Difference between revisions of "Team:XMU-China/Poster"

Tea is deeply rooted in Chinese culture. For a long period, a large amount of glyphosate has been used as a herbicide, which raises a severe problem of pesticide residues in tea and tea products.

This year XMU-China aims at developing an efficient glyphosate detection and degradation system.

There are two pathways found to degrade glyphosate:

►Glyphosate generates AMPA and glyoxylic acid through the C-N cleavage.
The product, glyoxylic acid, could be further converted into measurable signal, thus for the detection system, glyphosate is degraded by several enzymes through C-N cleavage and then transformed into a measurable fluorescence signal caused by the NADPH.

►Glyphosate generates sarcosine and phosphate acid through the C-P cleavage.
The product by C-P cleavage pathway, sarcosine, could be rapidly oxidized to become formaldehyde, a non-toxic substance. Therefore, glyphosate degradation method through C-P cleavage is preferred in our design which plans to disintegrate glyphosate to minimize the toxicity.


►Two kill switches controlled by different inducers are also proposed.

It is hoped that this project could provide new ideas for the detection and degradation of pesticide residues. Taking care of the earth by tiny bacteria, we here promise a better future of tea.

Design

Glyphosate is first converted to glyoxylate by GOX and then to glycolic acid while NADPH is consumed by GRHPR. Then, the amount of NADPH is determined by iNap.

For the sake of avoiding the interference of intracellular NADPH, the surface display system was employed (Fig 2) to anchor the enzymes mentioned above on the surface of engineered bacteria. Notably, this strategy subtly solves the difficulty that there are few natural regulators which could sense glyphosate or its derivatives in the glyphosate metabolic pathways, as scarcely reported.

Results
Every enzyme (GOX, GRHPR and iNap) and its fusion protein forms were characterized separately. The value of optical density at 340 nm (OD340) was used to represented the amount of NADPH, except for the experiments related to iNap.

The single GOX could slightly catalyze the conversion of glyphosate (data not shown), while the second reaction catalyzed by GRHPR has a faster kinetic behavior (Fig 3c). When tested with GRHPR, the GOX showed moderate enzymatic activity to catalyze the conversion of glyphosate to the substrate of GRHPR, which indicates the conjugation of the second reaction could actually accelerate the conversion of glyphosate (Fig 3a). INPNC-GOX and INPNC-GRHPR retain the enzymatic activity in a large part (Fig 3b and 3d), and the latter (BBa_K3332057) is our FAVORITE.



iNap manifests excellent SENSITIVITY and a large dynamic range to NADPH (Fig 4a). Compared to iNap-AIDA, INPNC-iNap has a larger dynamic range since the fluorescence intensities distinguished more significantly among different groups (concentrations of NADPH) (Fig 4b and 4c). Regretfully, BrkA-iNap has not been obtained due to the limit of time.

Proof of concept

Three types of E. coli BL21 (DE3) respectively carrying INPNC-GOX (BBa_K3332052), INPNC-GRHPR (BBa_K3332057), and INPNC-iNap (BBa_K3332047) were mixed to react with glyphosate and NADPH. The decrease of fluorescence intensity suggested that the detection system could work well.

Design

►Efficient phosphonate acid transmembrane transport
PhnE1E2 (encoded by phnE1 and phnE2, from Sinorhizobium meliloti 1021) could obviously enhance the transport capability of engineered bacteria to glyphosate.
►Cleavage of glyphosate
PhnJ (encoded by phnJ, from Enterobacter cloacae K7) is an essential subunit from C-P lyase which could break the C-P bond.
►RNAi
Downregulate ENDOGENOUS phnJ and phnF gene in engineered bacteria.
►AMPA derivation and degradation
PhnO (encoded by phnO, from Salmonella enterica LT2) could enhance the ability of engineered bacteria to degrade aminomethylphosphoric acid (AMPA), which would further decrease the toxicity of the derivative of glyphosate.

Result

Every section of our design was characterized separately in E. coli BL21 (DE3). The concentration of glyphosate determined by HPLC indicates the engineered bacteria could absorb more glyphosate than the control group did (Fig 2a). And the siRNA we designed worked well to hinder the endogenous phnJ and phnF from expression, proved by RT-qPCR (Fig 2b). The kinetic constants of PhnO was obtained through the in vitro test to the double-substrate reaction (Fig 2c). Regretfully, the PhnJ hasn’t been characterized due to both the difficulty of purification and limit of time.

Proof of concept
Based on the results above, the degradation capability of engineered bacteria to glyphosate was determined. While the RNAi system existed, the repression to the whole phn cluster was RELIEVED so that the degradation capability was greatly enhanced.

A. Kill Switch in Detection System

The toxin MazF, which is encoded by mazF, is designed to kill the engineered bacteria once they escape from the hardware to the environment lacking the inducer (L-arabinose). The number of E. coli colonies carrying BBa_K3332081 without induction decreased significantly after 8 hours, compared to the induction group.

B. Kill Switch in Degradation System
Formaldehyde (HCHO) will be generated during the process of degrading glyphosate. MazF would be expressed to kill the engineered E. coli when the amount of glyphosate is little in the application situations. Since all the pBAD-related parameters' sensitivity of OD600 is very small, the replacement of promoter from pBAD (BBa_I0500) to pHCHO (BBa_K1334002) would not affect the results largely.

Glyphosate detection system is composed of GOX, GRHPR, and iNap. GOX converts glyphosate into AMPA and glyoxylic acid; GRHPR converts glyoxylic acid into glycolic acid and consumes NADPH at the same time; iNap detects the amount of NADPH and emit fluorescence. Thus, all reactions can be concluded as following:

And those reactions can be described by the following ODE equations:

Due to the fast reaction of iNap and NADPH, the rapid equilibrium assumption was used to fit in with the experimental data, which is compared with the acquired simulation data in Fig 1.


In order to confirm the glyphosate threshold in detection system, the calibration curve was calculated by running different initial concentration of glyphosate and was obtained in the Fig 2, where the detection range of glyphosate is from 0.0 mM to 0.5 mM.

Modular Cloning, or MoClo, is a relatively new assembly method based on Golden Gate introduced in 2011 by Ernst Weber et al., which uses Type IIS restriction enzymes (we used BpiI and BsaI) to generate 4bp overhangs. These 4bp overhangs are called fusion sites, which must match between parts in order to ligate them together.

This allows the user to ligate up to six DNA parts together in a one-pot reaction, cutting down the time it takes to build large circuits dramatically (shown below).

There are three Levels of MoClo Parts (shown below):

Level 0: Basic part (ex: promoter, RBS, CDS, etc.)
Level 1: Transcriptional unit (up to 6 Level 0 Parts)
Level 2: Composite of up to 6 Level 1 parts



Reference: Weber et al., PLoS One 2011 

We converted 31 BioBricks and 4 new parts into Level 0 MoClo parts to create a library of MoClo parts.


All parts listed in Table 1 were confirmed with sequencing

All of our parts and primers are stored in a Clotho database (Xia et al., Methods in Enzymology 2011).




New Parts
We have cloned a new copper sensitive σ⁵⁴-regulatory system: pMmoR is induced by mmoR, which is repressed by copper (J. Scanlan et al., 2009).

Two new fluorescent proteins, EBFP2 and iRFP, have also been cloned.

To connect lab and tea garden, we also designed our own hardware and software. Hardware


The whole hardware consists of three parts:
►Cultivating Device: For resuspending the lyophilized engineered bacterial powder.
►Detection Chamber: For mixing bacterial liquid with sample liquid and testing.
►Data Transmission Device: For synchronizing data to the mobile terminal.



Software
It is our own software that matches the detection hardware. Users can download it on Android phones. Before using our detection hardware, the phone need to be connected with hardware via bluetooth. In another word, our hardware supports bluetooth data transfer.

After opening the detection page, users can follow the steps (Fig 4) provided on the screen preparing for detection, which means that the operation progress is easily-using to every group. While the hardware is working, users can learn the time left on the screen.


A report will be given when hardware finishes working, which users can save or share as they like. All the information is stored in the database which is on the cloud server in case of data loss.

Background
We interviewed with the Quality Inspection Institute, retailers and tea farmers and got useful information for our project. We have learned a lot about glyphosate, tea planting and herbicide residues. And we use the information to enrich our project background.
Design
We interviewed experts in environmental ecology and biology, and got some practical suggestions from them. These suggestions helped us to improve the biosafety of the project and guide our hardware design.
Model Improvement
We interviewed experts in chemical biology and cooperated with CSU to improve our mathematical and physical modeling.
Results of Our Questionnaire
By analyzing the questionnaire, we knew the attitudes of tea farmers and consumers towards our project, and their understanding and attitude towards the problem of herbicide residues in tea. Communicating Meetings
We attended CCiC and Southern China Regional Meeting, and gained many useful suggestions.
Safety Supporting
We accepted the guidance of professional and learned the importance of safety in Human Practices works.

New Methodology on Glyphosate

►Parts
Contributed 103 new parts, 44 of them are basic parts. All of the parts are designed by ourselves, and most of them have been tested to insure their reliability.
Formulated a Part Collection related to SURFACE DISPLAY SYSTEM, which includes the characterization to three kinds of anchor proteins (Fig 1). Users can also combine the anchor proteins with different proteins to verify the function of the anchored proteins and treat it as a subtle way to avoid the influence of components inside the cell.

►Detection System
✦Developed a convenient method based on synthetic biology to determine the concentration of glyphosate in tea food.
►Degradation System
✦Developed a highly efficient glyphosate degradation system based on synthetic biology
►Kill Switch
✦Proposed several systems to meet the needs of different scenarios in the actual application (simulate the needs of biosafety in the future).

New Hardware and Software

Developed a prototype device for the portable analysis of glyphosate.
►Hardware: Consisted of three parts: cultivating device, detection chamber, data transmission device.
►Software: Connected with hardware via bluetooth.

Business Cooperation

Reached a follow-up intention of deep cooperation with Qingshiyan Tea Co., Ltd. and Wuyishan Ruizhuang Tea Co., Ltd..


Future Plan

►Detection System
✦Test the enzymatic activity in the real environment.
✦Further develop and test the hardware and software (Version 2.0) to achieve the deep cooperation with companies in Mount Wuyi in Fujian province.
✦Extend the scope of herbicide residue using synthetic biology to enhance the detection capability of the device.
►Degradation System
✦Test the enzymatic activity in the real environment.
✦Develop the enzymatic activity to a higher level based on further protein engineering.
►Kill Switch
✦Further characterize the proposed design and test the switch and toxin activity in the real environment.