Team:METU-Ankara

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Methan<b>E.COLIc</b> : Decreasing the Greenhouse effects and Saving the workers life in one system

MethanE.COLIc : Decreasing the Greenhouse effects and Saving the workers life in one system

Firedamp explosions are frequently seen cases at all mines over the world. In Turkey every year, 50 miners lose their lives because of firedamp explosions. Firedamp is a flammable gas found in coal mines and it mainly contains methane. Beside its explosive property, methane is also the main contributor to global warming. However recent mine mechanisms release obtained methane into air. By offers of Synthetic biology, we aimed to design a device which will work on E.coli that provides solutions for side effects of methane. Device that we are planning to construct involves the genes of bacteria (Methylococcus capsulatus) and insect (Drosophilia melanogaster). Our compact system in E.coli is fabricated as sensation of methane, the conversion of methane to methanol and then entrapment of methanol to handle for biofuel and death of bacteria at 42 C by kill switch mechanism.

When project is analyzed in stepwise, there are successive 4 steps of our modelled system in modified organism. As mentioned in subtitles, methane is sensed, then converted to methanol and methanol is entrapped and to elute methanol the cells are dead.

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Introduction

MethanE.COLIc project is designed to solve one of the problems on worker security in mines of Turkey , by constructing the natural parts of organisms. The main reason for us to choose such a project is that in Turkey- and also in many countries- each year, huge numbers of workers in mines lost their life due to deficiencies and conditions in their working areas (mines). The release of Grizu gas leads to subsidence of mines and so death of workers.This project is also designed to solve one of universal problem; global warming. Methane gas is a potential greenhouse gas.This project focuses on sensing the ambient methane gas and converting to one of the bio-fuel sources, methanol. Methanol is entrapped by product of one of the constructs of project, and then cells are dead by kill switch device of project to elute methanol.

Team

Hello everyone!We are Metu Ankara IGEM team.Our team consists of high motivated,social and enjoyable “undergraduate and graduate scientists” from departments of physics, chemistry ,biology, computer engineer and computer education & instructional technology. We are such a team that complete each other so that we all experienced team spirit in IGEM. This makes us happy while doing experiences even in the hot summer.Our slogan is “ all for one, one for all”!!Then, who are we?

Instructors

Advisors

Team leader :Ceren Seref

Ceren has just finished the junior year in biological sciences department in METU. It’s her second year in iGEM competition. She’s planning to study about neuroscience-especially on Alzheimer’s disease. She believes synthetic biology approaches can be a very good way of drug development in neuroscience field. She enjoys climbing, listening to music and singing.

Sibel Ataol

Sibel is first semester student in Master's program of Biomedical engineering at Middle East Technical University indeed she is graduated from Biology Department at METU. This is the second year of her in IGEM team from METU.She is one of the experienced in team and elder sister:) As a vice team lead, planing the experiments and cloning strategies and motivating team are the responsibilities of her. Studying in bioengineering and regenerative sciences is her desire for future.

Funda Guzey

She is a senior student of Biological Sciences at Middle East Technical University. This is her third year in iGEM competition. She gained a lot of experiences in this summer. One of the her interests is Synthetic Biology. However, her main interest is Biomedical and Tissue Engineering. She feels lucky to have such a wonderful team and is glad to share a wonderful summer with her team.

Gökcan Aydoğan

Gökcan has just finished the third year of the B.Sc. Chemistry degree at METU. This is his first year in IGEM team. He has been a huge fan of Synthetic Biology ever since he understands nothing about the concept. Everyday, he tries hard to get the picture. In his free time, he likes gaming, drawing cartoon and eating, eating, eating...

Filiz ORMANCI

She is a fourth year student at the Department of Physics at Middle East Technical University. This is the first year of her in iGEM team. She wants to study on the usage of optical instruments in biophysics. She likes team working and sharing the success. She is always positive and happy... ehm..also a “soothsayer”. She enjoys playing musical instruments mainly violin and learning different languages.

G. Simge Yüz

She is a recent graduate student of Chemistry and a fourth year student of Biological Sciences. This is the first year of her in iGEM team. She wants to study on neuropharmacology in the field of neuroscience. She is glad to join to METU-Ankara-iGEM team. She enjoys dancing especially Argentine tango and swimming. She is also a member of Flashmob Ankara Society

Yunus Alpağu

Yunus is a third year student in biology department at Middle East Technical University in Turkey. This is his first year in iGEM team.He is interested in regenerative medicine, stem cells, biomaterials and tissue engineering. His favourite TV series is ‘Fringe’ and he likes playing tennis and volleyball.He is glad to join to METU-Ankara-iGEM team.

Pelin İspir

Pelin is currently studying the biology department of Middle East Technical University in Turkey. She is interested in stem cell, developmental biology,tissue engineering, and conservation biology.This is the first year of her in İGEM.She really enjoys with being this team and dealing with the mystery of synthetic biology.She enjoys listening latin musics and flamenco.

İbrahim Kutay Ozturk

Kutay is a graduate of Molecular Biology and Genetics, and now doing his M.Sc. in Biotechnology department at METU. This is his first year in IGEM team. He is interested in plant-based drugs, plant-pathogen interaction, biofuel production and bioremediation. He enjoys playing basketball, watching movies, and listening to music.

Advisor:Burak Yılmaz

He is a recent graduate of METU Molecular Biology and Genetics department and now studying towards his masters degree on Molecular Bioengineering at METU. His interest in synthetic biology did start during his undergraduate years and after graduation he started up the Sentegen company which is the first biotechnology based company focused on synthetic biology in Turkey. He continues his research and training in synthetic biology while also contributing to the development of the field in his country. He needs new scientific revolutions to solve huge problems of life and emerging field of synthetic biology is best candidate for biotechnological revolution. He is interested in synthetic biology applications, along with Lab-on-a-Chip devices for molecular biology techniques, and he is designing gene synthesis chips to produce biobricks - raw materials of garage biology- faster and cheaper. he enjoys snowboarding, cycling and writing poems.

Software Group

Muhammed Akif Ağca

Akif is a graduate of Computer Education and Instructional Technologies department. In bachelor he has worked on Software Development, Database Management, Instructional Technologies, and Bioinformatics.
He was software developer and team leader of BioGuide project and Synthetic Biology Online Lecture.
Currently he is mastering in Software Engineering and developing scientific applications, he will continue to work on Science and Technology Management after becoming master of Software Engineering.

Miktat Aktaş

He graduated from Computer Education & Instructional Technology Department of Middle East Technical University last semester and he contributes this genius team as a coder for website. He wants to be a successful academician in his department at METU. He likes swimming and everything related to computers and automobiles.

Ceyhun Kerimov

Ceyhun was born in Azerbaijan. He is studying in METU Computer Engineering Department.
He is Java and Salesforce developer now. He was in web side of the project. He tried to help the team to manupilate and store the data.
He is working in web and cloud computing technologies.

Huseyin Lutin

Huseyin is a senior student in METU Computer Engineering Department.
He was helping the team during software requirements specification step of the project.
He is working on computer graphics in this year.

WET LAB

Whole summer in lab work we aimed to design 7 new E.coli compatible Biobricks and 1 composite part from 2011 kit plate distributions, also we aimed to characterize each part by protein analysis and fluorescence protein measurements.

We achieved design and compatibility of parts in E.coli, however not all parts’ characterization is achieved. Only the genes of designed composite part from kit plate is characterized by GFP measurements.

In this section you can find out more details on all our wet lab works. You can also find our data under results subtitle and information about design of our parts-biobricks with explanations in literature. It also contains materials and protocols, and finally there is safety questions list on our safety considerations both in the lab and on a wider environmental scale.

Overview

MethanE.COLIc projec t arises from sustainability and conservancy of human health and safety. Therefore while we were constructing the organismic device we also wanted to inform people from all age and we wanted to increase the public awareness on our project; methane gas based (firedamp) explosions and the greenhouse effect of methane gas.

Our aim is to inform people in each age. After brainstorming and searching, we planned the content of information and activities according to following subtitles.

Future Plan

Methane explosions called as grisou are one of the main problems from the beginning of the coal mines. All countries have tried different systems to solve this methane problem. When we joined iGEM 2011 competition, our aim is to find a solution to that problem for humanity by cheaper and energy saving way. After a long literature research, we found how seriously dangerous the methane gas is and how people deal with this big problem on coal mines. Below are the countries that have faced with serious methane explosions throughout their history.

Todays companies deal with this problem by;
Regenerative Thermal Oxidizer from MEGTEC capable of handling 60,000 scfm of VAM. At a methane concentration of about 0.7% a unit this size generates 50,000 carbon credits per year. This unit costs about US$ 1,5 million Elizabeth Obediente from CO2IMPACT at a methane upgrading facility at a coal mine in Alabama. The gas is stripped of impurities and upgraded to 96% methane per volume and then compressed and then sold to the natural gas pipeline. Ventilation Air Methane (VAM): VAM units cost about $20-$30 USD per scfm.

At some mines, downtime is considerable, causing expensive losses to the mine. Ventilation cost cuts can be up to half of the original cost of the ventilation system. In one study, a mine with 400 ft3/ton of methane using vertical wells to pre-drain will result in an estimated US$ 11 million over 20 years in reduced ventilation costs, not including carbon and energy revenue[iii] (U.S. Environmental Protection Agency, 1999). Like many solution system for methane problem, the tree examples of solution of methane problem did not satisfied us, so we created our simple and very effective way to deal with the methane.( http://www.co2impact.com/cmm.html )

According to our methane E colic project, we aimed:

Save the coal workers’ life
Save the nature
Process a new energy
Obtain a cheap system

Save the coal workers’ life

We aimed to collect the methane gas from coal mines to the tanks which are filled with water. When the methane passes the legal percent that is 1%, according to our project, the gas is dissolved in water in tanks. Then, inside the tablets containing our bacteria and that are already present on the tanks the molecular process for conversion of methane to methanol begins. Below the number of workers dead by years are given according to Prof. Dr. Mustafa ÖZTÜRK’s research,Vice President of Parliamentary Commission for the Environment in Turkey:

Years	Death workers on Turkey
1983	103
1990	330
1995	40
2003	17
2004	3
2005	18
2006	21
2007	7
2008	43

Methane explosions:

Save the nature

Methane is one of the greenhouse gases in the atmosphere. Methane is continuously released from coal mining, livestock, landfills and natural gases to the atmosphere. The accumulation of these greenhouse gases causes one of the greatest problems of Earth: global warming. Our project aims to prevent release of methane gas by the conversion of it to methanol in bacterial system in coal mines.

Greenhouse gases:

Process a new energy

After the conversion of methane to methanol, methanol can be used as a precursor of bio-fuel so that accumulated methanol can be recovered. Ethanol is used as biofuel, also; however, methanol is more cheaper to obtain and use as a biofuel. According to Dr. Robert Zubrin, Methanol is cheaper than ethanol. It can also be made from a broader variety of biomass material, as well as from coal and natural gas. And methanol is the safest motor fuel.(American enterprise, February 13th, 2006).

Cheap system

There are many systems used to prevent or minimize methane explosions. However, they are so expensive to use and none of them are based on a biological system. The biological system that we build contains a bacterial cell having a molecular process of conversion of methane to methanol is completely harmless and cheaper since expensive equipments are not needed.

PartnerShip

Collaboration
Sponsors
METU
Acknowledgements

Extras

Hands on show!

Photos

Methane sensing

i) Introduction

In our methanE.COLIc project, the first critical point is to sense the ambient methane gas. While in literature search, we have focused on the methane and DNA or methane and regulator protein interaction which binds to DNA after binding to methane. In structure, methane consists of one carbon atom and four hydrogen atom which means it is smaller in size and weight. Therefore it is hard to find any study based on methane interaction with DNA or protein to regulate transcription. This made us search for carbon hydrogen bond interaction with any oligo or protein to trigger transcription of any metabolite product. So that we also searched the literature for any organism which utilizes any alkane chain and alkane which regulates the transcription of organismal metabolites by interaction with DNA or protein. Since bacteria are environmental adapting organisms, it is possible to find any alkane to regulate the transcription of degradation metabolism. Then we have found the organism, Pseudomonas oleovorans to analyze the alkane degradation of organism for its carbon source.

ii) Background

Many microorganisms live in the environments where the conditions changing frequently and so the evolution is inevitable for mechanisms to withstand unfavorable situations. Therefore microorganisms can use their specific and sensitive mechanisms for sensing the required nutrients for them or any pollution to affect their sustainability. Otherwise they can expose to mutations which change the gene expression and gain new functionalities. In case they have ability to survive in such conditions.

While we were scanning the literature based on methane and alkane degrading organisms we have found some organisms that sensitive to methane presence and have mechanisms to activate transcription of related gene clusters.

We analyzed the soil bacteria, Pseudomonas oleovorans. This strain can assimilate the alkane for its carbon source and one of the microbial whole cell –biosensor. They have a gene cluster which codes for degradative pathway and includes the activator which interacts especially with linear alkanes. This activator protein, AlkS, in the presence of alkanes, induces the transcription from PalkB promoter which initiates the expression of genes code for assimilation of alkanes. We have analyzed the related articles in which the promoter region is studied and showed that it is expressed in E.coli correctly because of the corresponding RNA polymerase binding regions.

REFERENCE:

Canosa, I. & Yuste, L. (2000). A positive feedback mechanism controls expression of AlkS, the transcriptional regulator of the Pseudomonas oleovorans alkane degradation pathway. Molecular Microbiology, 35 (4), 791-799.

iii) Modelling

Since the parts that we have found belongs to yeast organism, it was hard to us to manipulate it in bacteria E.coli. We designed the tests of this part by characterizing one of the subunits of methane monooxgenase (MMo). For the functional activity of monooxygenase enzyme, there are required 3 subunits as A,B and C. The A subunit of methane monooxygenase is 210 kDa protein that is supposed as the methane binding active site of monoxygenase complex. This protein consists of non-haem iron and contains p-hydroxo bridge structure. We planned the experiments of this step as characterization of protein A by PCR amplification with specific primers of full construct device to obtained the sequence of protein A. The methane gas is applied to cell culture with our modelled device then the cells were centrifuged and the gas concentration is measured from supernatant.

Clonning procedures

Getting the DNA parts

For parts in kit plates

With a pipette tip, punch a hole through the foil cover into the corresponding well of the Biobrick^TM-standard part that you want.
Pipette 10 uL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully re-suspended.
Transform 1 or 2 uL of the re-suspended DNA into desired competent cells, plate your transformation with the appropriate antibiotic and grow overnight.
Pick a single colony and inoculate LB medium (again, with the correct antibiotic) and grow for 16 hours.
Use the resulting culture to miniprep the DNA and make your own glycerol stock.

For new synthesized DNA parts

The DNA parts are sent in nmol values in each. These data are determined for all of them individually
As a stock solution preparation, these DNA parts are pipetted with PCR water or TE buffer which is 100 times of DNA amount. The final solution is prepared as 100uM.
For each transformation procedures, the 10uM of stock DNA part in water is transformed. 1uL is taken from stock DNA solution and is added to 9uL PCR water.

Preparation of Competent Cells
Transformation

Aliquot 10 ng of DNA in a volume 10 to 25 mL into a sterile 15 mL round bottom test tube and place on ice.

Plasmid DNA can be used directly from ligation reactions. When this is done , more DNA is usually used. However, if there is <1 mg of DNA in the ligation reaction, or if the ligation reaction is from low gelling/melting temperature agarose, it is wise to dilute the ligation mix.

Rapidly thaw competent cells by warming between hands and dispense 100 mL immediately into test tubes containing DNA. Gently swirl tubes to mix, then place on ice for 10 min.

Competent cells should be used immediately after thawing. Remaining cells should be discarded rather than refrozen

Heat shock cells by placing tubes into a 42 C water bath for 2 min

Alternatively incubate at 37 C for 5 min.

Add 1 ml LB medium to each tube. Place each tube on a roler drum at 250 rpm for 1 hr at 37 C.
Plate aliquots of transformatin culture on LB /ampicillin or other approprate antibiotic- containing plates.

It is convenient to plate several different dilutions of each transformation culture. The remainder of the mixture can be stored at 4 C for subsequent platings.

When plates are dry, incubate 12 to 16 hr at 37 C.

Plasmid isolation

This procedure is performed with Fermentas GeneJET™ Plasmid Miniprep Kit

Pick a single colony
Inoculate in 5 mL LB medium for high-copy or 10 mL for low-copy of LB medium supplemented with the appropriate selection antibiotic.
Incubate at 225 rpm not longer than 12-14 h at 37 C .
Centrifuge at 4000 rpm for 5 min at 4 C .
Discard the supernatant and keep pellet.
Resuspend the pellet with 250 ul Resuspension solution. (Bacteria should be resuspended completely by vortexing until no cell clumps remain)
Transfer the cell suspension to eppendorf.
Add 250 uL Lysis Solution and mix gently by inverting the tube 4-6 times until the solution becomes slightly clearPlate aliquots of transformatin culture on LB /ampicillin or other approprate antibiotic- containing plates.

Do not vortex!
Do not incubate for more than 5 min. (To avoid denaturation of supercoiled plasmid DNA!)

Add 350 uL Neutralization solution and mix immediately and thoroughly by inverting the tube 4-6 times.

(The neutralized bacterial lysate is cloudy and viscous) ("thoroughly" to avoid localized precipitation of bacterial cell debris)

Centrifuge at 13000 rpm for 5 min to precipitate cell debris and chromosomal DNA.
Transfer the supernatant to spin column (about 600 uL).

(Avoid disturbing or transferring the white precipitate)

Centrifuge at 13000 rpm for 1 min.
Discard the flow-through.
Place the column back into same collection tube.
Add 500 uL Wash solution to spin column.
Centrifuge at 13000 for 1 min
Discard the flow-through.
Repeat this step with using 500 uL Wash solution.
Discard the flow-through
Centrifuge at 13000 rpm for an additional 1 min to remove residual Wash solution.
(This step is essential to avoid residual ethanol in plasmid preps)
Transfer spin column into a fresh 1.5 mL epp.
Add 50 uL Elution buffer into center of the spin column membrane to elute the plasmid DNA

Do not contact the membrane with pipette tip!

Incubate for 2 min at 25 C.

To increase yield incubation is done for 2 min at heat block at 42 C

Centrifuge at 13000 for 2 min.
Discard the column and store the purified plasmid DNA at -20 C.

OPTIONAL= additional elution step with elution buffer or water. This step increases the yield by 10-20 %.
NOTE: For elution of plasmids ≥ 20 kb, prewarm Elution buffer to 70 C before applying.

Restriction digestion

This procedure is performed with NEB BioBrick™ Assembly Kit

Digest Upstream Part with EcoRI-HF and Spel
500 ng Upstream Part Plasmid
1 uL EcorI-HF
1 uL Spe I
5 uL 10XNE Buffer 2
0.5 uL 100X BSA
To 50 uL add dH2O
Digest downstream part will Xbal and PstI
500 ng Downstream Part Plasmid
1 ul XbaI
5 uL 10X NE Buffer 2
0.5 uL 100X BSA
To 50 uL add dH2O
Digest the destination plasmid with EcorI-HF and Pst I:

The destination plasmid DNA should either be prepared with PCR or contain the toxic gene (e.g, ccdB, sacB) in the cloning site to avoid the need for gene purification.
The Destination Plasmid should also have a different antibiotic resistance marker from both the plasmid containing the Upstream Part and the plasmid containing the Downstream Part to avoid the need to purify the Upstream and Downstream Parts.
500 ng Destination Plasmid DNA
1 uL EcorI- HF
1 uL PstI
15 uL 10X NE Buffer 2
0.5 uL 100X BSA
To 50 uL add dH2O

Incubate all 3 restriction digest reactions at 37 C for 10 min and then heat inactivate at 80 C for 20 min.

AGE (Confirmation)

Background

EtBr stains only dsDNA. You cannot see ssDNA on gel
Minimum amount of DNA load:

300 ng
• Each band of 1kb ladder is approximately 125 ng (2.5 ul loaded) and can be seen on gel

Preparation

50X TAE (Tris-Acetate) Buffer
121 g/0.5 L Tris
28.6 g/0.5 L Glacial Acetic Acid
7.31 g/0.5 L EDTA
pH 8.0
Sterile 1.5 mL eppendorf tubes
Sterile long pipette tips
DNA standards
- Promega 1.0 kb (1.0-10Kbp), 100 bp (0.1-1.0 Kbp)
TAE Buffer
EtBr sln (10 mg/mL). Purchase as solution ! Store at RT. Light-sensitive and carcinogenic !
Sample loading buffer. Store at 4 C.

Protocol

Prepare 360 ml, 1X TAE running buffer
Prepare gel compartment with tape (do not use parafilm)
Prepare 60 ml, 1% agarose gel (0.6 g agarose in 60 ml 1X TAE buffer)
- 1-2 min in microwave. Check and swirl after each 30 sec. It must be dissolved completely.
Add 1.0 ul EtBr (10 mg/ml) per 10 ml gel solution, swirl to mix
Add add 4 ul EtBr (10 mg/ml) per 100 ml running buffer.
Pour gel into gel compartment. Put comb.
After gel solidifies (30 min), take out the comb and load buffer to one well to measure its capacity.
Position the gel compartment and fill gel chamber with running buffer.
Prepare samples and standards (ladder)
Prepare samples by mixing 4 ul 6X Loading Buffer + 20 ul sample
Load the samples (for 24 ul sample, set pipette 24 ul).

Do not push the tip to the bottom of the wells. Make a 45 degree angle and support the pipette tip on the edge of the well. Don’t worry, gel will not brake when you take support. Slowly release the sample. Do not use second pipet push not to risk air injection.

Make the connections: Black (-) on sample side, Red (+) opposite side
Run at 60V
5-10V/cm (distance between anode and cathode) is recommended.
90V causes high heat generation for our unit. Use 60V maximum.
For overnight runs: 2V is too low, causes diffusion
35V / 4hrs / ¾ progress
Gel can be stored in 4 C until next day after completion

Gel Photo

Adjust zoom and position using visible light
Before turning on UV load your settings file which has the following parameters:
- Preview tab, all three options checked
- Active image
- Dynamic integration, auto exposure, 10 frames
- 50/50 brigthness/contrast
Maximize brigthness with camera knob (counterclockwise)
Turn on UV light
Lower brightness from camera knob if necessary

Gel extraction (For Fermentas Gel Extraction Kit)
Ligation

This procedure is performed with NEB BioBrick™ Assembly Kit

Ligate the Upstream and Downstream Parts into digested Destination Plasmid.
- 2 uL Upstream Part Digestion
- 2 uL Downstream Part Digestion
- 2 uL Desination Plasmid Digestion
- 2 uL 10X T4 DNA ligase bufferl
- 2 uL T4 DNA ligase buffer
- 11 uL dH2O
Incubate at RT for 10 min and then heat inactivate at 80 C for 20 min.
Transform 2 uL of the ligation product into 50 uL of competent E.Coli cells ( or other suitable host strain).
Select using the antibiotic corresponding to the Destination Plasmid.

Glycerol Stock Preparation
1. Centrifuge 5 mL overnight cells at 5000 rpm for 5 min.
2. Discard 4 mL supernatant, remain 1 mL with pellet.
3. Add 176 uL 15% glycerol and resuspend the pellet.
4. Aliquot 100 uL and immediately freeze at -70 C.
Cell imaging

Checklist Procedure

Suspension culture preparation

Dissolve a single colony in 10 mL LB+Amp
Incubate 14-15 hrs, shaking at 225 rpm

Scale-up of the suspension culture

Add 2-3 mL of the suspension culture to 100 mL LB+Amp
Incubate 5-6 hrs, shaking at 225 rpm

Equalizing OD readings

Take 10 mL samples from GFP/RFP & NC cultures
Centrifuge at 10000 rpm for 5 mins
Discard supernatant
Resuspend pellet in 500 uL 1X PBS
Take 250 uL & dilute to 3 mL with 1X PBS
Read in UV-Vis at 600 nm
Multiply the OD readings by 12
Dilute remaining 250 uL samples as required to make all samples have the same ODvalue, final volume should also be 250 uL

Fluorescent signal reading

Set parameters
[GFP]
Mode: Emission scan
Config: Digital
Slit: 1 turn / 2 nm
Blaze: 750 nm
Excitation: 395 nm
Emission range: 475-600 nm
Average: 3
Step size: 5 nm
[RFP]
Mode: Emission scan
Config: Digital
Slit: 1 turn / 2 nm
Blaze: 750 nm
Excitation: 570 nm
Emission range: 585-650 nm
Average: 3
Step size: 5 nm
Transfer samples to small 150 uL cuvette and scan

Data Analysis

Emission maxima:
GFP 515 nm
RFP 605 nm
Record intensity values at 509 nm for GFP & 606 nm for RFP
Subtract NC intensity value from GFP/RFP value
Result is the fluorescent signal of the protein in the bacteria

Protein expression

MAIN STEPS/TIME TABLE
- Pre-culturing/5 hours
- Culturing/5 hours
- IPTG induction/15 hours

Materials
- LB medium with a suitable antibiotic
- TB medium with a suitable antibiotic
- IPTG
- 2-liter flask
- Incubator with shaker
- Centrifuge

Check List Procedure
- Culture transformed E.coli BL21 for 5 hrs at 37 C in 10 LB medium containing antibiotic
- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask
- Culture it for 5 hrs at 37 C with a rotary shaker at 180 rpm!
- Add 0.5 mM IPTG in a final concentration
- Continue the cultivation for 15 hrs at 22 C.
- Harvest the cells with centrifuge.

Alternative Check List Procedure
- Inoculate single colony for 5 hrs at 37 C in 10 mL LB medium containing antibiotic
- Inoculate 4 ml pre-cultured cells into 400 ml of TB medium containing antibiotic in a 2-liter cultivation flask
- Culture it for 5 hrs at 37 C (OD600 to 0.5 - 0.6) with a rotary shaker at 180 rpm
- Add 1 mM IPTG in a final concentration ( 0.5- 2.0 mM recomended for pT7)
- Continue the cultivation for 3-4 h at 37 C (15 hrs at 22 C.)
- Harvest the cells with centrifuge.

Solutions
- TB (Terrific Broth)

Reference: http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/

SDS-PAGE
MATERIALS
- Vertical Electrophoresis apparatus
- Power supply
- pH meter
- Balance
- Silver staining shaker platform
- Transilluminator
- Filter paper

For Gel preparation
- Acrylamide
- Bisacrylamide
- Deionized Water
- Tris base
- SDS
- APS
- TEMED

For Silver Stanning
- Fixer
- %50 ethanol
- Pretreatment solution
- Silver nitrate
- Devoloping solution
- Stop solution

- Marker: Fermentas / Page Ruler Protein Ladder SM0661 (10-200kDa)

SOLUTIONS
Running Buffer (10L, 1X)

- 25mM Tris, pH 8.3
- 250mM Glycine
- 0.1% SDS
or
- 60 ml 10X stock + 6 ml 10% SDS + 534 ml dH2O
-----------------------
- 30.3g Tris
- 187.7g Glycine
- 10g SDS
- Final volume 10 L in demijon

Staining Solution (Coomassie Blue) ( 1 L, 1 X)
- 2 g brilliant blue (R-250)
- 450 ml methanol
- 450 ml dH2O
- 100 ml acetic acid
- store at 24 C

Destaining Solution ( 4 L, 1 X)
- Methanol:Acetic Acid:dH2O 40:10:50
- Prepare 4.0 L (1.6 L MeOH, 0.4 L AcA, 2.0 L dH2O) in 1 gallon amber bottle, cap tightly
- store at room temp.

4X Sample Loading Buffer
- 400 mM DTT
- 40 mM Tris
- 10% Glycerol
- 4% SDS
- 0.4% Bromophenol blue

- prepare 15 ml
- 925.2 mg DTT
- 72.6 mg Tris
- adjust pH to 6.8
- 1500 ul Glycerol
- 600 mg SDS
- 6 mg Bromophenol blue
- aliquot 50 x 600 ul
- store at -20 C

30% Acrylamide 1% Bis-acrylimide
- prepare in laminar flow
- 30 g acrylimide and 1 g bis-acrylimide in 100 ml
- 75 mL acrylimide solution ( 40% stock, Apllichem) and 25 mL bis-acrylimide solution ( 2% stock, Apllichem)to final volume 100 ml
- store at 4 C, stable for 1 month

Seperating Gel Buffer
- 1.5 M Tris pH 8.8
------------------------------------------
- for 100 mL Buffer solution
- 18,15 g Tris pH 8.8
- store at room temp.

Stacking Gel Buffer
- 0.5 M Tris
------------------------------------------
- for 50 mL Buffer solution
- 3 g Tris pH 6.8
- store at room temp.

APS (Ammonium Persulfate) Stocks (100 mg/ml)
- dissolve 0.6 g in 6 ml dH2O
- aliquot 10 x 600 ul and store at -20 C

1% Bromophenol Blue
- 0.01 g in 1 ml 1M Tris, pH 7.0
- store at room temp. in amber bottle

CASTING 13% GEL
- set heater to 100 C for sample prep step

Seperating Gel (30 ml)
- 13 ml 30% Acrylimide 1% Bis-acrylimide
- 7.5 ml seperating gel buffer
- 8,45 ml dH2O
- 500 ul %10 SDS
- 250 ul APS (initiator of polymerization)
- 25 ul TEMED (catalyst of polymerization)

- Load 5.4 ml separating gel between glasses

Stacking Gel (10 ml)
- 1.6 ml 30% Acrylimide 1% Bis-acrylimide
- 2.5 ml stacking gel buffer
- 5.85 ml dH2O
- 100 ul %10 SDS
- 15 ul 1% Bromophenol Blue
- 50 ul APS (initiator of polymerization) [add after resolving gel is casted]
- 10 ul TEMED (catalyst of polymerization) [add after resolving gel is casted]

- Load 1.7 ml stacking gel between glasses
- Inset the comb - make sure the comb has not been inserted in a tilted way. check from behind the apparatus

- load seperating gel
- add some butanol or isopropanol before resolving gel solidifies
- make sure gel stays on flat surface while solidifies to prevent tilted surface
- load stacking gel

- if bubbles form in the stacking gel after polymerization, press the plates between hands to push them out

SAMPLE PREPARATION AND LOADING

- Do not overload the the samples, purity check is difficult with overloaded samples.
- Sample volume: 5 ul sample+ 5 ul loading buffer + 10 ul dH2O
- vortex loading buffer before use
- put samples in heating block (100 C) for 5 min
- if possible, do not load into the first and last lanes
- load 5 ul marker
- load 17 ul samples

PREPERATION
- check the wire on running apparatus, clean and test

RUNNING
- never terminate the run early, lighter bands dont separate
- 600 ml running buffer is required for each run

Running Standards
- 5 mA > 3/4 of gel > 25hrs >>> overnight running
- max: 80 mA for both old and new gel systems
- For Lab 103 Tankı
   - for 145 min at 30 mA 120 V through seperating gel
   - amper constant

After Run
- wash electrophoresis unit after each use
- weekly cleaning of power connections recommended to prevent oxidation<

STAINING
- Stain the gel for 30 min

DESTAINING
- load the tray fully with destaining buffer
- do not put too many (over-destaining) or too less (under-staining) paper sheets
- destaining takes 2-3 hrs

Device Experiments

MAIN STEPS/TIME TABLE
- Pre-culturing/7 hours
- Culturing to 250ml flasks/3 hours

Materials
- LB medium with a suitable antibiotic (Ampicillin)
- 250 ml flasks
- Incubator with shaker
- Centrifuge

Check List Procedure
- Cell culture (with ROSE regulated kill switch) transformed E.coli BL21 for 7 hrs at 37 C in 10 ml LB medium containing antibiotic
- Inoculate 100ul pre-cultured cells into 100 ml of LB medium containing antibiotic in a 250 ml cultivation flask
- Culture it for 3 hrs at 37 C with a rotary shaker at 220 rpm up to OD reading reaches to 0.3
- Continue taking OD readings up to 0.7 at 37C.
- Harvest the 5 ml of cells with centrifuge for 5 min at 5000rpm.
- Resuspend in 5ml PBS and take fluorescence readings.

Online Lecture

Anybody interested and novice in Synthetic Biology can use the lecture;

Here is our animation; Industrial Revolution 2 After Synthetic Biology, to introduce the basics of the field;

Here is our Online Lecture, Click the picture to go;

Collaboration

Collaboration with Turkish teams in iGEM 2011

Since iGEM 2007, there is a team to research and prepare a project for iGEM competition from Middle East Technical University(METU). On behalf, there are advisors and instructors who are familiar with competition and content of it. In this year there are 4 registered teams from Turkey and 3 of them are new participants. Therefore in order to come together and criticize our projects and any deficiencies or ambugity related with project. So we organized and arranged a meeting on Synthetic Biology and iGEM competition which is the first in our country.The participants of this meeting were teams;Fatih Turkey, Bilkent_UNAM, METU-BIN and researchers who related with Synthetic Biology and company owners in Biotechnology field also there was special guests via online talk, Drew Endy, he is an assistant Professor of Bioengineering, Stanford University and Scott Mohr Professor of Biological Chemistry, Boston University This meeting was held in September 10, that was close to Regional Jamboree in order to also check the presentation preparations of teams. The news agencies were also invited and the interviews were done with all teams publicity of teams were enhanced.

Except from this, we established a collaboration with METU-BIN Software iGEM 2011 team on their software program. This year their project, M4B: Mining for BioBricks is on enhancement and simplification of parts registry and gene library usage to ease the wet lab researchers job. They requested us to use and try their software program. One of our parts in this year, kill switch is composite of this year distributions. By the way we tried their program to search and try on this composite. we gave them the following as feedback and also according to our comments on visual of program, we had collaborated this year.

“We are constructing a device that is induced by IPTG and that gives lysis enzymes and GFP as the outputs because, in order to elute methanol we need to kill our modified cells that convert ambient methane to methanol and the reason why we want GFP as one output is to be sure that it works. Our modelled device has promoter induced by IPTG, RBS, gene coding GFP and lysis cassette ending with cell death and we are glad to have it and it's working how we expected. So then, we use M4B to see that if it finds the exact device or the device that can do the same job. Since the software has only one place for output, we queried IPTG as an input and GFP as an output on the software and got corresponding 117 results. These results contain devices exactly what we have one for the same operation. However, we couldn't find the lysozyme output for the IPTG input in the software.”

We had requested them to support us on wiki page design and same tricks on loading the links on wiki page. We had prepared the self evaluating lab safety questionnaire and they made them manipulable on wiki page and the methane release meter on home page is their work.

Helping to other new Turkish teams in the future

Our university, METU has an online lecture application; METU OpenCourseware to support the open information source not only members of METU and to reach people to inform in all ages. In this online application, For Synthetic Biology field, last year the sessions were loaded and this year it is upgraded. The protocols for Synthetic biology methods were downloaded and supported with tutorials of procedures that taken during experiments. We established this page and enhanced for Synthetic Biology to reach more students to meet with Synthetic Biology in any region of Turkey. We had requested from President of METU made people to reach this webpage from universities with related departments in an official way. By the way, we believe to reach more students or researchers to iGEM competition and Synthetic Biology.

Collaboration with 2011 Uppsala Sweeden iGEM Team

We asked them to use and give feedback to our software tool on our wiki. This tool is named as BioGuide which was first studied on last year’s competition iGEM 2010 and gained silver medal. This year this team joined with wet lab team. While they were progressing the BioGuide project, this software tool also supported the web page and experiments of METU_Ankara team. We publish this software program on our wiki page to made other iGEM teams to reach and benefit from program on part and path choices. Uppsala-Sweeden team was one of the teams that use and try the BioGuide program. They tried to reach their used parts in kit distributions and then gave us feedback on them.

They tried this software tool according to parts of their project As an input parameters they tried to type red light 'cph8', blue light 'LovTAP', green light 'Ccas', IPTG(PT5-Lac), YF1 and as output parameters they typed amil GFP, amil CP and mCHERRY. However they found the library of program restricted and they could not reach all parts because BioGuide software tool library is designed and programmed on parts that are in 2010 and 2011kit plate distributions. By the way, they found this software program managable. Given part names or input and output data results with Biobrick codes of parts in parts registry library. Therefore, it made easier their job on longstanding assembly plans. For mutual collaboration, we read and evaluated their Biosafety web page and gave feedback below

“You replied the first question having main title “materials used in your project” as bacteria strain used in your project. We suggest that you should focus on chemical or any other material to answer this question.However we also used the E.coli Top 10 as host strain and found that these strains are classified as hazard group 2 pathogen by the UK Advisory Committee on the Dangerous Pathogens (ACDP).
In the question 3 that you explained “Institutional Biosafety Committee”, you described the threats of Synthetic biology in 3 main items based on your interview, we think you should expand content of items.

And lastly, it would be better if you give more detail on your last question to be more clear for future iGEM teams.”

https://2011.igem.org/Team:Uppsala-Sweden/Feedback

Software Tool

BioGuide is a Biological System Design tool. It has been developed in 2010 by the team METU TURKEY SOFTWARE. You can check the page for detailed information about the project :
https://2010.igem.org/Team:METU_Turkey_Software

Here is rough draft and setup of BioGuide Paper you can read for technical details and download the software;

BioGuide.pdf

Download the Software

This year;

We have completed biological system design part of BioGuide and enabled wet lab teams to use it.

You can watch the "how to" video of BioGuide to see what and how you can do with it;

Here is how METU ANKARA wet lab team is using BioGuide;

This year we are using it while building one of our devices, kill switch composite. Kill switch composite consists of 4 main parts which are in 2011 kit plate distributions; T7 promoter, RNA thermometer, GFP and lysis casette. The upstream part of this composite is T7 promoter (BBa_I712074) which is strong promoter from T7 bacteriophage mostly used expression system and so for strong transcription T7 promoter expression system is chosen.

We also tried the inducible promoters (pLac-IPTG inducer) to design and express the lysis casette. We desired to use a promoter that could be induced and enhance the expression to control and manipulate the translation of device. When we typed IPTG on input box of Bioguide we had reached the possible promoters that could be induced by IPTG. This program gave their standard biobrick codes and finally we got these parts by knowing their location in kit plate.

In downstream of promoter there is RNA thermometer(ROSE) coding region which determines the transcription of our lysis composite part. We had reached this part by coding the temperature 42 C in input box of BioGuide. RNA thermometer (BBa_K115001) is temperature sensitive at which 42 C translation initiates.

In downstream of RNA thermometer, green fluorescence protein(BBa_E0040) is ligated. This ligation is done to measure expression level and to comment on RNA thermometer efficiency. We typed GFP in output box since we wanted to measure fluorescence.

We had designed this composite and it's parts mostly by processing the BioGuide program that made our job easier in intense lab works. We reached the shortest path with our input and output parameters for this device to construct by BioGuide.