3+ Net

Bioremediation

Ⅰ Background and definition of 3+ net Ⅱ Division of duty Ⅲ Application+ Ⅳ Biosafety+ Ⅴ Society+

Ⅰ Background and definition of 3+ net Ø Background

The use of genetically engineered bacteria for environmental

bioremediation is currently the most economic means, but due to

limited application scenarios and biosafety concern, it’s often

difficult to put these genetically engineered bacteria into practical

use. In addition to uncontrollable factor in science, the public and

government also have their concern. To better understand the

present situation, ECUST raised the concept of “3+ net” which

consists of Applications+, Biosafety+ and Society+, and by

collaborating with SCUT, FJNU, SHPH and WHU, we finished the

guidelines which can serve as a reference for future teams.

Ø Components

The “3+ net” is composed of 3

main factors, Applications+,

Biosafety+, and Society+.

The “+” means combination

of biology with other fields,

better project improvement,

thinking further and working

together. We aim at putting

our projects into practical use, setting up universal programs or

guidelines, and collecting public opinions on bioremediation.

Ø Relationship among the three components

The three components are

not isolated, but closely

related to each other.

Better application design can

lead to higher biosafety, which will result in higher public

acceptance, and higher public acceptance will make more

applications possible.

Ⅱ Division of duty Ø Applications+: WHU

Ø Biosafety+: SCUT and FJNU

Ø Components: ECUST and SHPH

Ⅲ Application+ Ø Overview

² Scientific Methods

Design better hardware for engineered microbial detection and

non-proliferation, and use controllable technology to make

genetically engineered bacteria applicable to more scenarios.

² Business Methods

Provide commercial services for transforming resistant systems:

services such as elimination of resistance genes, insertion of

essential genes, genome integration, expression optimization, and

risk prediction.

Provide bacterial bioremediation services.

Develop a GE bioremediation application standard of the industry.

Ø Work done by WHU

As a member of 3+net, WHU took on the task of hardware. This is

somehow a big problem. Every year, many “environment track”

teams develop amazing biological pathways, which provides the

possibility to solve some practical problems. However, time is

limited. As for the platform of the application-hardware device,

these teams will encounter difficulties more or less. Many teams are

taking the detours that others have already traveled over and over

again. We hope to solve this problem - in fact, many

teams' projects are consistent in the application scenario, we can

classify these application scenarios and provide an optimal

hardware template for each situation. In the future, the team can

find a template and modify it to suit their projects.

According to the environmental media, we can divide the

application scenario of these projects into air, water and soil.

According to the purpose of the device, it can be subdivided into

large bioreactors and small biosensors. Thus we divided the

environmental track project into six categories.

We searched iGEM's previous environmental projects and

summarized each category. In each category, we searched many

teams and found out the outstanding devices or safety

considerations, and summarized them on this basis to create a

consensus template

1. Water- bioreactors

1) Aachen – 2017

They considered four key aspects of safety: Release, Organism and

Parts, Antibiotic Resistance, Dialogue

Release: A membrane system would definitely keep that back. If

you want to be 100%, or 120% sure, you would probably follow that

up by another membrane, but actually the membrane, if it is leak-

proof, can hold it all back. Dr. Palmowski

2) Exeter - 2017

The most highlight point of this team is the amazing device. The

three areas of their installation are very distinct, and the water

pump allows molecules of a certain size to enter the bioreactor,

which allows the bacteria to effectively contact and react with the

sewage. Finally, the safety pool ensures that the bacteria are

impermeable, which is a very standard and amazing large

bioreactor device in water. They also used ultraviolet light to

prevent the escape of bacteria.

3) NCKU-China – 2017

Regulation box of their project to make the N content back to

normal level. It is also a model of water- bioreactor, the highlight of

theirs is “that is water input hole, output hole, “motor and

filter system” and replaceable grooves. When regulation box

starting to work, it pumps water into the box by motor system. Then,

water flows through filter system for physical water cleaning and

goes into grooves to do biological transformation. Consequently,

clean water appears and the whole aqua system can stay

healthy.”

In conclusion, we can see water filter, UV light, water pump,

membrane module in those fantastic project and device to solve

the safety problem.

2. Water-biosensor

NCKU-China – 2017

A sensor should have many essential components, and if you want

to find one, look at their great job!

- Sample Collection

We constructed a multiple sensing boat to collect fundamental

information of water in the fish pond. It’s a woody boat with three

sensors and two solenoid valve. Firstly, the water will enter the boat

and flow through two solenoid valves. With the specific controlling,

we can gather quantitative water for nitrate sensing. Then, the

sample of water will react with our E. coli in a tube fixed in a light

absorber.

- Detection

We use a pH meter and a thermometer to detect some date. Also,

we use 450nm laser to excite our E. coli. As you can see the picture

below, there are a light sensor, a detecting box and a light source

placed from left to right. Then, E. coli will emit 510nm green light

and the green light will be received by light sensor. By analyzing

different fluorescence intensity. We will get concentration value of

nitrate in the water.

- Processing

The signal then is sent to the processor. We chose Arduino as the

processor, because Arduino is an open-source prototyping

platform featuring as easy-to-use hardware and software. We

coded our Arduino to initialize our detection, to filter our input

signal and to measure the result kinetically.

- Transmission

We change digital data to analog. Then combine all the data

sensing boat got to a single string and transmit the data via 2.4G

radio from boat to remoter. The remoter would send the to our

sever. To send data immediately, we choose to use get request

which is much faster than post request. And the user can use their

mobile app to get the latest data. The mobile app follows the

same path our boat did. It can show the data in chart or even the

location of the boat

All the components are very great, and for a special safety track,

a delicate collection box will account so much!

3. Air reactor

1) ICT-Mumbai - 2017

We introduced an element in our design to keep track of the

amount of ammonia assimilated. Indigoidene, a blue-colored

compound, is formed by non-ribosomal peptide synthesis (NRPS)

from glutamine in a single step reaction catalyzed by the product

of the bspA gene. A colored compound that is produced in

proportion to the amount of ammonia assimilated would indicate

when cells would have to be replenished.

We designed a device to house the engineered cells. As

proteorhodpsin is a light-driven proton pump, a light source is also

incorporated in the device. We envisage a battery-

powered, wall-mounted device that contains the engineered cells

in a BioCassette, which can be replaced at regular intervals.

This cassette is a device to solve the NH3 problem and use

engineered bacteria to fix NH3. Their highlight is , using liquid or

water to dissolve the gas first, and then all the steps are

the same as water reactor and even more safe than water reactor.

For those gas cannot fit this situation, you must find another way to

capture the gas first.

2) Pasteur Paris - 2017

Two scenarios have been designed. The first one is about working

with small groups of people - beta-testers - that will help Æther’s

service to provide adapted filters, information and advices to the

end user. The second one is about providing these adapted

solutions to everyone, thanks to these beta-testers.

Beta-tester scenario:

Æther’s services will send early air purifier kits to volunteers

that would apply for beta-testing the product, app and service for

free. In exchange, they would help Æther to build a strong

knowledge about close environment and local indoor air pollution

by sharing their data - location, habitat description, etc.-. Plus,

beta-testers will receive an additional filter - activated carbon for

a wide range pollutants trapping - to send back to Æther’s

laboratory for analysis. Thanks to these data, Æther will be able to

design adapted filters for various environments and will be able to

build and to provide verified information and advice for an end

user living in similar environments.

End user scenario:

A future user could order Æther’s device and apply to the

service thanks to a dedicated website. After completing his profile,

the user will be about to receive a kit including:

- a self assembly kit of the air purifier;

- an envelope containing an adapted filter and gloves;

- a smartphone/digital tablet app.

Through the app, the user will be guided through the next stages.

Once the product is assembled, the filter introduced and the

flexible photovoltaic cells connected, Æther’s device starts

trapping and degrading indoor pollutants.

When the filter’s lifetime is over, the user receives a push

notification on his smartphone: an invitation to check his mailbox

containing a brand new filter. Using provided gloves, he will

replace the used filter by the new one. Once done, the device will

start again purifying the indoor air and the used filter will be sent to

collection centers in order to recover the degraded pollutants as

an input for various industries, such as metal industries.

In a nutshell, Æther’s service and product would benefit:

-users’ health, by providing an affordable and efficient way to fight

indoor air pollution through an innovative product, its integrated

filter and a service;

-environment, by feeding industries with pollutants recycled into

useful raw materials - metals -, rather than using polluting

and harmful extraction/transformation processes.

4. Soil

1) AshesiGhana bioreactors

This would not only separate the bacteria from the natural

environment in the event of a contamination but it would also

allow for the proper termination of the bacteria before they are

disposed.

A picture of a bioreactor that could be used to hold the bacteria

This is a standard soil bioreactor and carefully designed in bio-

safety. For more details, you can see their wiki.

Conclusion: we screened all the 2017 team in

environment track, and found the most carefully-designed

hardware. Most of teams worked in water-reactor, and this is also

the most dangerous one. We have concluded many methods for

hardware solution, filtration, membrane, UV light device, pump …

Additionally, for those sensors or detectors, a test strip can be used

in many cases, and even though we use a biological device, the

scale of exchange from environment and engineered organisms is

very small. We should pay attention to those reactors device, and

if you have any questions, please contact us!

WHU-China 2018 (1070388373@qq.com)

Ⅳ Biosafety+ Ø Overview

1. Suicide (Kill switch, auxotroph…)

2. Elimination of antibiotic genes

3. Genomic integration

4. Visual identification

5. Hardware design

6. The Internet

Ø Work done by SCUT

In biological projects, genetically modified organisms (GMOs) are

widely used in research, industrial systems, and even

environmentally restoration applications, which leads to potential

bio-safety risks. As iGEMers, we have the responsibility to minimize

such risks. Since various efforts to avoid bio-safety risks have been

made by past researchers, we now try to organize the information

from previous researchers to write about how to make our projects

most harmless.

handbook

1. Methods of building up antibiotic-free strains

An essential requirement in GMOs is to select strains with defined

genotypic alterations. The common strategy is attaching a marker

gene, which is usually a gene providing antibiotic resistance to its

host, to a genetic engineered vector. In this way, researchers can

easily distinguish whether the colonies of their engineered strains

are the ideal genotypic alterations.

However, antibiotics are used liberally in many areas of medicine,

agriculture. The use of antibiotic resistance genes as markers may

result in potential biosafety and clinical hazards, such as horizontal

spread of resistance genes or accelerating the emergence of

multidrug-resistant pathogens. In iGEM, Escherichia coli,Yeast and

Bacillus subtilis are the most commonly used in various projects,

including Environment track, in which many projects may apply

their engineered organism in the an open environment. Therefore,

here we try to collect some feasible examples of building up

antibiotic-free hosts.

2. Escherichia coli

A simple strategy to stabilize the antibiotic-free plasmids in E. coli,

is to destroy the function of an essential gene on the genome of

the host, and place this gene on the plasmid carrying the target

genes. This method has been generally developed and applied in

previous researches. [1,2,3]

Another perhaps more elegant way is using Antisense peptide

nucleic acids (PNAs) as the substitute for antibiotics in bacterial

strain selection. In this regard, treatment of a mixture of E. coli wild-

type cells and cells carrying a binding-site altered copy of acpP

(acpP-1) with anti-acpP PNA completely killed wild-type cells. In

this process, the PNAs play a role in screening the positive strains

instead of an antibiotic marker. [4]

An RNA based method, using constitutive expression of sacB as a

counter-selectable marker during growth on sucrose was reported

to be able to bring about antibiotic-free selection and highly

productive fermentation while not being restricted to ColE1

vectors. [5]

The Operator-Repressor-Titration (ORT) strategy is based on

negative regulation of an essential chromosomal gene by an

operator sequence allowing the binding of a constitutively

expressed repressor protein. [6]

3. Yeast

For yeast, auxotrophic selection is a comprehensive solution wide-

ranging applied in building up antibiotic-free engineered strains

The auxotrophic yeast strains of -Trp, -Leu, -His, -Ura, -Met, -Ade

have now been built up and commercialized. These strains cannot

synthesize specific compounds which are essential for growth.

Therefore, only if the DNA fragments with the specific essential

gene carrying the target genes have been successfully

transformed into the host, can the colonies grow on the yeast

selective media.

Besides of auxotrophic selection, using the Cre/mutated lox system

to knock out the antibiotic marker after selection is also a method

to build up an antibiotic-free engineered yeast strain [7]. At the

beginning, the Cre/mutated lox system, resistance marker and

homologous arms are spliced together by fusion PCR to generate

the gene disruption cassettes, which could be integrated into the

yeast’s genome via homologous recombination. After selection,

researcher can excise the Cre-marker cassette from the yeast’s

genome by the induction via Cre.

4. Bacillus subtilis

Similar with Yeast, Cre/lox System is also used to build up Bacillus

subtilis antibiotic-free strain [8].

Another way is to introduce the E. coli mazF cassette into the

genome of Bacillus by double crossover homologous

recombination.The mazF gene codes an endoribo- nuclease that

cleaves free mRNAs as a couter-selection tool [9].

5. Reference [1]Selvamani R S V, Telaar M, Friehs K, et al. Antibiotic-free segregational plasmid stabilization in Escherichia coli owing to the knockout of triosephosphate isomerase ( tpiA )[J]. Microbial Cell Factories, 2014, 13(1):1-13. [2]Vidal L, Pinsach J, Striedner G, et al. Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli.[J]. Journal of Biotechnology, 2006, 134(1):127-

136. [3]Hägg P, de Pohl J W, Abdulkarim F, et al. A host/plasmid system that is not dependent on antibiotics and antibiotic resistance genes for stable plasmid maintenance in Escherichia coli.[J]. Journal of Biotechnology, 2004, 111(1):17-30. [4]Dryselius R, Nekhotiaeva N, Nielsen P E, et al. Antibiotic-free bacterial strain selection using antisense peptide nucleic acid[J]. Biotechniques, 2003, 35(5):1064-0. [5]Luke J, Carnes A E, Hodgson C P, et al. Improved antibiotic-free DNA vaccine vectors utilizing a novel RNA based plasmid selection system[J]. Vaccine, 2009, 27(46):6454-6459. [6]Cranenburgh R M, Lewis K S, Hanak J A. Effect of plasmid copy number and lac operator sequence on antibiotic-free plasmid selection by operator-repressor titration in Escherichia coli.[J]. Journal of Molecular Microbiology & Biotechnology, 2004, 7(4):197-203. [7]Pan R, Zhang J, Shen W L, et al. Sequential deletion of Pichia pastoris, genes by a self-excisable cassette[J]. Fems Yeast Research, 2011, 11(3):292-298. [8]Yan X, Yu H Q, Li S. Cre/lox system and PCR-based genome engineering in Bacillus subtilis[J]. Appl Environ Microbiol, 2008, 74(17):5556-5562. [9]Morimoto T, Ara K, Ozaki K, et al. A new simple method to introduce marker-free deletions in the Bacillus subtilis genome[J]. Genes & Genetic Systems, 2009, 84(4):315-8.

Ø Work done by FJNU

We summarize the current common suicide switches for different

types of environmental conditions.

PARTS NAME TEAM

BBA_K733004 YDCE 2012HKUST

BBA_K2350021 HOLIN-ENDOLYSIN-NRTA 2017NYMU-TAIPEI

BBA_K2407301 URA3 2017TIANJIN

BBA_K1061002 RIP 1 2013SYSU-CHINA

BBA_K2365508 BAX INDUCED PART

2017 NAU-CHINA

BBA_K2232025 MAZEF SWITCH 2017SZU-CHINA

BBA_K2493004 ANTISENSE SOK MCMASTER_II

BBA_K1631003 COLICIN LYSIS PROTEIN 2015UT-TOKYO

BBA_K1660008 BACE16 2016BNU-CHINA

BBA_K1727005 SIGNIFERIN 2015TCU_TAIWAN

BBA_K1405008 MAZF 2014 BNU-CHINA

BBA_K1378031 HOLIN FROM LAMBDA PHAGE 2014PEKING

BBA_K1510233

A BLUE LIGHT REGULATED

CCDB APOPTOSIS

2014NYMU-TAIPEI

BBA_K628006 PROTEGRIN-1 KILL SWITCH 2011ST_ANDREWS

BBA_K1172904 RNASE BA 2013BIELEFELD-GERMANY

Part:BBa_K733004

Designed by: WANG, Yuqi Group: iGEM12_HKUST_Hong_Kong (2012-09-16)

RBS+ydcE

ydcE which is also named as ndoA, encoding an RNase, namely

EndoA. Being in the same RNase family with MazF/ChpAK/PemK,

YdcE - the endoribonuclease ydcE encodes - can inactivate

cellular mRNAs by cleaving them at specific but frequently

occurring sites (i.e. UAUAAU↓AC). Thus, overexpression of EndoA

can lead to programmed cell death or unhealthy conditions,

depending on the amount of EndoA. Note that ydcE(ndoA) is

demonstrated functional both in E. coli and B. subtilis. (Pellegrini et

al., 2005)

Applications of BBa_K733004 - UCAS 2016

We are iGEM team from University of Chinese Academy of

Sciences. In this year's program, we used this part to build a kill-

switch. The gene is induced by IPTG or aTc. When identifying

interaction between toxin and antitoxin, toxin is induced by aTc.

Growth curve of E. coli expressing toxin EndoA

We also measured the growth curve of wild type E. coli harboring empty plasmids.

Pellegrini O, Mathy N, Gogos A, Shapiro L, and Condon C. "The Bacillus subtilis ydcDE operon encodes an

endoribonuclease of the MazF/PemK family and its inhibitor.." Molecular microbiology. 56.5 (2005): 1139-1148. Print. Part:BBa_K2350021

Designed by: YA-XUAN YANG Group: iGEM17_NYMU-Taipei (2017-10-22)

R0010-B0034-Holin-B0010-B0012-J23106-B0034-Endolysin-B0010-

B0012-J23118-B0034-NrtA-B0015

Holin-Endolysin-NrtA

This part combines holin, endolysin, and NrtA. NrtA protein can stick

to periplasmic membrane through a flexible linker to capture nitrite

or nitrate in the periplasm. Therefore, we can make use of NrtA in

nitrogen starvation to kill E.coli to prevent contamination. When the

lactose is added into the environment, the suicide mechanism,

holin and endolyin, is induced.

- Result

As the figure shows, the trend of the relative absorbance is

downward as the lactose is added to induce the suicide

mechanism. The concentration of lactose is also positively

correlated with the declining degree of relative absorbance.

Part:BBa_K2407301

Designed by: Xinyu Chen Group: iGEM17_Tianjin (2017-10-12)

Ura3 gene

This is the part regulatory region from the URA3 gene coding for

OMP decarboxylase, an essential protein in the uracil synthesis

pathway in S. cerevisiae budding yeast. It is widely used as a

nutrition tag in Saccharomyces cerevisiae. URA3, a gene

on chromosome V in Saccharomvces cerevisiae, is widely used in

researches concerning yeasts as a “marker gene” (systematic

name YEL021W. URA3) and used as a label for chromosomes

orplasmids. URA3 encodes Orotidine 5'-phosphate

decarboxylase—an enzyme that catalyzes one reaction in the

synthesis of pyrimidine ribonucleotides.

Principle of operation

1) Pyrimidine biosynthetic pathway of S. cerevisiae

In Saccharomyces cerevisiae, the biosynthesis of pyrimidines

involves the de novo synthesis of UMP from glutamine. Carbamoyl

phosphate, derived from glutamine, undergoes a condensation

reaction with aspartic acid, resulting in the formation of N-

carbamoyl aspartic acid. Both the formation and subsequent

condensation of carbamoyl phosphate are performed by Ura2p.

The pyrimidine ring of N-carbamoyl aspartic acid is closed by the

elimination of water to form dihydroorotic acid (DHO), which is

subsequently oxidized to form orotic acid (OA), and a ribose-

phosphate group is then added to form orotidine 5′-

monophosphate (OMP). The formation of OMP is performed by

two isoenzymes, Ura5p and Ura10p. OMP is

then decarboxylated to yield UMP, which may subsequently be

processed to form other pyrimidines. Regulation of this pathway

occurs at several levels. First, UTP down-regulates the enzymatic

activity of Ura2p and transcription of the URA2 gene. Second,

under conditions of pyrimidine starvation, transcription of

the URA1, URA3, URA4, and URA10 genes (the URA genes) is

increased some three- to eightfold. This increase in transcription is

dependent on a transcriptional activator, Ppr1p.

2) Usage in yeast research

The URA3 gene in the yeasts used in lab has already been

deleted. Hence the loss of ODCase activity leads to a lack of cell

growth unless uracil or uridine is added to the media. The

presence of the URA3 gene in yeast restores ODCase activity,

facilitating growth on media not supplemented

with uracil or uridine, thereby allowing selection for yeast carrying

the gene. In contrast, if 5-FOA (5-Fluoroorotic acid) is added to

the media, the active ODCase will convert 5-FOA into the toxic

compound (a suicide inhibitor) 5-fluorouracil causing cell death,

which allows for selection against yeast carrying the gene.

Since URA3 allows for both positive and negative selection, it has

been developed as a genetic marker for DNA transformations

and other genetic techniques in bacteria and many fungal

species. It is one of the most important genetic markers in yeast

genetic modification. While URA3 is a powerful selectable marker

it has a high background. This background is because cells that

pick up mutations in URA3 may also grow on 5-FOA. Colonies

should be verified by a second assay such as PCR to confirm the

desired strain has been created.

Reference

[1] Wikipedia-URA3 gene. https://en.wikipedia.org/wiki/URA3.

[2] Flynn, P. J.; Reece, R. J. (1999). "Activation of transcription by metabolic

intermediates of the pyrimidine biosynthetic pathway". Molecular and Cellular

Biology. 19.

Part:BBa_K1061002

Designed by: mengyi sun Group: iGEM13_SYSU-China (2013-09-11)

RIP 1

Receptor interacting protein 1, a serine/threonine kinase that play

an important role in cell apoptosis and necrosis. Overexpression

of RIP 1 in cancer cells may induce cell death

Functional experiment

The death-induce effect of RIP 1

References

[1]Olivier Micheau, Jürg Tschopp et al,2003, Induction of TNF Receptor I-Mediated Apoptosis

via Two Sequential Signaling Complexes,Cell,114:181-190

Part:BBa_K2365508

Designed by: HangYu Duan Group: iGEM17_NAU-CHINA (2017-10-20)

Bax induced part

Bax, a member of the Bcl-2 family, is a mammalian derived pro-

apoptotic protein that regulates apoptosis by promoting cell

death. Bax can undergo different conformational changes, and

its site of action appears to reside in mitochondria. BI-1 is an

evolutionarily conserved integral membrane protein containing

multiple membrane-spanning segments and is predominantly

localized to intra-cellular membranes, similar to Bcl-2 family

proteins. When over-expressed in yeast cells, BI-1 suppressed

apoptosis included by Bax protein. In the kill switch design we will

utilize the toxin-antitoxin (TA) system to functionally connect the

two agonistic protein together to achieve our biosafety.

This part is used to as the key part of our biosafety device. Coding

sequence of Bax protein is linked to the Gal1 promotor. It could

be inserted to your device as kill switch when your chassis fungal

is yeast (in the most broadly yeast).

Part:BBa_K2232025

Designed by: Wenkai Hu Group: iGEM17_SZU-China (2017-10-18)

mazEF switch

Encodes a stable non-specific ribonuclease toxin (mazF) and its

inhibitory antitoxin (mazE) in Bacillus Subtilis. These genes are used

in Bacillus Subtilis to provide a toxin-antitoxin kill switch in various

stressful conditions. When expression of both genes is turned off

(as both are under contol of same promoter) mazE will be

degraded faster than mazF. There is then no inhibiton of mazF,

killing the cell. Contains Sucrose sensitive inducer that will only

allow coding sequence translation in the presence of sucrose.

These genes are used in Bacillus subtilis to provide a toxin-antitoxin

kill switch in various stressful conditions. When translation of both

genes is turned off by sucrose limitation mazE will be degraded

faster than mazF. There is then no inhibiton of mazF, killing the cell.

Part:BBa_K2493004

Designed by: Yu (Peter) Zeng Group: iGEM17_McMaster_II (2017-10-27)

Antisense Sok

The Sok RNA serves as the antitoxin within the Hok/Sok toxin-

antitoxin system. The system facilitates plasmid maintenance

within growing bacteria.

In particular, the system is designed to kill the cells that lack the

plasmid containing the Sok gene. The Hok protein is very stable

and acts as a toxin to the cell when it is expressed. Specifically,

Hok depolarizes the cell membrane, leading to cell death.

However, the Sok RNA post-transcriptionally regulates the

expression of Hok. Sok is complementary to the leader region of

the Hok mRNA. Through its association with the leader region, Sok

inhibits the translation of the toxic Hok protein, preventing cell

death. However, the Sok RNA is very unstable and it is degraded

quickly. Therefore, the plasmid containing the Sok gene is

necessary for the survival of the cell, if it contains the Hok gene.

Using this toxin-antitoxin system, the maintenance of particular

plasmids can be controlled.

References

Gerdes, K., Thisted, T. & Martinussen, J. Mechanism of post-segregational killing by

the hoklsok system of plasmid R1: sok antisense RNA regulates formation of a hok

mRNA species correlated with killing of plasmid-free cells. Molecular Microbiology

4, 1807-1818, doi:10.1111/j.1365-2958.1990.tb02029.x (1990).

Part:BBa_K1631003

Designed by: Yuto Yamanaka Group: iGEM15_UT-Tokyo (2015-09-13)

Tlanslational unit of Colicin Lysis Protein (for colicin-E3)

Colicins are a cytotoxins which are released to environment and

kill other related strains.Release of colicin involves one protein;

Colicin Lysis Protein. Colicin lysis protein allows colicins to be

released. The mechanism how the colicin lysis protein allows

colicin release has not been fully elucidated, but it is sure that this

protein raise membrane permiability and cause quasilysis. After

Colicin released, they diffuse through the medium and bind to

the receptor on the target cell membrane. Then, they are

imported to the cytoplasm or cytoplasmic membrane of target

cell by Tol-system or Ton-system.

Reference

[1] Cascales, E., Buchanan, S. K., Duché, D., Kleanthous, C., Lloubes, R., Postle, K., ... & Cavard, D. (2007). Colicin biology. Microbiology and Molecular Biology Reviews, 71(1), 158-229.

Part:BBa_K1660008

Designed by: Dai Yuanyi Group: iGEM15_BNU-CHINA (2015-09-08)

J23100-B0034-bace16

A serine protease bace16 was first reported as a pathogenic

factor against nematodes, whose accession number is AY708655.

It was identified by methods such as ammonium sulfate

precipitation. In vitro assay demonstrated that the recombinant

protease Bace16 expressed in Escherichia coli presented a

nematotoxic activity, and it has been verified by experiments that

Bace16 has the ability to degrade a nematode cuticle, leading

to the nematode’s death. And both Bace16 could degrade a

broad range of substrates including casein, denatured collagen,

and nematode cuticle. Bace16 could be considered as a core

component to kill the nematode.

Reference

[1] Huang X W, Niu Q H, Zhou W, et al. Bacillus nematocida sp. nov., a novel bacterial strain with nematotoxic activity isolated from soil in Yunnan, China[J]. Systematic and applied microbiology, 2005, 28(4): 323-327. [2] Niu Q, Huang X, Zhang L, et al. Functional identification of the gene bace16 from nematophagous bacterium Bacillus nematocida[J]. Applied microbiology and biotechnology, 2007, 75(1): 141-148. [3] Day R M, Thalhauser C J, Sudmeier J L, et al. Tautomerism, acid-base equilibria, and H-bonding of the six histidines in subtilisin BPN′ by NMR[J]. Protein Science, 2003, 12(4): 794-810. [4] Qiuhong N, Xiaowei H, Baoyu T, et al. Bacillus sp. B16 kills nematodes with a serine protease identified as a pathogenic factor[J]. Applied microbiology and biotechnology, 2006, 69(6): 722-730.

Part:BBa_K1727005

Designed by: Ying Kuan Group: iGEM15_TCU_Taiwan (2015-09-05)

Signiferin (Crinia signifera)

Signiferin is a kind of antimicrobial peptide (AMPs) and it is a

stable peptide that has extensive abilities to kill or inhibit the

growth of bacteria. It plays a role in defense mechanism

for Crinia signifera to against microbes. Signiferin use its

chargeability to interact with bacteria cell membrane. Than use

hydrophobic region interfere the membrane structure. This leads

to cell lysis and bypasses bacterial antibiotic drug-resistance

mechanisms. Signiferin has demonstrated effectiveness in killing

Methicillin-Resistant Staphylococcus aureus (MRSA), and has

been proven by the TU-Delft 2013 iGEM team.

References

[1] Yeaman, M.R. and N.Y. Yount, Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev, 2003. 55(1): p. 27-55. [2] Lai, Y. and R.L. Gallo, AMPed up immunity: how antimicrobial peptides have multiple roles in immune defense. Trends Immunol, 2009. 30(3): p. 131-41.

Part:BBa_K1405008

Group: iGEM14_BNU-China (2014-10-16)

Our kill switch is able to be “off” for a certain time for the bacteria

to perform its function and then trigger the suicide progress

spontaneously at a certain time.

In the medium, the bacteria are easily controlled by adding or

removing regulatory factors. However, when the bacteria perform

its function in an unregulated environment, the suicide progress

needs to be activated spontaneously. Moreover, the kill switch is

supposed to be “off” for a certain time, so the bacteria will gain

enough time to perform its function. For these reasons, toxin protein

MazF is the best candidate to kill the bacteria for us.

MazEF is a toxin-antitoxin module composed of mazE and mazF

locating on the chromosome of E. Coli and other pathogens

(Hanna et al, 2005). The expression product of mazF is a stable toxin,

while that of mazE is a labile antitoxin of MazF (Hazan et al, 2004;

Schusteret al., 2013). MazF is a sequence-specific mRNA

endoribonuclease that initiates a programmed cell death

pathway in response to various stresses. The mazEF-mediated

death pathway can act as a defense mechanism that prevents

the spread of bacterial phage infection, allowing bacterial

populations to behave like multicellular organisms.

Part:BBa_K1378031

Group: iGEM14_Peking (2014-09-28)

Holin from lambda phage

Holin is a 105-amino-acid-residue cytoplasmic membrane protein

with three transmembrane domains, naturally expressed by

double-stranded lambada phage. Holin will oligomerize and form

a hole on the inner membrane of host bacteria at a certain time

at an allele-specific time. And then the formation of hole will help

Endolysin, a kind of lysozyme, come out from cytoplasm to

periplasm to degrade peptidoglycan and inhibit the respiration by

eliminating proton gradient.

Part:BBa_K1510233

Group: iGEM14_NYMU-Taipei (2014-10-07)

A blue light regulated ccdb apoptosis gene

This is a composite of two units, a blue light promoter and a ccdb

killing gene. The blue light promoter may turn on the transcription

of ccdb.CcdB poisons the gyrase-DNA complex, blocking the

passage of polymerases and leading to double-strand breakage

of the DNA. Alternatively, in cells that overexpress CcdB, the A

subunit of DNA gyrase (GyrA) has been found as an inactive

complex with CcdB. The lethal effect of CcdB is most probably

due to poisoning of the gyrase-DNA complex.

Part:BBa_K628006

Designed by: Charles Thompson Group: iGEM11_St_Andrews (2011-09-20)

Protegrin-1 Kill Switch

Protegrin-1 is an antimicrobial peptide (AMP) first derived from

porcine leukocytes. These peptides are part of the innate immune

system and function by attacking the membranes and intracellular

processes of invading bacteria. Protegrin-1 functions to protect the

body against non-host cells by integrating itself into the

phospholipid bilayer of prokaryotic bacteria, destabilizing the

membrane and causing pore formation (Lam, 2006). These pores

inhibit the cell’s ability to control transmembrane ion and water

movement, resulting in cell death via either osmosis or cytosol loss.

The kill switch would be to induce bacteria to produce AMPs

intracellularly, and allow these peptides to integrate into the

membrane. As the concentration of AMPs builds, pores will form in

the membrane, inevitably leading to cell death.

References

[1] Steinberg et al (1997) 'Protegrin-1: a broad-spectrum, rapidly microbicidal

peptide with in vivo activity Antimicrobial Agents and Chemotherapy', Aug 1997,

1738-1742, Vol 41, No. 8

[2] Lam et al (2006) 'Mechanism of Supported Membrane Disruption by

Antimicrobial Peptide Protegrin-1' J. Phys. Chem. B, 2006, 110 (42), pp 21282–21286

[3] Bierbaum, G. & Sahl, H.-G (1985) 'Induction of autolysis of Staphylocci by the

basic peptide antibiotics pep5 and nisin and their influence on the activity of

autolytic enzymes.' Arch. Microbiol. 141, 249 ± 254

Part:BBa_K1172904

Designed by: Tore Bleckwehl Group: iGEM13_Bielefeld-Germany (2013-09-19)

Rnase Ba (Barnase) from Bacillus amyloliquefaciens

The Barnase (EC 3.1.27) is a 12 kDa extracellular microbial

ribonuclease, which is naturally found in the Gram-positive soil

bacteria Bacillus amyloliquefaciens and consists of a single chain

of 110 amino acids. The Barnase (RNase Ba) catalyses the

cleavage of single stranded RNA, preferentially behind GpN. In the

first step of the RNA-degradation a cyclic intermediate is formed

by transesterification and afterwards this intermediate is

hydrolyzed yielding in a 3'-nucleotide (Mossakowska&nscbet al.,

1989).

In Bacillus amyloliquefaciens, the activity of the Barnase (RNase Ba)

is inhibited intracellular by an inhibitor called barstar. Barstar

consists of only 89 amino acids and binds with a high affinity to the

toxic Barnase. This prevents the cleavage of the intracellular RNA

in the host organism (Paddon et al., 1989). Therefore the Barnase

normally acts only outside the cell and is translocated under

natural conditions. For this BioBrick we modified the enzyme by

cloning only the sequence responsible for the cleavage of the RNA,

leaving out the N-terminal signal peptide part.

References

[1] Mossakowska, Danuta E. Nyberg, Kerstin and Fersht, Alan R. (1989) Kinetic

Characterization of the Recombinant Ribonuclease from Bacillus

amyloliquefaciens (Barnase) and Investigation of Key Residues in Catalysis by Site-

Directed Mutagenesis Biochemistry 28: 3843 - 3850..

[2] Paddon, C. J. Vasantha, N. and Hartley, R. W. (1989) Translation and Processing

of Bacillus amyloliquefaciens Extracellular Rnase Journal of Bacteriology 171: 1185

- 1187..

[3] Voss, Carsten Lindau, Dennis and Flaschel, Erwin (2006) Production of

Recombinant RNase Ba and Its Application in Downstream Processing of Plasmid

DNA for Pharmaceutical Use Biotechnology Progress 22: 737 - 744..

Ⅴ Society+ Ø Overview

² Legal Regulation Methods

Develop biosafety regulations on remediation by genetically

engineered bacteria (RGEB) based on opinions from iGEMers in

China.

² Interview or Questionnaire

1. The government’s attitude towards RGEB.

2. Attitudes of pollution-producing enterprises for RGEB.

3. Individual attitude towards RGEB.

Ø Work done by ECUST and SHPH

1. Worldwide Convention on Biosafety

- Convention on Biological Diversity

The Convention is the most important biosafety convention in the

world whose Its main content is related to biosafety management.

- Cartagena Protocol on Biosafety

The protocol builds a set of operational frameworks around the

world. And its main content is to manage the environmental and

health problems that may occur in the international circulation of

genetic modifiers under the precursor of prevention.

- United Nations Convention on the Ocean

The Convention requires concerted measures around the world to

prevent pollution of marine species by new species.

2. China Biosafety Regulations

(1) Agricultural genetically modified organism safety regulations

①Strengthen the management of domestic research, testing,

production and other activities for agricultural genetically

modified organisms.

②Establish an inter-ministerial joint meeting system for the safety

management of agricultural genetically modified organisms.

③Implement a hierarchical management evaluation system for

the safety of agricultural genetically modified organisms.

④Establish a safety evaluation system for agricultural genetically

modified organisms.

⑤Implement a labeling system for the safety of agricultural

genetically modified organisms.

(2) Implement a unified laboratory biosafety standard. The

laboratory shall comply with national standards and requirements,

and shall establish and improve safety management systems,

inspection and maintenance methods.

(3) Legal provisions to prevent the invasion of alien species.

①Establish an entry and exit animal and plant quarantine system.

②Implement a new plant variety protection system.

③Implement a germplasm resource protection system.

④Implement marine species protection regulations.

⑤Implement legal protection provisions for aquatic germplasm

resources.