E. coligy: Plants with BanAphids

Introduction

Robbert Boudewijns, Laurens De Backer, Pieter De Bruyn, Flore De Wit, Aurelie Lenaerts, Lukas Marcelis, Frederik Meynen, Bert Mortier, Michaël Poorthuis, Tina Smets, Sabine Thieren, Saar Vandekeere,

Tomas Van Pottelbergh, Su Wang, Sylvie Weckx, Sander Wuyts Ingmar Claes, Veerle De Wever, Johan Robben, Sam De Vlieger – contact.igem@kuleuven.be

1)  cpram promotor constitutively expresses EBF which repels aphids (BBa_K1060011). 2)  When habituation to EBF occurs, aphids populate your plant and produce honeydew. 3)  Honeydew (high glucose) represses TetR expression (BBa_K1060010), releasing

expression of bsmt1 and pchBA to produce MeS (BBa_K1060012); LacI to inhibit EBF synthase and AroG* (P150L; L175D) production to increase intracellular levels of a MeS precursor (chorismate) (Hu et al., 2003) (BBa_K1060013).

4)  MeS repels aphids, honeydew levels reduce, pCaiF promoter switches on – back to 1).

Our two systems Glucose model

+ Interactive system: system responds to aphid presence + Avoid habituation: alternating use of two compounds + Just in time production: each pheromone produced

only when its effect is required + E. coli: proof of principle

Erwinia herbicola: on plant bacterium

Acknowledgements Thanks to our sponsors for their generous support throughout the past months!

Human practices - Ethics Outreach

Our network creates oscillations in the following way: 1)  A high level of TF A induces production of both TF C and TF X. 2)  After a little while, the level of TF C initiates repression of the top AND gate (and

stimulation of the bottom AND gate). 3)  Once the top AND gate is significantly repressed, the level of TF A starts to decline.

When TF A drops underneath its threshold, TF X levels start to decline too. At this point, the level of TF C does not decline yet. Only when TF X also drops below its threshold the level of TF C will start to decline. This prolonged expression of TF C results in an extended repression of the production of TF A. This ensures that the level of TF A drops sufficiently.

4)  Simultaneously, the extended expression of TF C results in extended activation of TF B production, which reaches a high level.

5)  The same explanation as before can now be applied to TF B, D and Y. This will result in a decrease in TF B levels and a high level of TF A, once again resetting the system to its initial state.

Our second model prevents direct contact between our BanAphids and the environment (using stickers from Groningen iGEM 2012). We changed our promoter system from a glucose (honeydew) responsive system to an autonomous system. Habituation is prevented by enforcing oscillation within and between each bacterial cell in the population. For lack of a suitable model, we designed our own, transcription factor (TF) based network. Its characteristics have been validated through modelling, results are shown in the “Modelling results” section.

Oscillator model

MeS is naturally produced by the plant upon aphid infestation. It activates plant defence systems and attracts natural aphid predators (e.g. the ladybug). EBF is produced by aphids as an alarm pheromone. It repels aphids off the plant.

Aphids are a pest. An outbreak leads to crop losses worth hundreds of millions of euros!

Current approach to aphids (insecticide use) has negative consequences:

MeS and EBF are natural parts in the signal interplay between plants, predators and aphids. Our BanAphids will enhance the plants’ natural communication and protection mechanism. Compared to insecticides, BanAphids should only have a minimal impact on the ecosystem, an improvement to the current state.

We tested MeS production by our b r i c k ( B B a _ K 1 0 6 0 0 0 3 ) & BBa_J45700 (MIT, 2006) under different conditions via a blind smell test (11 participants). The MeS (wintergreen) smell got stronger from 8 to 24 h incubation time. Growing bacteria at 37 °C also produced a more pronounced smell than those at room temperature. Addition of salicylate positively affected the smell, independent of the incubation time. The chorismate effect became only noticeable after 24 hours.

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Favourite BioBricks Functional characterisation of BioBrick BBa_K1060009. Aphids were placed on a leaf in the middle of a Petri dish, EBF-producing bacteria were placed on the left, a control on the right. The general trend of aphid movement was away from the EBF-producing bacteria. This suggests our EBF synthase producing bacteria works. Below are three stills of our video. Aphids (orange/brown) dots at the base of the leaf move between 2nd (78 min) and 3rd (176 min) time point.

51 min 78 min 176 min

Wetlab results Aphids experiments

Smell test Ecology experiments

Glucose model Spraying our BanAphids on plants requires the incorporation of a kill switch. This is not a contained area so auxotrophy is _

Safety

Protein expression

E. coli strain BL21(DE3) was transformed with the respective BioBricks (EBF-Synthase under a Lac operator; medium or strong constitutive promoter); grown to mid-exponential phase and proteins extracted. SDS-PAGE analysis and colloidal coomassie stain showed 2 bands with different expression between BioBricks (arrows). The l o w e r b a n d ( ~ 6 6 k D a ) corresponds with the expected EBF synthase protein size.

Modelling results Ecology model

We developed a model to predict the effect of our pheromones on the environment. We modelled the transport of pheromones in the air as a convection-diffusion problem using a realistic wind profile in crop fields and linked this to our pheromone production rates. The outcome can be used to calculate optimal spacing of the BanAphid containing stickers or define BanAphid concentrations for given sticker distances.

Flux Balance Analysis A Flux Balance Analysis was performed with the COBRA Toolbox. This analysis predicts E. coli growth rates after introducing the reactions belonging to our MeS system under default conditions. We also analysed the predicted flux towards chorismate, a MeS precursor. Results suggest that LB medium conditions are favourable for E. coli growth but not for the flux towards chorismate. The introduction of BBa_K1060013 (AroG*) should help overcome this problem.

We modelled the pathway from the MeS precursor chorismate to MeS using ODEs and a SimBiology model. From a predicted enzyme concentration, we estimated kinetic parameters of these reactions. The enzyme concentration was obtained from the balance between protein translation (Penn State University RBS calculator) and degradation rates. We planned to couple this to the wetlab by obtaining the required mRNA concentrations via a qPCR.

Oscillator model

Here, the KU Leuven iGEM 2013 team presents an ecological, insecticide-free, aphid-controlling system. It builds on the naturally occurring defence signalling systems, present in plants and insects. Our bacteria (BanAphids) produce two substances, methyl salicylate (MeS) and E-β-farnesene (EBF), a phytohormone and a pheromone, respectively.

§  increasing aphid resistance §  harms natural predators §  accumulation in food chain

(Minks and Harrewijn, 1987)

0%

10%

20%

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50%

60%

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90%

SA SA CA CA SA SA CA CA SA SA SA SA SA SA SA SA

RT 37°C RT 37°C RT 37°C RT 37°C RT 37°C RT 37°C RT 37°C RT 37°C

Bba_J45700 Bba_K1060003 Negative  Control

percen

tage  of  p

eople  sm

elt  M

eS

SA: salicylic  acidCA:  chorismic  acid

Remark:  In  all  conditions  showed 0.2  mM of  IPTG  was  added

8h

24h

 

C  A  X  

B  Y  

D  

AND  

AND  

OR  

OR  

A è  EBF  synthase

Integrated modelling

Slice through the 3D pheromone concentration profile during an oscillation cycle

   EBF

synthase  RBS  cpram   dTerm  lac

operator  

RBS   lacI  pTetR          

RBS   AroG*   dTerm  

       RBS  pTetR   bsmt1   pchBA  RBS  pTetR   dTerm  dTerm  

   

RBS   tetR  pCaiF   dTerm  

BBa_K1060011

BBa_K1060013

BBa_K1060012

BBa_K1060010

BBa_K1060002: an ORF coding for EBF synthase from Artemisia annua. converts farnesyl diphosphate into EBF.

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BBa_K1060009: const i tu t ive expression of EBF synthase under a medium strength promoter and RBS.

BBa_K1060011: constitutive EBF expression controlled by a lac operator. Controls EBF production via a LacI construct, responsive to honeydew levels.

All BioBricks are sequence confirmed.

not an option. We propose the use of a kill switch which will induce a lethal cascade upon addition of a well-chosen signal. We would like to reuse one of the constructs already made in iGEM, such as the inducible BamHI system contributed by Berkeley iGEM 2007. Sticker model We will contain our BanAphids with the sticker of Groningen iGEM 2012 and make the BanAphids tryptophan auxotrophic.

EBF synthase

1 : BBa_K1060011 2 : BBa_K1060009 3: BBa_K1060014

1 2 3 130

100 70

kDa

55

100

70

kDa

55

MeS operon

0.1 0 1

0.5

2

E. coli strain BL21(DE3) was transformed with each MeS B i o B r i c k ( B B a _ J 4 5 7 0 0 ; BBa_K106003 (data not shown); grown to mid-exponential phase and proteins extracted. SDS-PAGE analysis and colloidal coomassie stain showed that changing the IPTG induction concentration does not change the band pattern.

IPTG(mM) 0.1 0 1 2

Induction @ OD600nm

1.0

In our method, we searched for a realistic parameter space. We randomly probed between realistic lower and upper boundaries for the different parameters. For the K values, which can vary tremendously (even a lot more than over the three decades that we allowed), our results (not shown) indicate that lower values of K benefit oscillations. The reason is that K can be seen as a threshold for the concentration to have an effect. If that threshold would be higher than the concentration can possibly be, there is of course no occurrence of oscillations. The most important characteristics we tested for were rapid resynchronisation of desynchronised subpopulations and the robustness of oscillations against cell-to-cell variability.

Figure 2: The oscillations of C in an example in which 50% relative variability is allowed. Figure 1: Example of rapid resynchronisation in which the first 40% of the cells start out of sync.

Table 1: Confidence intervals of the fraction that rapidly retake synchronised oscillations when 40% of the cells is oscillating out of sync and when a relative variance of 50% for the production rates is taken into account. Details on the ranges can be found on our wiki. Table 1 shows confidence intervals for

the fraction of the parameter space that exhibits these properties. Figure 1 and figure 2 illustrate these features. The high fraction of parameter sets that exhibit satisfactory behavior shows the robustness of the model.

In a series of behavioural experiments, our model was validated by investigating the effects of EBF and MeS on aphids and their natural predators. Interestingly, 10 days after MeS induction via the roots, a decrease in aphid population as well as an increase in amount of winged aphids was observed. This suggests that the activation of the plant defence mechanism motivates aphids to leave the plant and decreases reproduction.

When induced via the root plants show reduced growth because they activate the whole plant de fence sys tem and therefore more resources are spent.

We went to the industry to prove that our project is viable. We worked together with major industrial players in the field of crop protection: Biobest and pcfruit. They were both very enthusiastic and we were able to perform experiments in co-operation with these companies.

•  We asked the opinion of the end user as well. 94% of the general public would use our genetically modified bacteria in their gardens or eat food derived from plants treated with them.

•  We gave workshops in high schools using our own designed ‘Bricks’.

The experiments at Biobest and pcfruit prove the feasibility of our project. We investigated the behaviour of insects (ladybugs Adalia bipunctata and aphids Myzus persicae) on EBF and MeS. Interview with iGEM students Panel members of the debate

An innovative way to integrate ethics into the iGEM competition!

Range to which the Ks for

proteins belong!

95% confidence interval of the fraction that rapidly

resynchronises!

95% confidence interval of the fraction that oscillates

despite cell-to-cell variability!Range 1 {0.89;0.99} ! {0.59;0.77} !Range 2 {0.89;0.99} ! {0.64;0.82} !Range 3 {0.95;1.007} ! {0.91;0.99} !