"
Team:METUHS-Ankara/project.html
From 2014hs.igem.org
CO-sense
METU HS iGEM 2014
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.
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.
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
