Difference between revisions of "Team:Newcastle/Notebook/Endophyte"

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   <title>Alternative Roots/Notebook</title>
 
   <title>Alternative Roots/Notebook</title>
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                 <h1>
 
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                     Endophytic Chassis Notebook
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                     Root Colonisation
 
                     <br><br>
 
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                 <h3 class="subhead">NOTEBOOK</h3>
 
                 <h3 class="subhead">NOTEBOOK</h3>
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                 <h1 class="display-2">Developing <I>Pseudomonas</I> as a new endophytic chassis</h1>
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                 <h1 class="display-2">Root Colonisation</h1>
 
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                     <h3 class="h2">Week Beginning 16/07</h3>
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                     <h3 class="h2">Week Beginning 20/07</h3>
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<p><font size="3">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.</font></p>
 
<p><font size="3">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.</font></p>
  
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<p><font size="3">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.</font></p>
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<p><font size="3">The team firstly attempted to plant <i>Arabidopsis thaliana</i> seeds in a range of agar concentrations. Groups of 8 replicates were made at concentrations; 0.4 %, 0.6 %, 0.8 % and 1 % in bottomless microcentrifuge tubes and were left to chill over the weekend.</font></p>
  
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                    <h3 class="h2">Week Beginning 23/07</h3>
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<p><font size="3">The seeds from last week were taken from the fridge and placed in a pipette-tip box filled with water to approximately 1 mm below the microcentrifuge tube bottoms. After 5 days these seedlings were examined and all but the 1 % agar replicates had swollen and fallen through the microcentrifuge tubes into the bottom of the pipette-tip box. This showed that 1 % agar was appropriate for our uses.</font></p>
  
 
                     <h3 class="h2">Week Beginning 30/07</h3>
 
                     <h3 class="h2">Week Beginning 30/07</h3>
  
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<p><font size="3">Development of our new endophytic chassis began on the 1st of August with the arrival of root colonising <i>Pseudomonas</i> sp. CT 364 (DSM25356 from DSMZ in Germany). The strain arrived in a glass ampoule and was inoculated onto tryptone soy agar (TSA) plates using <font color="blue">methods outlined by DSMZ.</font> 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.</font></p>
 
  
 +
<p><font size="3">The team planted a 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 for lab-based plant growth.</font></p>
  
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<img class="NBimg" src="https://static.igem.org/mediawiki/2018/9/9f/T--Newcastle--AT.tube.jpg">
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<p class="center"><font size="2"><center>Figure 1. An <i>Arabidopsis thaliana</i> seedling growing in 1 % agar inside a microcentrifuge tube.</center></p>
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                    <h3 class="h2">Week Beginning 06/08</h3>
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<p><font size="3">In order to select transformants when introducing foreign DNA, we first needed to determine antibiotics active against <i>Pseudomonas</i> sp. We initially planned to transform <i>Pseudomonas</i> sp. with the one of the InterLab test devices we modified to contain mNeonGreen instead of GFP. Creating a mutant <i>Pseudomonas </i> sp. with strong fluorescence would be useful for identification when carrying out microscopy on roots inoculated with <i>Pseudomonas</i> sp.. As the test devices are in a pSB1C3 backbone, activity of chloramphenicol against <i>Pseudomonas</i> sp. had to be characterised. To test activity 200 µl of <i>Pseudomonas</i> sp. TSB overnight culture was spread onto TSA containing 50 and 100 µg/ml of chloramphenicol.</font></p>
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                    <h3 class="h2">Week Beginning 13/08</h3>
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<p><font size="3">As <i>Pseudomonas</i> sp. was found to be resistant to chloramphenicol, a selection of four further antibiotics (kanamycin, gentamycin, streptomycin and carbenicillin) were tested for activity against <i>Pseudomonas</i> 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. Stocks were distributed into 1 ml aliquots and frozen at -20 °C.</font></p>
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<p><font size="3"><center>Table 1. Antibiotic concentration and solvent of stock solutions prepared.<center></font></p>      
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<img src="https://static.igem.org/mediawiki/2018/d/da/T--Newcastle--endo-table1.png">
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<p><font size="3">TSA plates were prepared containing 50 µg/ml and 100 µg/ml of each antibiotic for screening of activity against <i>Pseudomonas</i> 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.</font></p>
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                     <h3 class="h2">Week Beginning 27/08</h3>
 
                     <h3 class="h2">Week Beginning 27/08</h3>
  
  
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<p><font size="3">After identifying streptomycin and gentamycin as active against <i>Pseudomonas</i> 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. To do 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, <i>Pseudomonas</i> 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.</font></p>
+
<p><font size="3">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 <i>Pseudomonas</i> 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 tryptone-soy agar plates.</font></p>
  
−
<p><font size="3">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 <i>Pseudomonas</i> 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 <i>Pseudomonas</i> sp. was re-isolated in pure culture.</font></p>
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<img class="NBimg" src="https://static.igem.org/mediawiki/2018/1/1f/T--Newcastle--AT.window.jpg">
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<p><font size="2"><i><center>Figure 2. Arabidopsis thaliana</i> seedlings growing in a pipette-tip box</center></p>
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      <img src="https://static.igem.org/mediawiki/2018/thumb/2/2e/T--Newcastle--Pseudomonas_re-isolate_plate_1.jpeg/730px-T--Newcastle--Pseudomonas_re-isolate_plate_1.jpeg">
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<p><font size="2"> Figure 1. <i>Pseudomonas</i> sp. on a TSA plate that has been re-isolated from <i>Arabidopsis thaliana</i> seedlings which have been inoculated by the seed-coating method suggested by GrowUp Urban Farms.</p>
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                     <h3 class="h2">Week Beginning 03/09</h3>
 
                     <h3 class="h2">Week Beginning 03/09</h3>
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<p><font size="3">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.</font></p>
 
  
 
<p><font size="3"> The team decided that the most valuable way to assess endophytic relationship would be to use microscopy to visualise <i>Pseudomonas</i> sp. inside the plant. As a positive control a set of 96 Arabidopsis seeds were surface sterilised and coated in wild type <i>Pseudomonas</i> sp. liquid culture. These seeds were planted in 1 % agar and allowed to germinate on the laboratory windowsill.</p>
 
<p><font size="3"> The team decided that the most valuable way to assess endophytic relationship would be to use microscopy to visualise <i>Pseudomonas</i> sp. inside the plant. As a positive control a set of 96 Arabidopsis seeds were surface sterilised and coated in wild type <i>Pseudomonas</i> sp. liquid culture. These seeds were planted in 1 % agar and allowed to germinate on the laboratory windowsill.</p>
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                     <h3 class="h2">Week Beginning 10/09</h3>
 
                     <h3 class="h2">Week Beginning 10/09</h3>
  
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<p><font size="3">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 <i>Pseudomonas</i> sp. This plasmid was selected as it was the only plasmid found that was isolated from a <i>Pseudomonas</i> sp. and contained either a gentamycin or a streptomycin resistance gene. The plasmid arrived from Addgene in <i>E. coli</i> 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 <i>E. coli</i> 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.</font></p>
 
  
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<p><font size="3">Electroporation was chosen as the first method of transformation for testing as it was the most documented method for <i>Pseudomonas</i> sp. An overnight culture of <i>Pseudomonas</i> sp. were made electrocompetent by washing with 300 mM 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).</font></p>
 
  
−
<p><font size="3">During this week the team also designed a streptomycin resistance gene that could be used in <i>Pseudomonas</i> 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.</font></p>
+
<p><font size="3">Following successful transformation of <i>Pseudomonas</i> sp. a smorgasbord of <i>Arabidopsis thaliana</I> and <i>Eruca sativa </i>seeds were sterilised and coated in either transformed <i>Pseudomonas</i> sp. liquid culture or <i>E. coli</i> DH5α liquid culture (as a negative control) allowed to germinate on the windowsill ready for microscopy. The wild type <i>Pseudomonas</i> sp. inoculated seedlings from the previous week were taken for microscopy with Dr Vasilios Andriotis where we found DAPI staining seedlings that had been gently washed and mounted on 15 % glycerol was a suitable way to visualise endophytes.</p>
  
−
<p><font size="3"> Using the seedlings inoculated with wild type <i>Pseudomonas</i> sp. last week, bright field microscopy was used to visualise the relationship between plant and bacteria. Our strain of <i>Pseudomonas</i> sp. produces fluorescent siderophores, causing green fluorescence, therefore we attempted to visualise the bacteria through a GFP filter. This was not successful as the auto-fluorescence of the plant cells was so much that bacteria were not distinguishable. Therefore staining was implemented, DAPI stain was added to 15 % glycerol and this was used to stain nucleotides meaning that bacteria and wounded/dead plant cells will be stained.</p>
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<img class="NBimg" src="https://static.igem.org/mediawiki/2018/8/86/T--Newcastle--Windowsill-Rocket.jpeg">
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<p><font size="2"><center>Figure 3. <I>Eruca sativa</i> seedlings growing in a contained environment on the laboratory windowsill.</center></p>   
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                    <h3 class="h2">Week Beginning 17/09</h3>
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<p><font size="3">The streptomycin resistance gBlock was assembled into a pSB1C3 backbone by Gibson assembly. The gBlock 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.</font></p>
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<p><font size="3">Prior to transformation <i>E. coli</i> 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.</font></p>
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<p><font size="3">Heat shock transformations were performed using commercially competent <i>E. coli</i> 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.</font></p>
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<p><font size="3"> Following successful transformation of <i>Pseudomonas</i> sp. a smorgasbord of <i>Arabidopsis thaliana</I> and <i>Eruca sativa </i>seeds were sterilised and coated in either transformed <i>Pseudomonas</i> sp. liquid culture or <i>E. coli</i> DH5α liquid culture (as a negative control) allowed to germinate on the windowsill ready for microscopy.</p>
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<img src="https://static.igem.org/mediawiki/2018/8/86/T--Newcastle--Windowsill-Rocket.jpeg">
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<p><font size="2"> Figure 3. <I>Eruca sativa</i> seedlings growing in a contained environment on the laboratory windowsill with a nice view of sunny Newcastle.</p>   
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                     <h3 class="h2">Week Beginning 24/09</h3>
 
                     <h3 class="h2">Week Beginning 24/09</h3>
  
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<p><font size="3">The assembled plasmid containing the streptomycin gene was isolated from <i>E. Coli </i>DH5α cells again using a Qiagen QIAprep spin miniprep kit and sent for sequencing using primers verification forward (VF2) and verification reverse (VR).</font></p>
 
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<p><font size="3">The streptomycin resistance part was characterised by comparing growth rates of the transformant and wild-type <i>E. coli DH5α</i> 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 600 nm.</font></p>
 
  
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<p><font size="3"> A selection of <i>Pseudomonas</i> sp. transformant-inoculated seedlings were taken for microscopy, again seedlings were washed and DAPI stained prior to visualisation.</p>
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<p><font size="3"> A selection of <i>Pseudomonas</i> sp. transformant-inoculated seedlings were taken for microscopy with Dr Andriotis, again seedlings were washed and DAPI stained prior to visualisation. An experiment was set up to compare the effects of the wild type and transformed <i>Pseduomonas</i> sp. on germination so a large agar plate containing 70 micrograms per millilitre gentamicin was made [1]. This will show if the transformation has made <i>Pseudomonas</i> sp. detrimental to plant growth. </p>
  
  
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<p><font size="3">The gentamycin resistant transformant <i>Pseudomonas</i> sp. was characterised to quantify the strength of resistance. Transformant growth rates were compared to wild-type <i>Pseudomonas</i> 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 600 nm.</font></p>
 
  
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<p><font size="3"> A selection of seedlings were again selected for microscopy, this time negative control <i>E. coli DH5α</i> inoculated seedlings were examined with bright field microscopy and DAPI staining. </p>
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<p><font size="3"> A selection of seedlings were again selected for microscopy, this time negative control <i>E. coli</i> DH5α inoculated seedlings were examined with bright field microscopy and DAPI staining. This opportunity was also used to capture more images of the transformed <i>Pseudomonas</i> sp. chassis in <i>Arabidopis thaliana</i> roots.</p>
  
  
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                 <h1 class="display-2">REFERENCES & Attributions</h1>
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                 <h1 class="display-2">References & Attributions</h1>
 
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<p class="about-para"><font size="3"><b>Attributions: Frank Eardley and Lewis Tomlinson<b></p>
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<p class="about-para"><font size="3"><b>Attributions: Frank Eardley, Luke Waller and Lewis Tomlinson</b></p>
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<p class="about-para"><font size="3">References: Conte S, Stevenson D, Furner I, Lloyd A (2009) Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein. <i>PLANT PHYSIOLOGY</i> 151:559-573.</p>
 
                      
 
                      
 
    
 
    
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Latest revision as of 19:30, 17 October 2018

Alternative Roots/Notebook

Alternative Roots

Root Colonisation

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NOTEBOOK

Root Colonisation

Week Beginning 16/07

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 firstly attempted to plant Arabidopsis thaliana seeds in a range of agar concentrations. Groups of 8 replicates were made at concentrations; 0.4 %, 0.6 %, 0.8 % and 1 % in bottomless microcentrifuge tubes and were left to chill over the weekend.

Week Beginning 23/07

The seeds from last week were taken from the fridge and placed in a pipette-tip box filled with water to approximately 1 mm below the microcentrifuge tube bottoms. After 5 days these seedlings were examined and all but the 1 % agar replicates had swollen and fallen through the microcentrifuge tubes into the bottom of the pipette-tip box. This showed that 1 % agar was appropriate for our uses.

Week Beginning 30/07

The team planted a 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 for lab-based plant growth.

Figure 1. An Arabidopsis thaliana seedling growing in 1 % agar inside a microcentrifuge tube.

Week Beginning 27/08

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 tryptone-soy agar plates.

Figure 2. Arabidopsis thaliana seedlings growing in a pipette-tip box

Week Beginning 03/09

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.

Week Beginning 10/09

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. The wild type Pseudomonas sp. inoculated seedlings from the previous week were taken for microscopy with Dr Vasilios Andriotis where we found DAPI staining seedlings that had been gently washed and mounted on 15 % glycerol was a suitable way to visualise endophytes.

Figure 3. Eruca sativa seedlings growing in a contained environment on the laboratory windowsill.

Week Beginning 24/09

A selection of Pseudomonas sp. transformant-inoculated seedlings were taken for microscopy with Dr Andriotis, again seedlings were washed and DAPI stained prior to visualisation. An experiment was set up to compare the effects of the wild type and transformed Pseduomonas sp. on germination so a large agar plate containing 70 micrograms per millilitre gentamicin was made [1]. This will show if the transformation has made Pseudomonas sp. detrimental to plant growth.

Week Beginning 01/10

A selection of seedlings were again selected for microscopy, this time negative control E. coli DH5α inoculated seedlings were examined with bright field microscopy and DAPI staining. This opportunity was also used to capture more images of the transformed Pseudomonas sp. chassis in Arabidopis thaliana roots.





References & Attributions

Attributions: Frank Eardley, Luke Waller and Lewis Tomlinson

References: Conte S, Stevenson D, Furner I, Lloyd A (2009) Multiple Antibiotic Resistance in Arabidopsis Is Conferred by Mutations in a Chloroplast-Localized Transport Protein. PLANT PHYSIOLOGY 151:559-573.