Team:WHU-China/Notes

Brief Answers to Safety Questions

Welcome to our Safety Page. We will first answer the safety questions asked by iGEM headquarters briefly, and then discuss safety issues associated with our project in detail. In addition, we will provide our ideas and practice on guaranteeing and developing biosafety.

Q1. Would any of your project ideas raise safety issues in terms of: Researcher safety, public safety, or environmental safety?
No. Our design is based on the commonly used nonpathogenic E. coli K.12 strain and genes we manipulated are original genes in E. coli. The protein products, at least from current understanding, will cause no harm to researchers, the public and environment. In addition, strict lab practice is executed to further ensure safety.

Q2. Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues? If yes,
Did you document these issues in the Registry?
How did you manage to handle the safety issue?
How could other teams learn from your experience?
Yes, we will discuss this question in latter part of this page (Safety Considerations of Our Biobrick parts and Our Project).

Q3. Is there a local biosafety group, committee, or review board at your institution?
If yes, what does your local biosafety group think about your project?
If no, which specific biosafety rules or guidelines do you have to consider in your country?
Yes. All materials obtained have received the approvals from the department's laboratory management committees. We are also obliged to observe the regulations of Teaching Centre of Experimental Biology and apply for approval for materials before we start our project.

Q4. Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
Some classified measures should be taken according to the safety of the material. For example, plasmids that may harm the safety, when submitted, should receive more attention and have a stricter package. Some harmful byproducts during experiments should be eliminated properly.
We have done our human practice aiming at understanding attitude of the public towards genetically modified baceria and publicizing biosafety ideas to the public, which we think should be popularized to other teams in future iGEM competitions.

General Safety Issues

In this part, we will illustrate organisms, reagents and equipments we use that may cause safety problems, and introduce our operation standard, management and trains protecting the team members, public and environment.
As above, all the organisms and DNA hosts are not of high individual or community risk. Until now, the only used organism is E. coli K.12 (and some of its varieties, for example, DH5α, a RecA mutated strain). Our current lab of basic-biosafety level 1 is safe enough to manipulate this strain. Only the genome (a generous present from University of Dundee iGEM team), but not the living Salmonella enterica was manipulated in the lab, and the genes from it are homogeneous of E. coli K.12, not associated with pathogenesis. We will not implement our future experimental plan with microorganisms of risk group 2 or vertebrates in our current lab, for too low is the biosafety level and no animal facilities.
To manipulate microorganisms, an ultra clean cabinet is used and strict aseptic technique is followed. All experimenters have been trained on foundation microbiology technique and biosafety. All microorganism contacting vessels are sterilized before and after experiments in appropriate protocols. Also, all microorganism materials will be sterilized before discarded. In this way, we believe that no public or environmental harm will be caused by the experimental organisms. In addition, no one in our team will be hurt by the experimental organisms.
The genetic modifications we make will change metabolism of bacteria. However, there is no evidence both theoretically and experimentally that these modifications will improve the pathogenicity of the bacteria or cause damage to environment even taking the risk of horizontal gene transfer into consideration. The effects of expressing gene adrA regarding infectivity and pathogenicity on E. coli is not clear now, but it still can be easily controlled in the lab environment without human ingestion. Also, we will insert a death device into the bacteria cell when we construct the whole system (still far from now) to avoid the proliferation out of control. For more information about gene safety, please read the next section and documents of our relevant parts on registry.

We have considered the potential harmful chemicals and equipments, as listed below:

• Basic molecular experiments: NaOH, HCl, SDS, acrylamide, TEMED, ethanol, IPTG, liquid nitrogen, β-mercaptoethanol, xylene cyanol FF.
• Bacteria culture: ampicillin, kanamycin, chloramphenicol.
Chemical analysis and measurements: acetone, cupric acetate, Sudan III, Congo red, Coomassie Brilliant Blue G-250, Coomassie Brilliant Blue R-250.
• Equipments: UV lamp, supercentrifuge, heating equipments (alcohol burner, PCR amplifier, water bath, dry bath), electrophoresis apparatus, -80℃ refrigerator, ultrasonic cell disruptor.

All of these are regular reagents and apparatus in a molecular biology laboratory. The risks of them come from inflammability, explosibility, irritation, corrosivity, toxicity, carcinogenicity and physical injury. But none of them raises special safety issue, with chemical hood, emergency shower, normal personal protection, safety management and safety training.
Only trace amount of antibiotics are used. Inactivated will they be before discared.

Emergency Shower

Safety Considerations of Our Biobrick parts and Our Project

In this section, we are going to answer safety question 2 in detail, taking the potential risk in the future into concern.
The main safety challenge we must face is that as a practical bacteria therapy our direction is, we shall demonstrate that the “E. coslim” will not harm its host when it is developed completely. As few experiments we can do in such limited time, we have done a series of theoretical work to solve problems in this field. Intestine-colonized pathogenic E. coli may cause immune responses, following by diarrhea, inflammation and fever (although the strain we use is considered non-pathogen). Thus we plan to transplant the whole synthetic system to another organism (for example, Bacillus subtilis, has proved safe in human intestine). We know that it is hard as the two organisms are very different on transcriptional mechanisms, but we believe the work of establishing model system (that is what we are doing) in E. coli will make it easier. Also, as the rapid development of synthetic biology and gut microbiology, we hope in the near future, we can modify the genome of E. coli to change it a safe intestine microbe.
Another risk is that when the engineered bacteria get higher efficiency on energy production, they could proliferate out of control. To forestall this situation, we design a “death” device (for more information, click on Death) using D-xylose as inducer. It means if you want to stop weight losing, what you have to do is to eat some D-xylose, and the “E. coslim” will die, shed from the intestinal wall and be poured out. What is more, designed as a two-plasmid system, it prevents all possible horizontal gene transfers.
Further safety issues will be raised and discussed in future experiments, including those operates in intestinal model and animals (For more information, click on Future Perspectives).

Safety Management and Practice

Our project has past a review of an expert committee, of which members are professors or associate professors of microbiology, genetics and bioengineering. Safety issues are considered seriously before they approved our design. We have consulted a few experts for safety questions about both artificial intestinal bacteria and experiments. Their advices help us improve the safety of the whole planning system. For that, we thank Dr. Yulan Wang and Dr. Tiangang Liu so much. Our lab is supervised by Teaching Centre of Experimental Biology, Wuhan University (TCEB, whose leader is our instructor Dr. Zhixiong Xie). An expert group of this centre formulates safety guidelines and guarantees their performance in all teaching labs. All experiments we do past its review. Our safety management system includes the followings: Management of Chemicals, Equipments and Experimenters: Chemicals and equipments are registered in TCEB before they are available for us. Equipments are checked and maintained regular. And an entrance guard system is used to ensure only the admitted could enter the lab.

• Responsibility distribution: All members worked in the lab are divided to three groups. The group leaders arrange schedules of experiments after assessing safety issues in the group (for example, the safety train of the experimental executer). Every member records his/her experiments in detail in the notebook of the group. Each group takes responses for cleaning and checking risks in the lab in turns. The group leader takes responses to the team leader. The team leader reports regular on safety to engineer Miss Long Yan, who is authorized by TCEB and manages the lab.
• Management of Chemicals, Equipments and Experimenters: Chemicals and equipments are registered in TCEB before they are available for us. Equipments are checked and maintained regular. And an entrance guard system is used to ensure only the admitted could enter the lab.
• Training: Our team members have been trained after Guidance of Student Experiments formulated by TCEB.

Guidence of Student Experiments

Biosafety and Publicity

We believe that biosafety is not an issue which should be only considered inside biological lab, but also around people and communities. Spreading knowledge of biosafety to the public will help eliminate misunderstanding and prejudice, from which the science and all the people will benefit. We hope that sharing this idea with all iGEM teams can be useful to take advantage on biosafety.
To investigate public attitude towards our project and to publicize our biosafety ideas are what we do for our human practice. As “eating bacteria” is somehow an unacceptable concept now, we would like to find whether people know the truth about biosafety issues raised by bioengineered bacteria, or they are just panicked by their imaginary “bacteria”. And then, after initiating basic knowledge of microbiology, gene engineering and synthetic biology, we shall take a look on if people’s opinion to biosafety issues will change. In this way, we will estimate the value of our scientific publicity.
The rational discussions of biosafety we take with the public do not limited on our project, but also hotspot issues such as transgenics and stem cell therapies. For more information, please click on Human Practice.

Introducing a Simplified Intestinal Model

Materials for cloning all the genes

Bacteria strain:

E.coli DH5α

Minimal Medium:

M9:

For 1L Medium add

Na₂HPO₄·12H₂O 15g

KH_{2PO4 3g}

NaCl 1g

NH_{4Cl 0.5g}

After autoclaving, add:

MgSO_{4(1mol/L) 2mL}

Bacteria strain:CaCl_{2 100μL}

Triton X-100 (as emulsifier) 2uL

Note：

1.For M9 medium using oleic acid as sole carbon source, various amount of oleic acid was first emulsified 1:1 with 10% Triton X-100. M9 medium was then slowly added with constant vortex. M9 medium with high concentration of oleic acid was diluted by M9 medium with triton to form various concentrations；

2. For M9 medium using glucose as sole carbon source, M9 medium with high concentration of glucose was diluted by M9 medium to form various concentrations.

Step:

1. Seed liquor which was activated over night was inoculated into M9 medium which contains different concentration of oleic acid. And it was then incubated at 37℃ for 24 hours;

2. After 24h of incubation in 24 well plates in 37℃, bacteria culture was centrifuged at 3000rmp for 5min, washed and resuspended in PBS;

3. We detected the OD600 and fluorescence of using SpectraMax M2 plate reader (Molecular Devices) .Excitation at 584 nm and emission at 607 nm were used. All fluorescence was normalized with cell density by measuring the absorbance at 600 nm.

Result:

Normalized using Fluorescence/0D600

Blue: Constitutive promoter J23110

Red: PfadR

Glucose Concentration gradient: 0.5, 1, 5, 10 mM

Fatty acid Concentration gradient: 0.025, 0.05, 0.1, 0.25, 0.5, 1, 1.5 mM

this part is finished

Protocols of Cupric-Soap Reaction:

To test metabolism of long fatty acids, we used oleic acid as sole carbon source in mediums, and used cupric-soap reaction to determinate oleic acid concentration preliminarily.

Modified M9 minimal medium with emulsified oleic acid as sole carbon

Minimal medium was the same with that in Materials and methods for PfadR

What's Important:

Slowly pour M9 minimal medium into mixture of oleic acid and Triton X-100 to get more homogeneous solution.

Analysis the concentration of the oleic acid in the medium by cupric-acetate method

1.Collect 5 ml medium which has been used to cultivate bacteria. Then centrifuge the medium at 3000rpm for 10 min to separate bacteria and medium;

2.Decant 3ml supernatant liquid into a 10ml EP. Add 3ml acetone to the liquid, 1ml at a time, shaking 10-20 times before adding another 1 ml in order to avoid the effect of the ions of the liquid during the extraction progress;

3.Add 3 ml isooctane once ,shaking for at least 90s, stand for 2min or longer until layering completely;

4.Collect 3 ml clear isooctane in a 5 ml EP, Add 800l cupric-acetate (5% m/v, adjust pH to 6.8 with pyridine), Shaking for 90 seconds, stand for 2min or longer until layering completely;

5.Detect OD of organic phase in a spectrometry at 715nm.

The Standard Curve:

We add 1.6ml, 3.2ml, 4.8ml, 6.4ml, 9.6ml, 11.2ml oleic acid into 3ml M9 medium to generate a gradient. The absorbency is measured as described in the protocol.

We slightly modified the methods provided by Kwon and Rhee, 1986, adding the extraction step, for it is dispensable when bacteria are in the medium. However, the standard curve is still very close to the paper’s, which proves that plausible and accurate

Krebs-Ringer phosphate（KRPD buffer solution）：

Solution A：

For 100 mL:

NaCl 7.5985g ; KCl 0.3727g ; CaCl2 0.1054g; glucose-H2O 0.9495g,

For 100 mL:

NaH2PO4·2H2O 0 .2964g ;Na2HPO4·12H2O 2.9011g，

After dissolving, add MgSO4·7H2O 0.3130g

Mix A solution and B solution, add 790ml dd H2O, then regulate pH to 7.2~7.4 using 1M NaOH/1M HCl, finally add dd H2O to a final total volume of 1L.

Procedures

1. cultivate cells in 1 liter of medium to late exponential phase;

2. harvest by centrifugation at 35℃;

3. wash twice with 2 liters of warm(35℃)0.05M potassium phosphate buffer (pH 7.4) containing 1%(v/v) Triton X-100;

4. resuspend in 0.05M potassium phosphate buffer (pH 7.4) containing mercaptoethanol;

5. add sufficient volume of buffer to give a concentration of about 50mg(dry weight) of cells/ml;

6. disrupt cells with a Bransor Sonifier for 1min. The treatment was applied for 15s intervals, under 4 ℃;

7. centrifuge at10,000×g for 30min;

8. decant supernatant liquid for enzymatic assay, the protein concentration was determined by the biuret method;

9.analyze fatty acid oxidation in cell-free extracts

Note: little oxidation was observed when less than 1mg.

Reaction mixture:

1.0ml of freshly oxygenated Krebs-Ringer phosphate(pH 7.4)

Palmitate-1-14C, 20nmole (80,000 counts/min)

CoA, 1 μ mole

NAD, 1μ mole

ATP, μ mole

Succinate, 10 n moles

Supernatant protein, 1~5 mg

dd H2O to a final total volume of 2.0ml

Cellulose biosynthesis

1. Inoculate the bacteria into liquid LB medium and then incubate it for 24 hours at 37℃;

2. After centrifugation, supernatant is preserved for use, while deposits are resuspended with PBS and adjust to OD600 1.0;

3. Exceed cellulase was used to digest cellulose in supernatant and deposits. After the cellulase is added, 1mL solution was sampled every 3 min and cellulose of the sample was inactivated immediately;

4. Ten minutes later, the reduce sugar is detected in all the samples with or without cellulose;

5. 2mL DNS solution is mixed with the sample, and incubated in boiling water for two minutes, then it is cooled rapidly;

6. After 9 mL ddH2O being added into the solution, record absorbance at 540nm;

The Standard Curve of glucose concentration

Materials and methods of SDS-PAGE:

Coomassie Blue Stain

-Coomassie brilliant blue G-250 50mg

-95% ethanol 25ml

-85%H3PO4 50ml

-H2O, adjust to 500ml

-filter

Destain solution

-methanol 250ml

-acetic acid 50ml

-H2O adjust to 500ml

2x SDS loading buffer

-0.5mol/l Tirs-HCl(PH6.8) 25ml

-10%SDS 8ml

-50%glycerol 20ml

-2-mercaptoethanol 2ml

-1%Bromphenol Blue 4ml

-H2O adjust to 100ml

10xSDS-PAGE running buffer

Tris base, 30.3 g

M glycine 144.1g

SDS 10 g

-H2O adjust to 1L

Steps

Protein expression

1.inoculate the liquid strains into LB medium supplement with 50μg/mL ampicillin, incubate the medium at at 37℃ until OD600 reaches 0.6;

2. Separate the culture into two test tubes. Add IPTG into one of two tubes at a final concentration of 1mM to induce the expression of

3. 1 mL of the culture is sampled every hour. After centrifugation, deposits was suspended with 300 μL PBS and 200 μL 2x SDS loading buffer, then incubated in boiling water for 15 min.

4. 10μl of samples was load in lanes, run the SDS-PAGE

5. Stained the SDS-PAGE 5 hours

6. Destain the SDS-PAGE until you can see the protein band.

team history

Dec. 2011

WHU iGEM team was established

Dec. 2011 to Feb. 2012

Every one presented their own idea, then we discussed the feasibility of these ideas.

Feb. 2012

The final project was determined, named E.coslim

Mar. 2012

We finished an outstanding presentation. Our reply was approved by the leaders of college of life sciences, Wuhan University.

Apr. 2012

Our iGEM team was divided into 3 groups—group of Sheng, group of Xia and group of Mei.

Apr. 2012 to prsent

Experiments started….

Apr. 2012

Group of Sheng: FADR was connected to pSB1A2 plasmid carrier

FADR was connected to RFP gene

Group of Xia: starting to connect genes of CI control system

Testing the function of CI control system

Group of Mei: YhjH/ FadE/ FadD/ FadL gene was connected to pSB1A2 plasmid carrier

May. 2012

Group of Sheng: FadB/ FadJ gene was connected to pSB1A2 plasmid carrier, then BBa_B0030(RBS) as well

Group of Xia: They devised two promoters p110 and p101, which were expected to be controlled by glucose. However, they failed.

Group of Mei: YhjH/ FadE/ FadD/ FadL gene was connected to BBa_B0030(RBS)

June 2012

G of S: FadR was connected to low-copied plasmid carrier.

sFadB and sFadA genes were connected to BBa_B0030 (RBS)

sFadE gene was connected to pSB1A2 plasmid carrier

G of X: the connection of genes, which were relative with cellulose, was finished.

Another two promoter were devised, p1 and p2

G of M: YhjH/ FadE/ FadD/ FadL gene was connected to BBa_B0024 (terminator)

July 2012

G of S: FadJ was connected to BBa_B0024 (terminator), sFadE gene was copied by PCR technology

On the front half of this month, they conducted several pre-experiments of oleic acid test

On the next half month, they started normal experiments of oleic acid test

G of X: started to test the function of p1 and p2

G of M: YhjH/ FadE gene was connected to BBa_J23100 (promoter)

Finish the standard curve of oleic acid test

Aug. 2012

G of S: sFadE was induced to point mutation, then it was connected to BBa_B0030 (RBS) and BBa_B0024 (terminator) respectively

sFadB was connected to BBa_B0030 (RBS) and BBa_B0024 (terminator) respectively.

G of X: test the function of cellulose-controlled genes, having got satisfying results

G of M: copied Adra gene and finished the connection of BBa_B0030 (RBS), BBa_B0024 (terminator) and BBa_J23107 (promoter), BBa_J23114 (promoter) respectively. FadE/YhjH/FadD/fadL were connected to BBa_J23107 (promoter), BBa_J23114 (promoter).

Sep 2012

G of S: Transferred all the parts in pSB1A2 into pSB1C3, Tested the function of PfadR, Characterized the effect of each gene on fatty acid consumption, started to set up the platform for in vitro experiments.

G of X: Transferred all the parts in pSB1A2 into pSB1C3, tested the function of cellulose system. Repeat the test of the function of cellulose-controlled genes.

G of M: Transferred all the parts in pSB1A2 into pSB1C3, test the function of Adra/YhjH.

About E.coslim by Kuanwei Sheng

In order to help people lose weight, besides our three devices, we also have come up with many other creative ways.

Ⅰ.Short chain peptides synthesis

Recent researches have indicated that some short chain peptides in intestine have effect on inhibiting appetites, therefore decrease the food intake. We thus formed the idea that we could synthesis a DNA chain that encodes those short peptides.

Ⅱ.Biosynthesis of L-carnitine

L-carnitine is a molecule that facilitates the progress of transporting fatty acids into mitochondria where these fatty acids will be disintegrated. We once considered use L-carnitine to help the host metabolize fatty acid better. However, the biosynthesis of L-carnitine has too many derivatives or the pathway is patented by others.

Ⅲ.Xylose isomerase

Xylose is a prebiotics that can hardly be absorbed by human. We consulted many papers and find that glucose can be converted into xylose by xylose isomerase. Therefore, we thought maybe we could lower the glucose available in intestine by expressing xylose isomerase. However, this process is shown to be reversible latter. Mutated the sequence of the protein may generate high converting rate, yet it is too laborious and risky for a short time project.

Other novel ideas

Tackle Water bloom by Tong Wang and Kuanwei Sheng

Since the detrimental effects caused by cyanobacteria to the environment such as making water carcinogenic have become a serve global problem, we therefore tried to employ E.coli as an expression system to eliminate these detrimental effects. When we first took over this project, we thought about limiting the growth of cyanobacteria. Along with the process we got to read a large amount of relevant papers about cyanobacteria, we found that not only the main detrimental effects caused by cyanobacteria is attributed to its product which is a cyclic peptide called microcystin, but also microcystin can regulate the population density. Then we came to realize the importance of microcystin and began to search information about it. Through over this process, we discovered a gene cluster which is responsible for the microcystin degradation pathway. It encodes four enzymes——MlrA、MlrB、MlrC and MlrD. Also, we found that some non-toxic cyclic peptides produced by cyanobacteria such as Anabaenopeptin B and Anabaenopeptin F can induce lysis of cyanobacteria. The latter finding can be utilized as an effective cell population density control mechanism. Thus we thought about constructing two independent systems to eliminate cyanobacteria, one about inducing lysis of cyanobacteria and the other about degrading microcystin. Finally we gave up this project because of the reasons that these gene clusters are too large to be expressed in E.coli and that E.coli cannot survive in the sea, however, we still feel proud of these fancy ideas.

Desalination of sea water by Kuanwei Sheng

We came up with the thought that engineering bacteria can intake ions like Na+, cl-, Mg2+ and etc. under special stimulus. Also, under another certain stimulus, the bacteria can export those salts out of cells for reuse. Therefore, we can use these bacteria to desalinize sea water and extract the salt the same time. However, there is no such ion channel that can meet our needs.

Sense the earthquake By Min Ye

We once tried to find proteins that can sense vibration and construct a pathway to report that vibration. However, we are not able to find the protein that can meet our needs.

Auto plasmid preps By Kuanwei Sheng

We thought about constructing a synthetic protein that combines zinc finger protein which can recognize the specific sequence of DNA and signal peptide that can make proteins be exported out of the bacteria by secretion pathway. Therefore, it is possible that plasmid can be exported out the cells together with the zinc finger protein.

Outer Membrane Vesicle (OMV) to treat cancer By Kuanwei Sheng

We thought to use Outer Membrane Vesicle (OMV) to treat cancer. Specifically, we thought about localizing antibody that can recognize certain cancer on the surface of OMVs via signal peptides. Also, we thought we may localize cell division inhibitor protein or protein that lead to cell death inside the OMVs. Therefore, we hoped that the OMV excreted by the bacteria can recognize and kill cancer cells.

Multicell Yeast By Wenxiong Zhou

Transform the yeast from a single-cell microbe to a multi-cell organism by setting bistability of gene expression among cells of yeasts adhered in amalgamation.

team

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