A Tale of Aptamers: Ghost and Elf ZJU-China iGEM 2013 aims to develop aptamer-based detector and cleaner

Introduction

W e build two aptamer-based devices this year - A bacterial ghost system by which detection for certain substance is facilitated and an elf system in which

two sorts of engineered E.coli eliminate targeted herbicide Atrazine by cell-cell col-laboration. The project dramatically revolutionizes the concept of detection and deg-radation of substance of interest. The applicable feature of our devices imparts to them a promising future to be virtually used in industry and agriculture. Accumulat-ing evidence is acquired which adds to the possibility of putting elves and ghosts into practice.

The following chapters aim to briefly elucidate how our devices are designed and how they function properly. Our tale about Ghost and elves is right ahead of us! Let’s get started!

Elves’ Cooperation: Defeat Atrazine!

A trazine is a widely-used herbicide. Though convenient and feasible, atrazine is report-ed detrimental to the endocrine system of human and other animals. We designed a

system to seek and destroy atrazine in soil by two sorts of cheZ knocked out E.coli called Detector and Cleaner.

Ghost Sensor

O ur ghost sensor project can be shown by the schematic diagram on the left side. This is a protein quick sensor based on aptamer-mediated membrane scaffolds dimerization

and we chose to detect thrombin as a proof-of-principle experiment.

Project design:

(Panel B) We expressed a unique lysis protein called protein E which could make orifices in the inner- and outer-membrane of E.coli by sealing the two layers. In this way, the mor-phology of cells and the periplasmic space were maintained. Proteins in the solution can freely diffuse in and be detected by our mem-brane scaffolds.

(Panel A) We constructed inner-membrane scaffolds with their cytoplasmic ends fused to streptavidin and periplasmic ends to split GFP or split β-lactamase. 15-mer and 29-mer ap-tamers targeting two sites of thrombin were bi-otinylated to render them strong affinity for streptavidin. So these aptamers acted like bridges linking thrombin and membrane scaf-folds. As a result, two aptamers targeting thrombin pull two molecules of membrane scaffolds together so the split proteins in the periplasmic ends integrate to form functional proteins of which the activity can be easily de-tected.

Results:

Characterize the function of protein E:

Protein E was put under the control of pBAD promot-er. We recorded the growth curve of transformed E.coli cells treated with different concentrations of L-arabinose varying from 0.2% to 1%(fig1). MgSO4 has been reported to allow the expression of protein E but inhibit lysis. MgSO4 was added 30min before the induction according to previous works by others. The results showed that even as low as 0.2% L-arabinose can efficiently in-duce the lysis E.coli. fig2 shows a SEM image of protein E lysed E.coli.

Characterize the function of aptamers:

We chose two aptamers, namely 15-mer and 29-mer, tar-geting two different sites of thrombin. We linked each of them with either short or long linkers.

As our aptamers were biotin modified, we used a strep-

tavidin beads pull down assay to see if these aptamers were capable of binding to thrombin.

Detect thrombin using membrane scaffold FA and FB:

We expressed the first set of membrane scaffolds with split EGFP fragments FA and FB fused to the periplasmic end. Then we tested the function of our membrane scaffolds by adding two sets of aptamers with different linker length. The concentration of thrombin in the solution was adjusted to 0.2nM. 29-mer aptamer and 15-mer aptamer efficiently induced dimerization of membrane scaffolds (Figure C, D ver-sus A) and led to green fluorescence emission under confocal microscopy.

What are we going to do?

For the second set of inner-membrane scaffolds, two parts of β-lactamase were fused to the periplasmic ends. When 19-mer and 25-mer biotin-modified ap-tamers are added to the solution and thrombin is pre-sent, aptamers act like a bridge linking streptavidin-fused membrane scaffolds and thrombin. In this way, two molecules of membrane scaffolds are pulled to-gether and the two residuals of β-lactamase will com-

bine to become an integrity part with enzymatic activity. This activity can be detected by commercial β-lactamase testing strips, with a total preparation time of about 10 min.

Detector:

An aptamer which specifically binds atrazine enables detectors to recognize and directly move towards atrazine. GFP is incorporated in this circuit as a reporter.

This circuit aims to control the bacteria density within a certain range. LuxI is plugged in to

produce AHL to recruit cleaner. Detector’s density is tightly controlled. (refer to Safety Part

Ⅱ1))

Cleaner:

AHL released by detector is sensed by cleaner. CheZ secure the motility of cleaners and thus spur them to move towards detectors. RFP is used as a reporter.

TrzN is expressed when bacteria respond to AHL signal, which enable cleaner destroy atra-zine. Detectors’ movement is controlled by atrazine:

Detector-cleaner communication under confocal microscope:

GFP and RFP were expressed when atrazine was added into LB medium, GFP was expressed when

detector recognized atrazine(B), Communication between detector and cleaner was characterized

by RFP expression (C). Negative control was performed without IPTG and atrazine, GFP was weakly

observed which indicated the existence of leakage(D/E), RFP was too weak to be observed(F).

A B C

Fig 1

Fig 2

Only very weak green fluorescence was visible when no aptamers were added to the reaction system (A). Bioti-nylated thrombin can readily induce membrane scaf-folds dimerization because there were typically 1-4 bi-ontin in one molecule leading to scaffolds crosslinking (B). 15-mer and 29-mer aptamer with short (C) or long (D) linkers were both effective in inducing crosslinking. But it seemed that long linkers (D) is more potent than short linker (C) in this system.

β-lactamase testing strip. Left shows β-lactamase posi-tive, right shows negative.

Meet Our Team!

O ur team consists of 12 members from different disciplines. we are all enthusiastic about iGEM!

Characterization

S ynthetic biology is about designing and constructing biological devices and sys-tems for useful purpose and usually involves standardized parts and devices. Thus we believe behavior descriptions of riboswitches and aptamers will be quite helpful for the future iGEM teams and synthetics biologists considering their high specificity and affinity.

Our intended characteristics describing the behavior of the riboswitches consist of the followings:

Static Relationship, which is about to measure the transfer function, details the rela-tion between device input(s) and output(s) and allows prediction of the behavior of composed devices.

Dynamic Relationship, we believe that it is of great importance to report the re-sponse time of the device since it will have to work with other devices in overall system and synchronism is definitely un-negligible.

Input Compatibility, which is intended to describe the behavior of our ri-boswitches when induced by some sub-stances having similar structure with their own inducers and offer us a pro-found understanding of riboswitches.

Fig.2 Dynamic Relationship of Atrazine Riboswitch

Fig.1 Static Relationship of Atrazine Riboswitch

Fig.3 Input Compatibility of Theophylline Riboswitch

(K411003)

Safety Part I: Artificial Shell

T o guarantee that the atrazine killer will not move around and pose potential threat to the environment, we come up with an idea to encapsulate our engineered bacteria

with CaCO3/ CaP (amorphous calcium phosphate) on the purpose of constraining their movement before using and extending the preservation time.

As biocompatible, stable, and nontoxic templates, calcium carbonate and calcium phos-phate have already received increasing attention. E. coli have polysaccharide on the surface, which provides negatively charged hydroxyl(-OH) to bind with Ca2+. Thus we designed a calcium phosphate shell.

Previous studies also pointed out that the electronic interaction is a key factor in biominer-alization, especially the carboxylates(-COOH) or other charged functional groups rich in proteins. In order to make a more concrete shell, we used polymers(PDADMAC/PAH) to modify the surface of E.coli.

Characterization

E.coli with artificial shells were viewed under SEM and TEM. From figure 1 we can tell that the artificial shells have successfully capsuled the E.coli.

When capsuled by LBL multilayer and mineral, a

spherical calcium phosphate also seems to be

formed.

Part II: E.coli Suicide

O ur engineered cells aimed to solve environ-

mental problems on the premise that they

themselves are safe. To assure that, a dual-

function system is constructed. It includes:

1) A population control module which serves to au-

tomatically limit the engineered bacteria concentra-

tion in a narrow range;

A previously well-designed plasmid in which ccdB,

luxR and luxI are plugged functions as a gene oscil-

lator by virtue of ccdB, a gene widely postulated as

an inhibitory factor during DNA replication.

We have experimentally proved that ccdB-circuit is

powerful enough to stabilize bacteria concentration.

2) A safeguard circuit by which the horizontal trans-

fer of engineered plasmids are prevented.

Antiholin-holin system has been characterized to

preclude horizontal gene transfer (HGT). By insert-

ing holin and anti-holin (comprising of LrgA and LrgB) to two plasmids in one cell respec-

tively, the system owns the capacity of fulfilling two critical functions: (1)To prevent HGT

(2)To discriminate intact engineered cells and those that have lost plasmids.

Figure 1. TEM image of biominerized E.coli: a)bare

E.coli; b) E.coli with Na2HPO4 shell, 0.05 M CaCl2/0.05

M Na2HPO4; c) E.coli with CaCO3 shell, 0.33 M

CaCl2/0.33 M Na2CO3

Figure 2. TEM and SEM

image of multilayer bio-

minerized E.coli: TEM

image

Figure. 3 Growth curve of BL21 E.coli cells induced by

different concentrations of IPTG

References [1] Yeast Cells with an Artificial Mineral Shell: Protection and Modification of Living Cells by Biomimetic Mineralization. Ben Wang et.al. Angew. Chem. 2008, 47:3560–3564

[2] Encapsulation of Living E.coli Cells in Hollow Polymer Microspheres of Highly Defined Size. Jennifer Flemke et.al. Bi-omacromolecules. 2013, 14:207 - 214

[3] Long-term monitoring of bacteria undergoing programmed population control in a microchemostat. Balagadde F K, You L, Hansen C L, et al Science, 2005, 309(5731): 137-140.

[4] The Staphylococcus aureus lrgAB operon modulates murein hydrolase activity and penicillin tolerance. Groicher K H, Firek B A, Fujimoto D F, et al. Journal of bacteriology, 2000, 182(7): 1794-1801.

[5] Canton B, Labno A, Endy D. Refinement and standardization of synthetic biological parts and devices[J]. Nature biotechnology, 2008, 26(7): 787-793.

[6] Joy Sinha, Samuel J Reyes&Justin P Gallivan. Reprogramming bacteria to seek and destroy an herbicide. Nat Chem. 464-470(2010)

[7] Shana Topp and Juatin P. Gallinvan. Guiding Bacteria with small molecules and RNA. JACS.6807-6811(2007)

[8] Andre Galarneau et al., β-Lactamase protein fragment complementation assays as in vivo and in vitro sensors of pro-tein–protein interactions, nature biotechnology 2002, 20, 619-22

Human Practice The Tale of Ghost and Elf

T o inherit our tradition, this year we write a story of 7 chapters, which is titled The Tale of Ghost and Elf. In

the tale, Little Prince and Summer try their best to save the planet E.coli equipped with the ghost sensor and atrazine seek & destroy system.

The creative tale describes the application of our project appropriately. It can be easily received by the public of dif-ferent walks of life. Since there are bacteria as adorable fig-ures in the fairy tale, they can help build transgenic prod-ucts and engineered bacteria a good image in public.

Its metaphors, personifications, analogies and other artistic techniques of expression will bring you a joyful tour of reading.

The Synbio Chess Game

i GEM is full of joy, including our chess game. This game is designed to educate the public about how synthetic biol-

ogy works at molecular level --- on the game board there are DNA, mRNA and polypeptides, describing the central dogma, a process revealing how information flows. There are also functional units, modifications, stem-loop struc-ture in the chess. From promoter the starting point, to poly-peptides the target, players travel from DNA through mRNA to proteins and thus savor the sense of how biology works in the cells.

Colorful Activities

T his year we take a big leap to practice synthetic biology and iGEM in various fields.

In April, Dr. Yuhua Hu from University of Edinburgh visited us and shared the experi-ences with us. In addition, we got involved in Sci-Tech Museum of Zhejiang Province to car-ry out a Science Fair.

In May, team NJU_China visited our lab and we shared our projects and process.

In August, we were invited by team NCTU_Formosa to join the iGEM Conference in Taiwan. We held lectures under Qiushi Summer Camp as well. Nowadays we have current activities under Synthetic Biology Club (SBC) in ZJU.

In September, we visited three herbicide companies. We interviewed managers and work-ers to gain facts of atrazine on the market and seek the opportunities of turning our devices into products. At the same time, we interviewed transnational corporations by email. We managed to build a database of atrazine, which could be helpful for iGEMers who want to know more about it.

D E F

Thrombin aptamers with Short linker:

5’-biotin-ttttttggttggtgtggttgg

5’-biotin-tttagtccgtggtagggcaggttggggtgact

Thrombin aptamers with Long linker:

5’-biotin-ttttttttttttggttggtgtggttgg

5’-biotin-tttttttttagtccgtggtagggcaggttggggtgact

Coomassie blue staining of thrombin digested products. 20µL of streptavidin beads and final concentration of 50nM thrombin were added to a 300µL PBS solution. A final concentration of 50nM 15-mer aptamer with short linker (lane2), 29-mer aptamer with short linker (lane 3), 15-mer aptamer with long linker (lane4) and 29-mer aptamer with long linker (lane 5) were added respectively. No aptamers were added to lane 1. Beads were washed and resuspended and moved to a new solution of purified proteins in which a thrombin cutting site is included to allow digestion.

Typical image of E.coli chemotacic to atrazine. Left: atrazine concentration is 0. Right: atrazine concen-tration is 2.0mM. Images were taken 12h later.

(Back row)

Jiasheng Wang / Clinical Medicine

Xihan Li / Mixed Class

Jin Huang / Computer Science; Pre-Med

Lifeng Zhang / Chemical Engineering

Congcong Xing / Qiushi Bioscience

Zukai Liu / Biotechnology

Yunlong Cao /Physics

(Front row)

Ke Ding / Qiushi Bioscience

Angli Xue / Plant Protection

Wenjia Gu / Applied Chemistry

Yujie Chen / Bioscience

Xiaoyue Yang / Applied Bioscience

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A gradient concentration of atrazine was added on the surface of semisolid medium, and moving diameter was measured by the diameter of bacteria colony after 12h in-cubation.

The red areas were treated with AHL solution be-forehand, which we acquire from sterilized su-pernatant of detectors.

Blue dots indicate where cleaners are initially spotted. The blue regions indicate the colonies of cleaners 18hrs after AHL treatment and sample dotting.

The left figure shows how cleaner colony grows without AHL induce; the right figure shows how cleaner colonies grow with anistrophy under AHL treatment.

The dots are placed at different distances to AHL and all of them receive positive induce signals.