Team:METUHS-Ankara/project.html

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Revision as of 03:26, 21 June 2014

Project

METU HS iGEM 2014

CO-sense

Project Description

Design

Results

Human Practices

Lab Notebook

Carbon monoxide (CO) is a colorless and odorless gas which can be very poisonous and dangerous. Since it is produced from commonly used household devices and industry, in the case of any leakage it can cause severe poisonings and deaths. Many people die in Turkey because of mishandled ascot and blast heaters. According to statistics 10,154 people, meaning 14 out of every 100,000 people, were intoxicated.

The aim of our project is to prevent these cases. In order to achieve this, we plan to develop a biological device which will include a detection and a conversion system. Firstly, our detection mechanism is based on light dependent sensors. These sensors gather data from the bacteria, in turn triggering an alarm system. Secondly, in the event of CO presence, our conversion system will be activated in order to convert carbon monoxide into carbon dioxide (CO2). To accomplish this transformation, we are using an enzyme called Carbon Monoxide Dehydrogenase (CODH). Finally, as a safety measure we will include a kill switch mechanism that aims to inactivate the system.

CO to CO2 Converter & CO Monitoring System

Carbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We’ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill-switch design based on the lac-operon. The kill-switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device.

Kill switch

we use an mRNA interferase, MazF, which cleaves mRNA’s at specific sequences. In our kill switch, we used anti-sense RNA principle as a template. According to this principle, MazF, which is constantly produced via a constitutive promoter, is got inactivated by Anti-MazF construct. In order to trigger this mechanism, we used IPTG, a harmless molecule for the bee which at the same time does not appear in the honey too. When IPTG is present in the environment, LacI is inhibited by IPTG and therefore promoter gets activated. With the activated promoter of it, Anti-MazF is produced and inactive MazF. As long as IPTG exists, MazF gets inactivated continuously; therefore, the bacteria maintain their lives. On the other hand, if bacteria exists in a IPTG-free environment, Anti-MazF producing stops, which leads to MazF producing and bacteria get killed by MazF. (Circuit image courtesy of METU iGEM Team 2013)

In this page, you can find out the computational modelling for our design and finished system.

Carbon monoxide

Wıth our research we were able to find out that 1500 ppm of CO gas means death in an hour. To make calculations we need to convert this value into something we can work with easily, such as moles.

1500 ppm = 0.15%

If we take a 30 meter squared room with 3 meter ceiling height, the overall volume of the room is 90m^3 which equates to 90000 liters.

By using Ideal Gas Law, we can say that the amount of moles of CO present in 135 Liters is around about 4 moles. So what we have learned? In a big size room, there should be at least 4 moles of CO molecules for it to be considered as deadly.

CODH Activity

Even though we were not able to find detailed parameters for this particular enzyme, we were able to work out a very simple formula to calculate the amount of CO we will be able to convert each second.

r = CODH activity rate

p = plasmid copy number

b = amount of bacteria

s = synthesis rate

[CO]=amount of CO (moles)

A = fraction of CO converted

*Lets call r.p.b.s: "c" since we can assume that it will stay constant.

*Our calculations show that "c = 31777209688000" or "3.2 x 10^14"

When we divide c by the Avagadro number we can find out what fraction of moles of CO we will be able to convert each time frame. Even though "c" seems like a huge number, the Avagadro number is much bigger.

3.2 x 10^14 / 6.02 x 10^23 = 0.5 x 10^-9

This mans that we are able to convert roughly 0.000000001 of the CO available each second.

Here is a little script we wrote with Python programming language with modules "matplotlib" and "numPy":

from pylab import *

timer = 0

f = 0.000000001

s = 7200 #seconds

co = 4 # moles

co2 =0 # moles

values = [ ]

for i in range(s):

co2 = co2 + co*f

co = co - co2

if co <= 0:

break

values.append(co2)

timer += 1

x = arange(0,timer,1)

plot(x,values)

xlabel('time (s)')

ylabel('CO2 (moles)')

title('CO2 - Time')

grid(True)

show()

Please note that, even though we are subtracting the amount of CO converted from the initial CO value, the graph still does not have a visible curve to it. It is obvious that even though we are able to convert a huge number of CO molecules each given second, there are way to much CO then our system can convert.

By increasing the surface are of the single-layer e-coli colonies, for example take a 10 x 10 grid of the same exact system, we can achieve 100 times more efficient converters which can shave of two 0's from the results.

Down below you can see the photos of the colonies we were able to get and the electronic circuit design.

Trying out the color sensor. Red pen on the sensor light up red. Blue ruler on top, blue light.

Final images of the design.

Today, improving technology and ideas lead students to be the constructors of better future. That’s why as METU DF High School iGEM 2014 team we made researches and experiments to widen the perspective of what synthetic biology can achieve. We know that synthetic biology is the way of constructing core components which can be modeled by merging them with the fundamentals of biology. We assume that by the efficient use of synthetic biology we can lighten the way through future and save many lives.

Therefore, this year we worked hard to put forth an experiment that can lead the way to our better future. As METU HS iGEM team, we believe that synthetic biology is a tool that can turn the world to a better place. And iGEM is an opportunity for us to show people that synthetic biology is helpful for the humankind. That's why we did many events to introduce people synthetic biology. We have given a bake sale which included bacteria & DNA muffins and x-ray crystallography cake.

The muffins got retweeted by the iGEM HQ!

Furthermore we interviewed some students in our school to see how much they know about synthetic biology world. We decorated our school with DNA's and posters of our team on DNA day. Also as METU HS iGEM team we have attended to a Synthetic Biology Day to expand our knowledge. It is important for the next generations to be familiar with synthetic biology, in order to achieve this we gave a presentation to 7 and 8th graders. We have informed them about iGEM and biology. And our aim is to present iGEM to everyone to convince people that synthetic biology is useful. So we have told people about our team and iGEM at the opening of an ODTU DF school in Denizli. In order to maintain our goal we believed that a sale of work will be a nice introduction of iGEM and synthetic biology. We cooked cupcakes in order to sale and we designed them as bacteria and everything about synthetic biology. Secondly, we also designed an enormous DNA shaped gumdrop. The most critical part was we recommended iGEM and talk with them about the project we were doing. Our main purpose was to make people know what synthetic biology is and create awareness about these topics. To conclude we have tried and we are still trying to spread synthetic biology in order to acknowledge human. As METU DF High School iGEM 2014 team we work hard to settle the importance of synthetic biology by referring various projects.

We believe that awareness and education is the key of action, acceptance and understanding. Our project contains lots of valuable experiments that can prove the useful remedies of the efficient use of synthetic biology.

You can find every photo and video we referred to at the fun page by following this link or find it at the extras page!

Competent Cell

• Take 1 µL cell (3 µL cell for us )

100 ml LB 37ºC 2h (OD 0.600 200 rpm)

• Divide 100 ml into 2 falcons & centrifuge for 10 minutes 5000 rpm +4ºC

• Discard supernatant

• Add 10 µL 0.1 M (molarity) CaCl2

• Dissolve pellet by gently shaking

• Incubate on ice 10 min.

• Centrifuge 5000 rpm for 5 min. +4ºC

• Discard supernatant

• Put 2 ml CaCl2 and dissolve pellet

• Put in ice for 5 min. & keep it -80ºC 80% glycerol

Transformation

• Thaw competent cells on ice

• Mix ligation mix (100 µL cells 5 µL plasmids)

• Incubate cells on ice 30 min.

• Heat shock at 42ºC 90 sec.

• Incubate cells on ice 5 min.

• Add 900 µL LB

• Incubate at 37ºC for 80 min.

• Centrifuge at 3000 rpm for 10 min.

• Discard supernatant

• Resuspend the pellet in 100 µL LB

• Spread cells on LB Agar with antibiotic

K34F - DT Chl

K34B - RFP Chl

K25J - GFP Chl

Transformation again!

Selin Hoca Transformation

Pet28a

Pet28a+gene

RFP+ DT

We cannot insert the plasmid!! Something’s wrong with the competent cells!!

Comp 1 -

Comp 2 - RFP + DT V Pet28a

Comp 3 - Amp, Kan Kan

The second transformation.

Competent cell, why didn’t they work?

1) Lack of CaCl2

2) CaCl2 wasn’t cold enough

3) Non homogeneous cells (The closest)

4) OD was not enough

Transformation, why didn’t it work?

1) Heat shock

2) Cell died in centrifuge

3) Cell died whilst spreading

• Heat

• Mechanic force

Plasmid Isolation

.Gel Electrophoresis 10 µL .Stock 15 µL

Gel Extraction

A1 K1 RFP

Needed the plasmids!

Plasmid Isolation - 2 µL cell pellet, lyse down the cells take plamids!

16.05.2014

Transformation

pCooM with changes of 50 µL cell

pCooF + 10 µL plasmid

Digestion

NEB Buffer 5 µL

BSA 0.5 µL

Enzyme 1 0.5 µL

Enzyme 2 0.5 µL µL

Plasmid 1000 ng/µL

To complete to 50 µL, add ddH2O/dH2O

Keep it in water bath for 2.5 hours at 37ºC

LIGATION

10 x T4 ligase buffer 2 µL

6:1 M ratio to invert to vector

Add dH2O

T4 ligase 1 µL

Incubate at room temtperature 1 hours and 15 min. at 65ºC for enzyme activation

Alternative: +4ºC over night

Calculatory Insert Amount

Insert mass in ng = 6 x insert length in 6 µL / Vector length 6 g...

...x vector mass in mg

-Gel

-Gel Extraction

Vector - 2500 bp

Insert - 500 bp

1300 - to prepare the agarose gel 1.95 g of agarose (nearly 2%)

-Gel Results

-PCR

-Gel

Why didn’t it result correct?

There was no DNA

Problem with gel

Part we don’t want in the gel

Lack of the amount

TAE?

Agarose

6x loading dye

Plasmid - Digestion, Ligation

EtBr

DNA degredation

Conditions

Initial denaturation - 94ºC -30 sec

Cycles 94ºC 10-30 sec

(30-35) 45-65ºC (Tm= 58 ºC) 15-60 sec 2000+1800 bp

(32-33) 65ºC 50 sec/kb (⁓2 min)

Final extension 65ºC 10 min

Hold +4ºC

8F 9R / 10F 11R - primers used!

Nutrient agar plate inoculation

RFP GFP

IPTG +

IPTG -

Nutrient agar plate inoculation

IPTG + and –

Inoculation on broth media

IPTG x2

MP x4

• IPTG induction in Broth Media (1)• Miniprep pCooM, pCooF (1) Holin- Antiholin• CODH from BCG PCR (2)• Kill Switch Transformation (3) MazF- AntimazF• Digestion & Ligation (?) BCG -primer(Rhodospirillum rubrum primer)

Transformation of kill switch (with changes of LB - 150 µL at last step)

Gel Extraction

Genomic DNA Isolation

Chl & Tet Inoculation

Empty: MazF, AntimazF (streak plates) (4 inoculations)

Genomic DNA Isolation

• Suspend colonies in 120 µL TEN Buffer

40 µM Tris, 1 µM EDTA, 150 µM NaCl

• Incubate at 100ºC for 10 min

• Centrifuge at 13.000 rpm

• Take supernatant store at +4ºC

Gel Extraction

• Slice the gel

• Add 1:1 volume binding buffer

• Incubate 50-60ºC for 10 min.

• Transfer up to 800 µL of the solubilized gel solution to the purification column

• Cent. 1 min, discard flow-through

• 100 µL binding buffer cent. 1 min, discard flow-through

• 700 µL wash buffer, cent. 2 min discard flow-through

• Column to eppendorf 50 µL Elution buffer dH2O, cent. 1 min.

• Discard column store it at -20ºC

1) PCR

2) Gel Electrophoresis - Genomic DNA + Gel Extraction

-PCR

L Uncut 2 2.2 2.1 Uncut 4 4 BA BK L 2T 4T 4L

= Gel Electrophoresis

Gel Extraction

Nutrient Agar plate inoculation

LB at the last step of transformation is 150 µL

Ligation:

Same with 13.06.2014 but overnight +4ºC incubation  prepared at 19.45

17.06.2014 - at 11:45, will be transformed.

17.06.14 - Transformation

100 - 10

50 - 5

Transformation - heat shock 75’’

100 LB at the last step

Selin inoculation - amount of LB was more than needed.

0.5 eppendorf - it was opened before centrifuge (contamination)

4F 9R - primers 1- 2 GE 3- 2.2

PCR - 2. Sample 2- 2.1

LIGATION

T4 ligase 1 µL

T4 ligase buffer 1 µL

Vector 4 6 2 5 3

Insert 4 2 6 3 5

1:1 3:1 1:3 5:3 3:5

16ºC 30’

RT 30’

+4ºC Overnight

Transformation:

Öykü: 1st: (3) 2 GE 2h A (3:1) - 2nd: 2 GE A 301

Can (Öykü): 1st: (4) 2 GE A 2h (1:3) - 2nd: 2 GE A 301

Almira: 1st: (2) 2GE A 2h 1:1 (3:5) - 2nd: 2 GE A 301 – (5) 2GE A 301

Martin: 1st: (1) 2GE A 2h 5:3 (1:1) - 2nd: 2 GE A 301

Transformation - +4 ONC

PCR - 2 - 2’

PCR Purification

Digestion

Gel

Gel Extraction

Ligation 16ºC 1h

RT 2h

+4 ONC

Gel

Gel Extraction

Transformation

23.04.2014

28.04.2014

29.04.2014

01.05.2014

16.05.2014

23.05.2014

25.05.2014

30.05.2014

09.06.2014

10.06.2014

11.06.2014

12.06.2014

13.06.2014

16.06.2014

17.06.2014

18.06.2014

19.06.2014

20.06.2014

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          <p id="u2687-2">CO to CO2 Converter &amp; CO Monitoring System</p>
          <p id="u2687-3">&nbsp;</p>
          <p id="u2687-5">Carbon monoxide is a highly toxic gas which is undetectable by humans and it is fatal when inhaled. We’ve developed a biological device that comprises of both a qualitative detector for this dangerous gas and a conversion system to transform it into carbon dioxide. As for detection, CO sensitive promoters pCooM and pCooF from Rhodosprillum rubrum will initiate the production of fluorescent proteins in the presence of CO. Optic sensors will be used to track the production of these proteins and if the sensors pick up data indicating that CO is present; an alarm will be triggered. Meanwhile, the conversion system of our device will utilize a Cyanobacteria enzyme called Carbon Monoxide Dehydrogenase (CODH), which converts CO into CO2. We also have a kill&#45;switch design based on the lac&#45;operon. The kill&#45;switch mechanism will be activated to avoid any contamination of the environment, in case the altered bacteria escape the device.</p>
+        <p id="u2687-6">&nbsp;</p>
+        <p id="u2687-7">&nbsp;</p>
+        <p id="u2687-8">&nbsp;</p>
+        <p id="u2687-9">&nbsp;</p>
+        <p id="u2687-10">&nbsp;</p>
+        <p id="u2687-11">&nbsp;</p>
+        <p id="u2687-12">&nbsp;</p>
+        <p id="u2687-13">&nbsp;</p>
+        <p id="u2687-14">&nbsp;</p>
+        <p id="u2687-15">&nbsp;</p>
+        <p id="u2687-16">&nbsp;</p>
+        <p id="u2687-17">&nbsp;</p>
+        <p id="u2687-18">&nbsp;</p>
+        <p id="u2687-19">&nbsp;</p>
+        <p id="u2687-20">&nbsp;</p>
+        <p id="u2687-21">&nbsp;</p>
+        <p id="u2687-22">&nbsp;</p>
+        <p id="u2687-24">Kill switch</p>
+        <p id="u2687-25">&nbsp;</p>
+        <p id="u2687-28">we use an mRNA interferase, MazF, which cleaves mRNA’s at specific sequences. In our kill switch, we used anti&#45;sense RNA principle as a template. According to this principle, MazF, which is constantly produced via a constitutive promoter, is got inactivated by Anti&#45;MazF construct. In order to trigger this mechanism, we used IPTG, a harmless molecule for the bee which at the same time does not appear in the honey too. When IPTG is present in the environment, LacI is inhibited by IPTG and therefore promoter gets activated. With the activated promoter of it, Anti&#45;MazF is produced and inactive MazF. As long as IPTG exists, MazF gets inactivated continuously; therefore, the bacteria maintain their lives. On the other hand, if bacteria exists in a IPTG&#45;free environment, Anti&#45;MazF producing stops, which leads to MazF producing and bacteria get killed by MazF. <span id="u2687-27">(Circuit image courtesy of METU iGEM Team 2013)</span></p>
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           <p id="u3195-134"><span id="u3195-133">By increasing the surface are of the single&#45;layer e&#45;coli colonies, for example take a 10 x 10 grid of the same exact system, we can achieve 100 times more efficient converters which can shave of two 0's from the results.</span></p>
           <p id="u3195-135">&nbsp;</p>
-          <p id="u3195-137"><span id="u3195-136">Down below you can see the photos of the colonies we were able to get and the elctronic circuit design.</span></p>
+          <p id="u3195-137"><span id="u3195-136">Down below you can see the photos of the colonies we were able to get and the electronic circuit design.</span></p>
           <p id="u3195-138">&nbsp;</p>
           <p id="u3195-139">&nbsp;</p>
@@ Line 240: / Line 263: @@
          <p>Trying out the color sensor. Red pen on the sensor light up red. Blue ruler on top, blue light.</p>
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+          <p>Final images of the design.</p>
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          <p id="u3206-2">Today, improving technology and ideas lead students to be the constructors of better future. That’s why as METU DF High School iGEM 2014 team we made researches and experiments to widen the perspective of what synthetic biology can achieve. We know that synthetic biology is the way of constructing core components which can be modeled by merging them with the fundamentals of biology. We assume that by the efficient use of synthetic biology we can lighten the way through future and save many lives.</p>
          <p id="u3206-3">&nbsp;</p>
@@ Line 266: / Line 299: @@
          <p id="u3206-21">&nbsp;</p>
          <p id="u3206-22">&nbsp;</p>
-         <p id="u3206-24">The muffins got retweeted by the iGEM HQ!</p>
+         <p id="u3206-23">&nbsp;</p>
-         <p id="u3206-25">&nbsp;</p>
+         <p id="u3206-25">The muffins got retweeted by the iGEM HQ!</p>
          <p id="u3206-26">&nbsp;</p>
          <p id="u3206-27">&nbsp;</p>
-         <p id="u3206-29">Furthermore we interviewed some students in our school to see how much they know about synthetic biology world. We decorated our school with DNA's and posters of our team on DNA day. Also as METU HS iGEM team we have attended to a Synthetic Biology Day to expand our knowledge. It is important for the next generations to be familiar with synthetic biology, in order to achieve this we gave a presentation to 7 and 8th graders. We have informed them about iGEM and biology. And our aim is to present iGEM to everyone to convince people that synthetic biology is useful. So we have told people about our team and iGEM at the opening of an ODTU DF school in Denizli. In order to maintain our goal we believed that a sale of work will be a nice introduction of iGEM and synthetic biology. We cooked cupcakes in order to sale and we designed them as bacteria and everything about synthetic biology. Secondly, we also designed an enormous DNA shaped gumdrop. The most critical part was we recommended iGEM and talk with them about the project we were doing. Our main purpose was to make people know what synthetic biology is and create awareness about these topics. To conclude we have tried and we are still trying to spread synthetic biology in order to acknowledge human. As METU DF High School iGEM 2014 team we work hard to settle the importance of synthetic biology by referring various projects.</p>
+         <p id="u3206-28">&nbsp;</p>
-         <p id="u3206-30">&nbsp;</p>
+        <p id="u3206-30">Furthermore we interviewed some students in our school to see how much they know about synthetic biology world. We decorated our school with DNA's and posters of our team on DNA day. Also as METU HS iGEM team we have attended to a Synthetic Biology Day to expand our knowledge. It is important for the next generations to be familiar with synthetic biology, in order to achieve this we gave a presentation to 7 and 8th graders. We have informed them about iGEM and biology. And our aim is to present iGEM to everyone to convince people that synthetic biology is useful. So we have told people about our team and iGEM at the opening of an ODTU DF school in Denizli. In order to maintain our goal we believed that a sale of work will be a nice introduction of iGEM and synthetic biology. We cooked cupcakes in order to sale and we designed them as bacteria and everything about synthetic biology. Secondly, we also designed an enormous DNA shaped gumdrop. The most critical part was we recommended iGEM and talk with them about the project we were doing. Our main purpose was to make people know what synthetic biology is and create awareness about these topics. To conclude we have tried and we are still trying to spread synthetic biology in order to acknowledge human. As METU DF High School iGEM 2014 team we work hard to settle the importance of synthetic biology by referring various projects.</p>
-         <p id="u3206-32">We believe that awareness and education is the key of action, acceptance and understanding. Our project contains lots of valuable experiments that can prove the useful remedies of the efficient use of synthetic biology.</p>
+         <p id="u3206-31">&nbsp;</p>
-         <p id="u3206-33">&nbsp;</p>
+         <p id="u3206-33">We believe that awareness and education is the key of action, acceptance and understanding. Our project contains lots of valuable experiments that can prove the useful remedies of the efficient use of synthetic biology.</p>
-         <p id="u3206-38">You can find every photo and video we referred to at the fun page by following this<a class="nonblock" href="https://2014hs.igem.org/Team:METUHS-Ankara/fun.html">&nbsp;link</a>&nbsp;or find it at the extras page!</p>
+         <p id="u3206-34">&nbsp;</p>
-         <p id="u3206-39">&nbsp;</p>
+         <p id="u3206-39">You can find every photo and video we referred to at the fun page by following this<a class="nonblock" href="https://2014hs.igem.org/Team:METUHS-Ankara/extras/fun.html">&nbsp;link</a>&nbsp;or find it at the extras page!</p>
+         <p id="u3206-40">&nbsp;</p>
         </div>
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