Team:METU Turkey/Alpha

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Cell Sensor Experiments

Fermentor studies

Objectives: To observe GFP signal;
- While the bacteria with the inserted construct are in their regular growth
- After bacteria are treated with CO sparging

Parameters:
- Culture volume: 750 mL LB + 136 ug/mL chloramphenicol + 50 ug/mL ampicillin
- CO brought in through a sample port.
- CO sparging starts after 3 hours of IPTG induction.
- CO sparging duration: For 5 min, 2.0L/min; for successive 30 min, 0.1L/min
- CO sparging cycle number: 2
- Air brought in through sparging ( Air flow 5.0L/min)
- IPTG induction point: OD=0.5 at 600nm
- IPTG concentration: 1.0 mM
- IPTG post induction time: NA

Sensor and controls:
- Sensor: pCooF - GFP - pCooM - RFP / pSB1C3 (chloramphenicol) + CooA/pTriEx (ampicillin) in BL21 DE3 (Our final construct in chloramphenicol vector and our transcription factor CooA in pTriEx ampicillin vector)
- Control: pSB1C3 + CooA/ pTriEx in BL21 DE3

Notes:

Depending on the volume and pressure of our gas tank we calculated the gas volume using the following equations:
Calculation of gas volume in cylinder
- 100 bar
- 10 L
- PV = nRT (under normal conditions)

Check list procedure:
- 10mL o/n subculture until OD=2.5 (at 600nm)
- OD is measured.
- Scale up in 2/100
- 1 mL sample is taken at each 30 min.
- OD is measured at 600nm.
- pH and remaining culture volume is measured.

To control the effects of CO in the cells, pre and post measurements were performed.
- Pre-sparge measurements:
- 1 mL sample is taken
- OD is measured at 600 nm.
- pH is recorded.
- Myoglobin assay (*)
- Viability assay (**)
- CO sparging started 3 hours later than IPTG induction with respect to the assumption of the expressed CooA amount in the cell.

Post-sparge measurements:
- 1 mL sample is taken
- Myoglobin assay

(*) Myoglobin assay

Myoglobin assays are used to measure the dissolved CO in the LB that the culture is grown in. Myoglobin binds to CO and forms carboxymyoglobin. With dithionate treatment, this carboxymyoglobin is reduced, forming a structure that gives absorbance at 600nm. This assay enables us handle CO in liquid media.

(**) Viability assay

Viability assays are used to measure the amount of alive cells in liquid media. We applied this assay before and after CO sparging as we know that CO has toxic effects on E. Coli.
- 1 mL sample is taken
- Measure OD at 600 nm
- Dilute the sample with LB to OD=0.1 at 600 nm
- Serial dilutions with LB
- 10-2 : 100 uL culture (OD= 0.1 at 600 nm) + 9900 uL LB
- 10-4 : 100 uL culture into 10-2 + 9900 uL LB
- 10-5 : 1000 uL culture into 10-2 + 9000 uL LB
- 10-6 : 1000 uL culture into 10-5 + 9000 uL LB
- Spread 100 uL from 10-4 / 10-5 / 10-6 tubes on LB agar plates w/ appropriate antibiotic

• Flask experiments

Sensor Test

Objective: This experiment is designed for easy manipulation of cell sensor module and for check of main sample with several parallel controls in same parameters.
This experiment was processed in 6 diffeent flasks. For the first sensor trial to work following samples were studied,
1. pCooM-RFP
2. pLac-CooA +IPTG
3. pCooM-RFP - pLac-CooA - IPTG , w/o CO
4. pCooM-RFP -pLac-CooA + IPTG , w/o CO
5. pCooM-RFP- pLac-CooA - IPTG, with CO
6. pCooM-RFP- pLac-CooA + PTG, with CO
The cultures of parts were inoculated as streak plate to agar plates with appropriate antibiotics (136 ug/ml Chloramphenicol ) from glycerol stocks. The plate growth cultures were then inoculated to 10 ml Luria Broth with antibiotics, incubated at 37 C for 9 hour. The absorbance values of samples were measured for determine the bacteria amount in liquid. After measurements, grown bacteria was inoculated to 400 ml flasks as to be in same OD values. Then IPTG induction is done (1mM ). The CO gas treatment was done by volumetric calculations . 80 ml culture was incubated in 400 ml flask. The 20% of remaining volume inside the flask above liquid was filled with carbon monoxide. With this measurements corresponding carbonmonoxide volume was 80 ml. The gas flashing was done via syringe. After carbonmonoxide flashing, the flasks were incubated overnight at 37C, 225 rpm shaking for 12 hours. The confocal laser microscopy and flourescence spectrophometric measurements were done afterby 12 hours.

For the second sensor trial to work following samples were studied in 4 different flasks with the same construction, For this trial aim was to differentiate the fluorescence signals of following samples
1. Empty vector (chloramphenicol p SB1C3) + CooA in pTrix (ampicillin vector)
2. pCooF-GFP (in chloramphenicol vector p SB1C3) + CooA in pTrix (ampicillin vector) + CO
3. pCooF-GFP (in chloramphenicol vector p SB1C3) + CooA in pTrix (ampicillin vector) - CO
4. pCooF-GFP (in chloramphenicol vector p SB1C3)

The cultures of parts were inoculated as streak plate to agar plates with appropriate antibiotics (136 ug/ml Chloramphenicol 50 ug/ml Ampicillin ) from glycerol stocks. The plate growth cultures were then inoculated to 10 ml Luria Broth with antibiotics, incubated at 37 C for overnight. The absorbance values of samples were measured for determine the bacteria amount in liquid. After measurements, grown bacteria was inoculated to 400 ml flasks as to be in same OD values. Then IPTG induction is done (1mM ). The CO gas treatment was done by volumetric calculations . 80 ml culture was incubated in 400 ml flask. The 20% of remaining volume inside the flask above liquid was filled with carbon monoxide. With these measurements corresponding carbonmonoxide volume was 80 ml. The gas flashing was done via syringe with tightly bounded to cork. After carbonmonoxide flashing, the flasks were incubated overnight at 37C, 225 rpm shaking for 12 hours. The confocal laser microscopy and flourescence spectrophometric measurements were done afterby 12 hours.

Growth Test

Objective: This experiment is designed for testing the bacterial growth enhancer factors, IPTG and ferric citrate (FC) effects in with or without conditions.
In this experimental part, for preliminary studies of fermentor system, parallel 4 flasks of subcultures from same culture are performed with the same parameters as,
1. +IPTG, +FC
2. +IPTG, - FC
3. - IPTG, +FC
4. - IPTG, - FC
The bacteria with full cell sensor module [pCooF-GFP-pCooM-RFP, in p SB1C3; p Trix with CooA , Amp] were inoculated from glycerol stocks in agar plates with appropriate antibiotics (136 ug/ml Chloramphenicol, 50 ug/ml Ampicillin). Then inoculated to Luria Broth, liquid culture, the flasks were incubated overnight at 37C 225rpm. After incubation, the absorbance values of subcultures were measured at 600nm. According to the obtained OD value, the culture divided into 4 flasks. Each flask were labeled as written above. When OD(600nm) value reached to 0.5 ; 1m M IPTG, 2 g/ml FC were added to control flasks. For 5 hour, the absorbance were measured at 600 nm at each half an hour intervals. No gas treatment was applied to this flask experiment. The results of OD values gave the conclusion of study.

• Solid culture experiments

Objective: This part of cell sensor experiments were studied to try the cell sensor module functions in growth colonies on agar plate with antibiotics (136 ug/ml Chloramphenicol, 50 ug/ml Ampicillin). Bacteria were inoculated as both streak plate and spread plate to agar plates with antibiotics(136 ug/ml Chloramphenicol, 50 ug/ml Ampicillin) from glycerol stocks of full construct of cell sensor as in fermentor studies [pCooF-GFP-pCooM-RFP, in chloramphenicol vector p SB1C3; p TriEx with CooA , ampicillin vector]. As following step,the inoculated plate was placed in anaerobic chamber. By adjusting the volume inside of chamber by the vacuum on the top of it, the flashed carbon monoxide volume was determined. The volume of chamber was nearly 2 L. The one third of chamber volume was filled with carbonmonoxide. The remaining volume was completed with air. The chamber was then incubated overnight at 37C,. The grown bacteria was then analyzed with flourescense spectroscopy and confocal laser microscopy with parallel control of sample with no gas treatment overnight inoculated bacteria on agar plate.

• Myoglobin assay optimization

Objective: To determine the dissolved carbon monoxide in culture grown LB myoglobin assays were performed.
In this experimental part, measurements were done based on blank, sample and control. Blank was phosphate buffer, control was myoglobin oxidized and sample was myoglobin reduced. Experiment was processed in two ways;
- Myg-Reduced + CO Sparged PB
- Myg-Reduced + PB
- Myg-Reduced + N2 Sparged PB
--
- Myg-Reduced + CO Sparged LB
- Myg-Reduced + LB
- Myg-Reduced + N2 Sparged LB
Assay volume was 1 ml. Myoglobin was dissolved in phosphate buffer (0.4M pH 6.8 (24 C)), 100 ul of myoglobin solution is taken in plastic cuvettes. Then 100 ul sodium dithionate was added. The volume is adjusted to 1 ml with CO sparged LB and without sparged LB as control.Then absorbance was measured at 540 n. Samples were prepared as follows

10X DT (Dithionate):
- 1.1% (64 uM) in dH2O
- 1 mL
10X Myg (Myoglobin)
- 10 mg/mL (66 uM) in PB
- 1 mL
This assay enables us handle CO in liquid media.

• Signal quantification with fluorescence spectroscopy

Objective: We would like to compare the responds of bacteria before and after CO sparging. Fluorescence spectroscopy enables us to observe the RFP and GFP signal coming from those bacteria.

Parameters:
- Excitation wavelength for RFP is set to 570nm and 395nm for GFP.
- Slit width is 2nm.
- Emission scan was taken from 585nm to 650nm for RFP and 475nm to 600nm for GFP.
- Readings were taken in 150 mL quartz cuvette.
- Reading made: steady state.
- All samples were set to the same bacterial concentration.
- All readings were taken in phosphate buffer solution.

• Imaging with confocal laser scanning microscopy

Objective: To visualize the cells with fluorescence expression at high magnification and resolution, confocal laser scanning microscopy is used.

Parameters:
- Excitation was done by 488nm laser for GFP and 543 nm laser for RFP.
- 10 uL bacterial phosphate buffer solution was smeared onto a glass slide and then fixed by flame.
- Objectives properties: 100x objectives, NA: 1.35 with immersion oil.

Cloning of pCooF, pCooM, G2C, C4G, C7G, C9G, H77T, P2H with pSB1C3 vector

1) Plasmid Isolation

- Subcultures have been prepared from pCooM-RBS-RFP-TT, PLacI-RBS and pSB1C3 vector colonies (5mL)
- Subcultures was incubated overnight
- Fermentas Plasmid Miniprep Kit is used for plasmid isolation
- Final elution was performed with 50 uL elution buffer
- Spectrophotometric measurements was performed to determine purity and the concentration of DNA samples.

2) Restriction Digestion

- For this procedure, Fermentas Fast Digest enzymes are used.

3) PCR purification
- Fermentas PCR purification kit was used
- Final elution volume was 30 uL

4) Agarose gel electrophoresis

- 1% 50 mL agarose gel was prepared with La Basica agarose

1- pSB1C3 vector

5) Gel extraction

- Fermentas Gel Extraction kit was used

6) Ligation

- Samples are incubated 5 min @ room temperature.

7) Transformation

- For first mixture 100 uL Top 10 competent cell is used.
- For second mixture 100 uL BL21 competent cell is used.
- Transformation was performed with heat shock method (method is explained at protocols under cloning session).

8) Confirmation

- Subcultures have been prepared from different 2 colonies.
- Subcultures was incubated overnight
- Fermentas Plasmid Miniprep Kit is used for plasmid isolation
- Final elution was performed with 50 uL elution buffer
- For Restriction Digestion, Fermentas Fast Digest enzymes are used

Cloning of pCooF-RBS-GFP-TT
1) Transformation
- pCooF –pMA vector was synthesized from GENEART
- RBS – GFP – TT- pSB1A2 was obtained from 2010 IGEM Kit plate
- pMA GENEART Backbone vector was used as backbone.
- 1 uL (10-15 ng) DNA was used for transformation
- Transformation was performed with heat shock method (method is explained at protocols under cloning session)
- After transformation, subcultures have been prepared for one colony for each vector (10mL)
- Subcultures was incubated overnight
2) Plasmid Isolation
- Fermentas Plasmid Miniprep Kit is used for plasmid isolation
- Final elution was performed with 50 uL elution buffer
- Spectrophotometric measurements was performed to determine purity and the concentration of DNA samples
-
-3)Vacuum Drying
- Samples are concentrated with Heto-Holten Model Maxi-Dry Lyo
- 50 uL samples are concentrated to 15-20 uL
- The pressure applied was 200 mBarr
- Duration of pressure application was 150 min.

4)Restriction Digestion
- For this procedure, Fermentas Fast Digest enzymes are used.



5) PCR purification
- Fermentas PCR purification kit was used
- Final elution volume was 30 uL

6) Agarose gel electrophoresis

- 1% 50 mL agarose gel was prepared with La Basica agarose



- Samples are incubated 5 min @ room temperature.

10)Transformation
- Transformation was performed with heat shock method (method is explained at protocols under cloning session).

11)Confirmation
- Subcultures have been prepared.
- Subcultures was incubated overnight
- Fermentas Plasmid Miniprep Kit is used for plasmid isolation
- Final elution was performed with 50 uL elution buffer
- For Restriction Digestion, Fermentas Fast Digest enzymes are used.
-


Cloning of pCooM-RBS-RFP-TT
1) Transformation
- pCooM –pMA vector was synthesized from GENEART
- RBS – RFP – TT- pSB1A2 was obtained from 2010 IGEM Kit plate
- pMA GENEART Backbone vector was used as backbone.
- 1 uL (10-15 ng) DNA was used for transformation
- Transformation was performed with heat shock method (method is explained at protocols under cloning session)
- After transformation, subcultures have been prepared for one colony for each vector (5mL)
- Subcultures was incubated overnight
2) Plasmid Isolation

- Fermentas Plasmid Miniprep Kit is used for plasmid isolation
- Final elution was performed with 50 uL elution buffer
- Spectrophotometric measurements was performed to determine purity and the concentration of DNA samples.


3) Vacuum Drying
- Samples are concentrated with Heto-Holten Model Maxi-Dry Lyo
- 50 uL samples are concentrated to 15-20 uL
- The pressure applied was 200 mBarr
- Duration of pressure application was 150 min.

4) Restriction Digestion
- For this procedure, Fermentas Conventional enzymes are used


5) PCR purification
- Fermentas PCR purification kit was used
- Final elution volume was 30 uL

6) Agarose gel electrophoresis

- 1% 50 mL agarose gel was prepared with La Basica agarose

7) Gel extraction
- Fermentas Gel Extraction kit was used

8) Ligation



9) Transformation
- For first mixture 100 uL Top 10 competent cell is used.
- For second mixture 100 uL BL21 competent cell is used.
- Transformation was performed with heat shock method (method is explained at protocols under cloning session).

10)Confirmation

- Subcultures have been prepared from different 4 colonies.
- Subcultures was incubated overnight
- Fermentas Plasmid Miniprep Kit is used for plasmid isolation
- Final elution was performed with 50 uL elution buffer
- For Restriction Digestion, Fermentas Fast Digest enzymes are used.