomycin. LB agar plates containing streptomycin at concentrations of 10, 50 and 100 µg/ml of streptomycin and a control plate containing no antibiotic were prepared. Each plate was inoculated with overnight culture and incubated at 37 °C for 20 hours.
Heat shock transformations were performed using commercially competent E. coli DH5α cells. Three separate heat shock reactions were carried out, the positive control, the streptomycin resistance gene assembly, and a negative control containing only commercial grade water and competent cells.
Following successful transformation of Pseudomonas sp. a smorgasbord of Arabidopsis thaliana and Eruca sativa seeds were sterilised and coated in either transformed Pseudomonas sp. liquid culture or E. coli DH5α liquid culture (as a negative control) allowed to germinate on the windowsill ready for microscopy (ROCKET PICTURE).
Week Beginning 24/09
The assembled plasmid containing the streptomycin gene was isolated from E. Coli DH5α cells again using a Qiagen QIAprep spin miniprep kit and sent for sequencing using primers verification forward (VF2) and verification reverse (VR).
The streptomycin resistant part was characterised by comparing growth rates of the transformant and wild-type E. coli DH5α at a range of streptomycin concentrations on a 96-well plate. One in two Serial dilutions were performed from a wells containing streptomycin (64 µg/ml) and inoculated with transformant and wild-type overnight culture. The plate was subsequently incubated at 37 °C for 24 hours with a slow double orbital shake during which absorbance was measured at 600nm.
A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed, and 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.
Week Beginning 01/10
The gentamycin resistant transformant Pseudomonas sp. was characterised to quantify the strength of resistance. Transformant growth rates were compared to wild-type Pseudomonas sp. at gentamycin concentrations of 0 µg/ml, 5 µg/ml and 50 µg/ml on a 96-well plate. The plate was subsequently incubated at 28 °C for 24 hours with a slow double orbital shake during which absorbance was measured at 600nm.
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.
REFERENCES & Attributions
Attributions: Frank Eardley