iGEM 2019 — Team NTHU_Taiwan

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NTHU Taiwan/Notebook

[![](https://static.igem.org/mediawiki/2019/4/42/T--NTHU_Taiwan--logo_nobackground.png)
NTHU\_Taiwan](https://2019.igem.org/Team%3ANTHU_Taiwan)

* Team

  [Team Members](https://2019.igem.org/Team%3ANTHU_Taiwan/Team)
  [Collaborations](https://2019.igem.org/Team%3ANTHU_Taiwan/Collaborations)
* Project

  [Description](https://2019.igem.org/Team%3ANTHU_Taiwan/Description)
  [Design](https://2019.igem.org/Team%3ANTHU_Taiwan/Design)
  [Experiments](https://2019.igem.org/Team%3ANTHU_Taiwan/Experiments)
  [Notebook](https://2019.igem.org/Team%3ANTHU_Taiwan/Notebook)
  [Contribution](https://2019.igem.org/Team%3ANTHU_Taiwan/Contribution)
  [Result](https://2019.igem.org/Team%3ANTHU_Taiwan/Results)
  [Demonstrate](https://2019.igem.org/Team%3ANTHU_Taiwan/Demonstrate)
  [Improve](https://2019.igem.org/Team%3ANTHU_Taiwan/Improve)
  [Attributions](https://2019.igem.org/Team%3ANTHU_Taiwan/Attributions)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ANTHU_Taiwan/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ANTHU_Taiwan/Basic_Part)
  [Composite\_Part](https://2019.igem.org/Team%3ANTHU_Taiwan/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ANTHU_Taiwan/Part_Collection)
* [Safety](https://2019.igem.org/Team%3ANTHU_Taiwan/Safety)
* Human practices

  [Human practices](https://2019.igem.org/Team%3ANTHU_Taiwan/Human_Practices)
  [Eduacation & Engagement](https://2019.igem.org/Team%3ANTHU_Taiwan/Public_Engagement)
* Awards

  [Entrepreneurship](https://2019.igem.org/Team%3ANTHU_Taiwan/Entrepreneurship)
  [Hardware](https://2019.igem.org/Team%3ANTHU_Taiwan/Hardware)
  [Measurement](https://2019.igem.org/Team%3ANTHU_Taiwan/Measurement)
  [Model](https://2019.igem.org/Team%3ANTHU_Taiwan/Model)
  [Plant](https://2019.igem.org/Team%3ANTHU_Taiwan/Plant)
  [Software](https://2019.igem.org/Team%3ANTHU_Taiwan/Software)

Notebook

### July 2019

| Sun | Mon | Tue | Wed | Thu | Fri | Sat |
| --- | --- | --- | --- | --- | --- | --- |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  |  |  |

### August 2019

| Sun | Mon | Tue | Wed | Thu | Fri | Sat |
| --- | --- | --- | --- | --- | --- | --- |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 | 31 |

### September 2019

| Sun | Mon | Tue | Wed | Thu | Fri | Sat |
| --- | --- | --- | --- | --- | --- | --- |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 |  |  |  |  |  |

### October 2019

| Sun | Mon | Tue | Wed | Thu | Fri | Sat |
| --- | --- | --- | --- | --- | --- | --- |
|  |  | 1 | 2 | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |  |  |

# July 1

Hello

# July 2

# July 3

# July 4

# July 5

# July 6

# July 7

# July 8

# July 9

# July 10

# July 11

# July 12

# July 13

# July 14

# July 15

# July 16

# July 17

# July 18

# July 19

# July 20

# July 21

# July 22

# July 23

# July 24

# July 25

# July 26

# July 27

# July 28

# July 29

# July 30

# July 31

# August 1

# August 2

# August 3

# August 4

# August 5

# August 6

# August 7

# August 8

# August 9

# August 10

# August 11

# August 12

# August 13

# August 14

# August 15

# August 16

# August 17

# August 18

# August 19

# August 20

# August 21

# August 22

# August 23

# August 24

# August 25

# August 26

# August 27

# August 28

# August 29

# August 30

# August 31

# September 1

# September 2

# September 3

# September 4

# September 5

# September 6

# September 7

# September 8

# September 9

# September 10

# September 11

# September 12

# September 13

# September 14

# September 15

# September 16

# September 17

# September 18

# September 19

# September 20

# September 21

# September 22

# September 23

# September 24

# September 25

# September 26

# September 27

# September 28

# September 29

# September 30

# September 31

# October 1

# October 2

# October 3

# October 4

# October 5

# October 6

# October 7

# October 8

# October 9

# October 10

# October 11

# October 12

# October 13

# October 14

# October 15

# October 16

# October 17

# October 18

# October 19

# October 20

# October 21

# October 22

# October 23

# October 24

# October 25

# October 26

# October 27

# October 28

# October 29

# October 30

# October 31

#### **FOLLOW US**

[![](https://static.igem.org/mediawiki/2019/9/93/T--NTHU_Taiwan--Facebook.png)](https://www.facebook.com/NTHU.iGEM/)
[![](https://static.igem.org/mediawiki/2019/f/fe/T--NTHU_Taiwan--Instagram.png)](https://www.instagram.com/igem_tw_nthu/?fbclid=IwAR1DB5OWM9-rSEoxWU95uJwIUaos3lcTa07citdUi1U3kJuYHInHiW3IwtE)

#### **CONTACT US**

![](https://static.igem.org/mediawiki/2019/3/3e/T--NTHU_Taiwan--email.png)

nthuxigem@gmail.com


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NTHU Taiwan/Experiments

[![](https://static.igem.org/mediawiki/2019/4/42/T--NTHU_Taiwan--logo_nobackground.png)
NTHU\_Taiwan](https://2019.igem.org/Team%3ANTHU_Taiwan)

* Project

  [Description](https://2019.igem.org/Team%3ANTHU_Taiwan/Description)
  [Design](https://2019.igem.org/Team%3ANTHU_Taiwan/Design)
  [Experiments](https://2019.igem.org/Team%3ANTHU_Taiwan/Experiments)
  [Characterization](https://2019.igem.org/Team%3ANTHU_Taiwan/Contribution)
  [Model](https://2019.igem.org/Team%3ANTHU_Taiwan/Model)
  [Safety](https://2019.igem.org/Team%3ANTHU_Taiwan/Safety)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ANTHU_Taiwan/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ANTHU_Taiwan/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ANTHU_Taiwan/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ANTHU_Taiwan/Part_Collection)
* Hardware

  [Overview](https://2019.igem.org/Team%3ANTHU_Taiwan/Hardware)
  [Fertilizer Tank](https://2019.igem.org/Team%3ANTHU_Taiwan/FertilizerTank)
  [Sensing System](https://2019.igem.org/Team%3ANTHU_Taiwan/SensingSystem)
  [Demonstrate](https://2019.igem.org/Team%3ANTHU_Taiwan/Demonstrate)
* Software

  [LoRa/TTN](https://2019.igem.org/Team%3ANTHU_Taiwan/LoRa_TTN)
  [Node-Red](https://2019.igem.org/Team%3ANTHU_Taiwan/Software)
  [Arduino Code](https://2019.igem.org/Team%3ANTHU_Taiwan/ArduinoCode)
* Results

  [Results](https://2019.igem.org/Team%3ANTHU_Taiwan/Results)
  [Improve](https://2019.igem.org/Team%3ANTHU_Taiwan/Improve)
* Human

  [Human Practices](https://2019.igem.org/Team%3ANTHU_Taiwan/Human_Practices)
  [Eduacation & Engagement](https://2019.igem.org/Team%3ANTHU_Taiwan/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ANTHU_Taiwan/Collaborations)
* Team

  [Team Members](https://2019.igem.org/Team%3ANTHU_Taiwan/Team)
  [Attributions](https://2019.igem.org/Team%3ANTHU_Taiwan/Attributions)

Experiments

* [Cloning](#Cloning)
* [Backbone](#backbone)
* [OD & Fluorescence](#OD_Fluorescence)
* [Western Blot](#Western)
* [Lipase Activity Assay](#Lipase)

# Cloning

PCR amplification

Normal PCR/ Gradient PCR (find best annealing temperature)

PCR mixture (YCL lab):

|  |  |
| --- | --- |
| YCL lab Phusion polymerase premix(dNTP 10mM, Phusion pol, ddH2O, 5X buffer) | 47ul |
| DNA template | 1ul |
| Primer forward 10uM | 1ul |
| Primer reverse 10uM | 1ul |

* DNA amount: 100ng
* primer concentration: 10uM

PCR program:

|  |  |
| --- | --- |
| 95&degC | Preheat |
| 95&degC | 5min |
| 95&degC | 30s |
| 60&degC (gradient determined best temp.) | 30s |
| 72&degC | 2-3min, go back to step 3 repeat for 40 cycles. |
| 72&degC | 10min |
| 4&degC | o/n |

Design:

Control: J04450 from miniprep

PCR cleanup (with Geneaid Gel/PCR Kit (DFH100, DFH300))

1. Transfer reaction product to a 1.5 ml microcentrifuge tube. Add 5 volumes of Gel/PCR Buffer to the sample then vortex.(\*If the mixture has turned from yellow to purple, add 10 µl of 3M sodium acetate (pH5.0) and mix thoroughly.)
2. Transfer the sample mixture to the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through.
3. Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the DFH Column and let stand for 1 minute. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Centrifuge again at 14-16,000 x g for 3 minutes to dry the column matrix.
4. Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 20-50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.
5. If the final concentration of purified DNA is too low (lower than 15ng/ul), we would put the elution buffer (in our protocol is ddH2O in 55°c) with purified DNA back to the column and stand for 10 minutes to elute the remained DNA.

Digestion (20ul reaction)

1. Set up the following reaction in a microcentrifuge tube on ice. (20ul reaction)

   |  |  |
   | --- | --- |
   | DNA (depends on concentration) | 1ug |
   | Cut Smart buffer (10X) | 2ul |
   | Enzyme 1 | 0.5ul |
   | Enzyme 2 | 0.5ul |

   * Digest for 1 to 2 hours
   * Use NED double digest finder [(https://nebcloner.neb.com/#!/redigest)](https://nebcloner.neb.com/#!/redigest)to find the best condition for digestion. EcoRI-HF, SpeI-HF, PstI and XbaI-HF are often used enzyme in iGEM.
   * Concentration of restriction enzyme should be lower than 5% to avoid star activity.
2. Heat inactivate at 65°C / 80°C for 20 minutes.
   Design：
   Control: J04450 (if success, there will be two bands, at about 2000bp and 1000bp)

Cleanup (Same protocol as above)

Same protocol as above

Gel electrophoresis (Before ligation to determine the amount of DNA)

1. Making 1.3% agarose gel (small)

   |  |  |
   | --- | --- |
   | TAE buffer (1X) | 25ml |
   | Agarose | 0.33g |
2. Loading sample
   DNA marker: 1ul of 6X (loading dye/ Novel juice) + 5 ul of DNA ladder
   DNA sample: 1.5ul of 6X (loading dye/ Novel juice) + 2 ul of DNA
3. Start the electrophoresis 100V for 30 minutes.

Ligation

* Method 1: Use Nanodrop to measure the concentration of DNA sample and determine the volume used to ligate.
* Method 2: Use the result of gel electrophoresis (brightness of DNA sample to determine the volume used to ligate.)

1. Set up the following reaction in a microcentrifuge tube on ice. (20ul reaction)

   |  |  |
   | --- | --- |
   | T4 DNA ligase | 1ul |
   | T4 DNA ligase buffer (10X) | 2ul |
   | Insert sequence | The volume left in tube |
   | Backbone | The volume of insert sequence /3 |
   | ddH2O | To 20ul |
2. Incubate at 16°C overnight
3. Heat inactivate at 65°C for 10 minutes

Design:

Control: the product prepared from digestion and gel extraction (psB1c3 backbone and the mrfp part.)

Transformation

1. Thaw a tube of DH5α Competent E. coli cells on ice.
2. Add 3 µl containing ligation product to the 30ul of the cells. (DNA cannot exceed 10% of the total volume of competent cells.)
   PS: We wondered that the low concentration of ligation product may be the reason failing our experiment, so one time we alternate the protocol. We did PCR clean up after ligation, and transform all the ligation product, but it is still unsuccessful.
3. Place the mixture on ice for 30 minutes.
4. Heat shock at exactly 42°C for exactly 45 seconds.
5. Place on ice for 5 minutes.
6. Pipette 900 µl of room temperature LB broth without antibiotic into the mixture. (for recovery)
7. Place at 37°C for 60 minutes.
8. Warm selection plates to 37°C.
9. Centrifuge at 400g to remove the excess LB and resuspend the cells with 200ul of LB with antibiotic. Spread onto a selection plate and incubate for 16 hours at 37°C.

Design:

Control: control group in ligation

Liquid culture

1. Pick single colony with a 20-200ul tip.
2. Put the tip into the tube with 5ml LB broth with antibiotic.
3. Place at 37°C for 60 minutes. Shake vigorously (250 rpm).

Miniprep (with Geneaid Miniprep kit)

1. **Harvesting**
   Transfer 1.0-1.5 ml of cultured bacterial cells to a microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute at room temperature to form a cell pellet then discard the supernatant completely.
2. **Resuspension**
   Add 200 µl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge tube containing the cell pellet. Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved.
3. **Cell Lysis**
   Add 200 µl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Close PD2 Buffer bottle immediately after use to avoid CO2 acidification. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.
4. **Neutralization**
   Add 300 µl of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Centrifuge at 14-16,000 x g for 3 minutes at room temperature. If using >5 ml of bacterial cells, centrifuge at 16-20,000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection Tube.
5. **DNA Binding**
   Transfer all of the supernatant to the PDH Column. Use a narrow pipette tip to ensure the supernatant is completely transferred without disrupting the white precipitate. Centrifuge at 14-16,000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
6. **Wash**
   Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column. Centrifuge at 14-16,000 x g for 30 seconds at room temperature. Discard the flow through then place the PDH Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PDH Column to a new 1.5 ml microcentrifuge tube.
   Perform Wash Buffer steps twice for salt sensitive downstream applications.
7. **Elution**
   Add 50 µl of water into the CENTER of the column matrix. Let stand for at least 2 minutes to allow Elution Buffer, TE or water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.

# backbone

Transformation from iGEM kit

Liquid Culture

Miniprep

Digestion

Gel electrophoresis

Gel Extraction (If backbone from iGEM kit is digested, for example J04450 = pSB1C3 + mrfp, gel extraction is needed)

1. Gel Dissociation
   1. Cut the agarose gel slice containing DNA fragments.
   2. Add 500 µl of Gel/PCR Buffer to the sample then mix by vortex.
   3. Incubate at 55-60ºC for 10-15 minutes or until the gel slice is completely dissolved. (During incubation, invert the tube every 2-3 minutes.) \*If the color of the mixture has turned from yellow to purple, add 10 µl of 3M Sodium Acetate (pH5.0) and mix thoroughly. This will adjust pH and the color will return to yellow.
   4. Cool the dissolved sample mixture to room temperature.
2. DNA Binding
   1. Place a DFH Column in a 2 ml Collection Tube.
   2. Transfer the sample mixture to the DFH Column.
   3. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through then place the DFH Column back in the 2 ml Collection Tube.
3. Wash
   1. Add 400 µl of W1 Buffer into the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube.

   \*The following steps are same as the third step onwards of PCR cleanup.

# Protocol for OD value and Fluorescence intensity Measurement

Preparation of 10mM Oleic acid medium in LB

1. Prepare 0.5% ethanol solution to dissolve fatty acid.
2. Transfer 1mL NaOH in to Eppendorf, and add 31.5ul Oleic acid in it. Gently inverse the Eppendorf until the solution is well mixed.
3. Transfer the Oleic-Alcohol solution 1ml into 9ml LB.
4. Leave 10-15 min in agitation.

OD value and Fluorescence intensity Measurement

1. Plate 180 ul Oleic acid LB into the wells of the 96-well plate.
2. Add 20 ul cell culture into each well.
3. Incubate the cells at 37°C.
4. Use plate reader to trace OD value and Fluorescence intensity in an hourly basis.

# Western Blot

CELL LYSIS

1. Before start, precooled the PBS and centrifuger at 4°C.
2. Prepare the lysis buffer composed of:

* 50mM Tris pH 7.4
* 50mM NaCl
* 1% SDS

3. Centrifuge the liquid culture of bacteria at 1000g for 5min and dispose the supernatant.
4. Wash the cells with precooled PBS and centrifuge at 1000g for 5min.
5. Aspirate the PBS and repeat the previous step for twice.
6. Remove the PBS and add lysis buffer, then wait for 30min.
7. Centrifuge with 16000g for 10 minutes at 4°C.
8. Collect the supernatant to new microcentrifuge tubes and stored the samples in -32°c refrigerator.

BCA PROTEIN ASSAY (BICINCHONINIC ACID PROTEIN ASSAY)

1. Take out the protein extraction kit and 96-well plate (can be reused)
2. Mix Reagent A and Reagent B in the ratio of 50:1 (The volume of the mixture depends on the amount of wells used.)
3. Drops each 100 μl of the mixed reagent into the wells.
4. Then, making the standard curves by different volume of BSA (Bovine Serum Albumin) with concentration of 2 μg/μl. The volume is as followed:

   |  |  |  |  |  |
   | --- | --- | --- | --- | --- |
   | 0μl of BSA | 0.5μl of BSA | 1 μl of BSA | 2 μl of BSA | Samples… |
5. Drops the left wells with 1 μl of samples.
6. Wrap with plastic wrap and then place it in oven at 37ºC for 30 minutes.
7. Measure the absorbance of samples for 562nm light.
8. According to the absorbance measured, dilute the sample, calculation needed
   * Minus the blank solution absorbance and make a standard curve.
   * By using the gradient and y-intercept of the curve to determine the concentration of protein in the samples.
   * Select the lowest concentration of protein samples as the baseline and calculate the volume of PBS needed to dilute the other samples.
   * Calculate the volume of dye and loading buffer to be added. (Loading buffer: dye = 5:1)
9. Heat at 95ºC for 10 minutes.
10. Centrifuge 13000rpm for 1 minute. If you continue the SDS-PAGE immediately, then you can proceed to step 9 and 10. If not, step 9 and 10 and be skipped and do it when you want to do SDS-PAGE.

SDS-PAGE (SODIUM DODECYL SULFATE-POLYACRYLAMIDE GEL ELECTROPHORESIS)

1. Set up the apparatus and make sure it is not leaking by adding ddH2O into it.
   The setup of apparatus is like this:
   Resolving gel 12% (lower gel)

   |  |  |
   | --- | --- |
   | **Solution** | **Volume used (μl)** |
   | ddH2O | 2000 |
   | 1.5M Tris-base (pH 8.6) | 5000 |
   | 10% SDS | 100 |
   | 30% acrylamide | 4000 |
   | TEMED | 5 |
   | 10% APS | 75 |

   \* ddH2O should be added first to prevent precipitation. TEMED must be added the last because it will cause the solution to condense.

   \*There are many other concentration of acrylamide gel which is suitable for different size of protein, please refer to the notes paste on the cupboard when making the gel.

   |  |  |
   | --- | --- |
   | **Protein size(kDa)** | **Gel percentage (%)** |
   | 4-40 | 20 |
   | 12-45 | 15 |
   | 10-70 | 12.5 |
   | 15-100 | 10 |
   | 25-200 | 8 |
2. Wipe off the ddH2O and add the lower gel. (\*Little tips: Isopropanol can be added to flatten the gel and polymerize the gel faster.)
3. Steaking gel

   |  |  |
   | --- | --- |
   | **Solution** | **Volume used (μl)** |
   | ddH2O | 2810 |
   | 1.5M Tris-base (pH 8.6) | 350 |
   | 10% SDS | 56.5 |
   | 30% acrylamide | 650 |
   | TEMED | 4 |
   | 10% APS | 37.5 |
4. Set up the apparatus.
5. Loading the marker and samples, the volume of samples loaded depend on your samples’ concentration.
6. Electrophoresis (20-30mA for one piece of gel).

COOMASSIE BLUE STAINING

1. Stain the gel with 5cc Coomassie blue for 10mins.
2. Wash the gel with ddH2O for 5-7 times or overnight.

(If Western Blot is needed, then proceed to the next step.)

TRANSFER

1. Immerse the PVDF membrane in methanol while the blot paper in 10x transfer buffer.
2. Transfer with 20V for 60 minutes. (We can modify the transfer speed by adjust the current to 30mA)

BLOCKING

1. 1. Prepared blocking solution, that is 5% non-fat dry milk in PBST and rock for 1 hour.
   (PBST is that PBS with 0.2% Tween 20, Tween 20 should be diluted into 20% first before prepared PBST)
    (The membrane can be rock for overnight but make sure in the -4°c refrigerator.)

INCUBATION

1. Cut the PVDF membrane into two pieces according to the location of protein interest.
2. Next, add in primary antibody in the ratio of 1:10000 to milk and then shake it for 1-2 hours under room temperature. To prevent wastage, we usually reuse the primary antibody. (time can be longer than that to get better effect.)
3. Wash with PBST by shaking it for 10 minutes. Repeat thrice.
4. Add in secondary antibody in the ratio of 1:10000 to PBST, shake it for 1-2 hours.

IMAGING

1. Wash away the unbounded secondary antibody with PBST for three times.
2. Prepared two types of ECL and mixed in the ratio of 1:1.

SUPPLEMENTARY

How to prepare reagents.

1. 10X transfer buffer

   |  |  |
   | --- | --- |
   | Tris base | 15.2 g |
   | Glycine | 72.1 g |
   | ddH2O | To 500 ml |
2. 10X running buffer

   |  |  |
   | --- | --- |
   | Tris base | 30.3 g |
   | Glycine | 144.1 g |
   | SDS | 10 g |
   | ddH2O | To 1 L |
3. 1.5 M Tris pH 8.8

   |  |  |
   | --- | --- |
   | Tris-free base | 90.75 g dissolved in 400ml ddH2O |
   | HCl | Titrate to pH 8.8 (about 40-50 ml) |
   | ddH2O | To 500 ml |
4. 1.0M Tris pH 6.8

   |  |  |
   | --- | --- |
   | Tris-free base | 60.54 g dissolved in 400ml ddH2O |
   | HCl | Titrate to pH 6.8 |
   | ddH2O | To 500 ml |

# Lipase Activity Assay

Material

1. 4-Nitrophenyl decanoate (pND)
2. Acetonitrile
3. HEPES buffer
4. CaCl2

Procedure

1. Substrate pND was dissolved in acetonitrile with concentration of 100 mM
2. Reaction mixture: 5 µM CaCl2, 100 mM HEPES (pH 9), 1.25 mM substrate (pND), and 0.02 mg of enzyme, total volume 1 ml
3. The amount of p-nitro-phenol released during hydrolysis was determined with spectrophotometric at 405 nm

#### **FOLLOW US**

[![](https://static.igem.org/mediawiki/2019/9/93/T--NTHU_Taiwan--Facebook.png)](https://www.facebook.com/NTHU.iGEM/)
[![](https://static.igem.org/mediawiki/2019/f/fe/T--NTHU_Taiwan--Instagram.png)](https://www.instagram.com/igem_tw_nthu/?fbclid=IwAR1DB5OWM9-rSEoxWU95uJwIUaos3lcTa07citdUi1U3kJuYHInHiW3IwtE)

#### **CONTACT US**

![](https://static.igem.org/mediawiki/2019/3/3e/T--NTHU_Taiwan--email.png)

nthuxigem@gmail.com

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:NTHU Taiwan/Results

[![](https://static.igem.org/mediawiki/2019/4/42/T--NTHU_Taiwan--logo_nobackground.png)
NTHU\_Taiwan](https://2019.igem.org/Team%3ANTHU_Taiwan)

* Project

  [Description](https://2019.igem.org/Team%3ANTHU_Taiwan/Description)
  [Design](https://2019.igem.org/Team%3ANTHU_Taiwan/Design)
  [Experiments](https://2019.igem.org/Team%3ANTHU_Taiwan/Experiments)
  [Characterization](https://2019.igem.org/Team%3ANTHU_Taiwan/Contribution)
  [Model](https://2019.igem.org/Team%3ANTHU_Taiwan/Model)
  [Safety](https://2019.igem.org/Team%3ANTHU_Taiwan/Safety)
* Parts

  [Parts Overview](https://2019.igem.org/Team%3ANTHU_Taiwan/Parts)
  [Basic Parts](https://2019.igem.org/Team%3ANTHU_Taiwan/Basic_Part)
  [Composite Parts](https://2019.igem.org/Team%3ANTHU_Taiwan/Composite_Part)
  [Part Collection](https://2019.igem.org/Team%3ANTHU_Taiwan/Part_Collection)
* Hardware

  [Overview](https://2019.igem.org/Team%3ANTHU_Taiwan/Hardware)
  [Fertilizer Tank](https://2019.igem.org/Team%3ANTHU_Taiwan/FertilizerTank)
  [Sensing System](https://2019.igem.org/Team%3ANTHU_Taiwan/SensingSystem)
  [Demonstrate](https://2019.igem.org/Team%3ANTHU_Taiwan/Demonstrate)
* Software

  [LoRa/TTN](https://2019.igem.org/Team%3ANTHU_Taiwan/LoRa_TTN)
  [Node-Red](https://2019.igem.org/Team%3ANTHU_Taiwan/Software)
  [Arduino Code](https://2019.igem.org/Team%3ANTHU_Taiwan/ArduinoCode)
* Results

  [Results](https://2019.igem.org/Team%3ANTHU_Taiwan/Results)
  [Improve](https://2019.igem.org/Team%3ANTHU_Taiwan/Improve)
* Human

  [Human Practices](https://2019.igem.org/Team%3ANTHU_Taiwan/Human_Practices)
  [Eduacation & Engagement](https://2019.igem.org/Team%3ANTHU_Taiwan/Public_Engagement)
  [Collaborations](https://2019.igem.org/Team%3ANTHU_Taiwan/Collaborations)
* Team

  [Team Members](https://2019.igem.org/Team%3ANTHU_Taiwan/Team)
  [Attributions](https://2019.igem.org/Team%3ANTHU_Taiwan/Attributions)

Results

* [Overview](#Overview)
* [Lipase](#Lipase)
* [Promoter](#Promoter)
* [Conclusion](#Conclusion)

# Overview

From previous descriptions, we know that lipase can convert lipid into fatty acid. And the lipase activity can change with the temperature. (see the design)

# Lipase

Here, we design several experiments to prove the feasibility of our project and analyze some data. The followings are our achievements:

Our vectors successfully constructed

The cold-adapted lipase A is a protein from an Antarctic deep-sea psychrotrophic bacterium Pseudomonas sp. 7323. Lipases are glycerol ester hydrolases that can hydrolyze ester to free fatty acid and glycerol. With overexpression of LipA, the bacteria can produce different concentrations of fatty acid under different temperatures.

We get the sequence of Lip A from NCBI. To check the expression of Lip A in cells and facilitate the purification of this protein, we attached the 6 X His tag on the C-terminal of this protein. This part was inserted into the iGEM provided expression vector psB1C3 through the restriction site EcoRI and SpeI (Fig1).

![](https://static.igem.org/mediawiki/2019/e/e1/T--NTHU_Taiwan--result1.png)

Figure 1. Construction of expression vector pSB1C3-LipA-6X His-tag. The insert sequence is flanked by EcoRI and SpeI restriction site.

This recombinant plasmid was further screened by ampicillin selection, colony PCR in the cloning E. coli, DH5α (Fig. 2) and the digestion of miniprep product (Fig 3). From those results, we can prove that the Lip A sequence synthesized by IDT was successfully integrated into the cloning vector psB1C3.

![](https://static.igem.org/mediawiki/2019/e/e6/T--NTHU_Taiwan--result2.png)

Figure 2. DNA electrophoresis with 1.32% gel was performed to screen the positive recombinant. The plasmid constructed was 4176bp and the predicted PCR result should be 2420bp (flanked by the VF2 and VR primer). Lane 1: DNA loading marker, Lane 2-7: VF2 and VR PCR product).

![](https://static.igem.org/mediawiki/2019/7/78/T--NTHU_Taiwan--result3.png)

Figure 3. DNA electrophoresis with 1.32% gel was performed to screen the positive recombinant. The plasmid extracted was digested with EcoRI and SpeI. The digested part (R0010-Lip A-6X His tag) should be 2129bp. Lane 1: DNA loading marker, Lane 2 to 5: Plasmid digested, Lane 6: J04450 control. The plasmid should be digested into 2 parts, one is R0010-Lip A-6X His tag, the other one is the psB1C3 backbone, both parts are about 2200bp. Thus, there will be only one band on the lane.

Lipase successfully produced

We have transformed E. coli BL21 strain with pSB1C3-LipA-His tag construct which has been previously proved successful. The positive transformants were screened with ampicillin and colony PCR. We liquid cultured the cells and collected after 16 hours. After washing with PBS, we lysed the cells by using lysis buffer (please refer to the Experiment protocol) to get the protein lysate. Western Blot has been performed to check the expression of Lip A by using an antibody against His tag.

![](https://static.igem.org/mediawiki/2019/9/98/T--NTHU_Taiwan--result4.png)

Figure 4. Western Blot analysis of total protein extracted from BL21 transformed with pSB1C3-Lip A-6X His tag by using antibody against His tag. Lane 1: BL21 control with no plasmid transform. Lane 2 and 3: Protein loading marker. Lane 4 to 13: BL21 transformed with our construct. According to the information on UniProt, Lip A from Pseudomonas sp.7323 was reported to be 64.555 kDa.

Our enzymes are functional

In order to verify the functionality of Lip A expressed, we have done a functional assay with the protein extract from BL21 by sonication. To determine the lipase activity, we utilized a spectrometry-based method by using 4-nitrophenyl decanoate as a substrate. The amount of 4-nitrophenol hydrolyzed and released was determined spectrophotometrically at 405nm. We compared the fluorescence and found that the change in fluorescence is greater in the protein in BL21 expressed Lip A compared to control (Fig. 5).

![](https://static.igem.org/mediawiki/2019/b/b3/T--NTHU_Taiwan--result5.png)

Figure 5. Lipase activity assay was performed to analyze the function of lipase A. The protein was first incubated at 30°c for 30 minutes at pH9.0. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm 30°c in continuous duration (0, 20, 40, 60 mins). The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

Enzyme activity matches our expectation over temperature change

Since our goal is to build a precise thermal-tunable promoter with a dynamic range of gene transcription, the lipase activity in a variety of temperature is very important to us. Hence, we evaluated the lipase activity at different temperature and the curve illustrated fitted to what reported in previous research of Lip A of Pseudomonas sp.7323 (Figure 6 and 7).

![](https://static.igem.org/mediawiki/2019/1/1c/T--NTHU_Taiwan--result6.png)

Figure 6. Lipase activity assay analysis was performed to check the activity of lipase at different temperature varies with time at pH9.0. The protein was first incubated at the experiment temperature (10, 20, 25, 30 and 40°c) for 30 minutes. Then 4-nitrophenyl decanoate (pND) mixture was added into the protein lysate. The mixture was then detected at 405nm in continuous duration (0, 20, 40, 60 mins) at the temperature required. The fluorescence level in the graph was subtracted with the background fluorescence of protein. Histograms represent normalized means±s.e.m. (n=3).

![](https://static.igem.org/mediawiki/2019/f/fe/T--NTHU_Taiwan--result7.png)

Figure 7. Lipase activity assay analysis was performed to check the activity of lipase at different temperature for 40 minutes at pH9.0. 40 minutes of data were chosen because this time duration is best fitted to the activity curve reported previously in the paper. Thus, our lipase is proved to be functional.

# Fatty acid sensitive promoter

We tried to improve the original fatty acid promoter, pFadBA, by increasing the strength and decreasing the leakage of the promoters. We tested several characteristics of these promoters:

1. Effect of fatty acid concentration on promoter
2. Decrease in leakage compare to pFadBA
3. Strength enhance compare to pFadBA

To compare our results, we divided our promoters into five groups based on their structure and design concept: (for detailed design of promoters, check parts)

Promoter pFadBA modification on consensus sequence

Though both pFadBA\_NTHU ([BBa\_K3040005](http://parts.igem.org/Part%3ABBa_K3040005)) and pFadBA\_NTHU ([BBa\_K3040006](http://parts.igem.org/Part%3ABBa_K3040006)) mutant have 1 to 2 fold increase in expression over native pFadBA as the concentration of fatty acid rises, pFadBA\_NTHU has greater performance than pFadBA\_NTHU mutant.

Thus, we could conclude that the modification of consensus sequence in -10 and -35 region could indeed improve the expression of the native promoter pFadBA, while the point mutation in advance had probably negative effects on its performance.

![](https://static.igem.org/mediawiki/2019/e/e8/T--NTHU_Taiwan--result8.png)

Figure 8. Relative protein expression of fatty acid promoter pfadBA-NTHU and pfadBA-NTHU mutant after 4 hours of induction under different fatty acid concentrations (n=3).

Hybrid pFadBA promoter with Lac and additional FadR binding sites

As we can see from fig.2, the expression of PFL1 ([BBa\_K3040011](http://parts.igem.org/Part%3ABBa_K3040011)) was greatly enhanced to 2 folds under IPTG induction. Yet, there is no difference between the inductions of IPTG in PFL2 ([BBa\_K3040012](http://parts.igem.org/Part%3ABBa_K3040012)). Therefore, PFL1 is proved to successfully express while PFL2 fails to function.

Here, we conjecture that two fadR binding sites might have been over repressing the expression of the promoter. Higher fatty acid induction might help, yet, the solubility of oil has limited us for further experiments.

![](https://static.igem.org/mediawiki/2019/2/27/T--NTHU_Taiwan--result9.png)

Figure 9. IPTG induction effects on protein expression of fatty acid pFadBA-Lac-2 (PFL2) after 16 hours of induction under 5mM fatty acid (n=3).

![](https://static.igem.org/mediawiki/2019/4/44/T--NTHU_Taiwan--result10.png)

Figure 10. Relative protein expression of fatty acid pFadBA-Lac-1 after 16 hours of induction under different fatty acid concentrations (n=3).

Replacement of pFadBA promoter with rrnD promoter, UP element, and additional FadR binding sites

The result turned out to be frustrated that none of them function. We had proposed some reasons that might lead to failure. UP element might have caused some conformational change of the promoter, there might be too many changes at the same time, or the fadR binding site might not function well with the rrnD promoter. Yet, we can’t figure out what has happened inside the cell. (Fad BS1-[BBa\_K3040008](http://parts.igem.org/Part%3ABBa_K3040008), Fad BS2-[BBa\_K3040009](http://parts.igem.org/Part%3ABBa_K3040009), Fad BS3-[BBa\_K3040010](http://parts.igem.org/Part%3ABBa_K30400010))

![](https://static.igem.org/mediawiki/2019/b/bc/T--NTHU_Taiwan--result11.png)

Figure 11. Protein expression of fatty acid promoter TP24-rrnD-fadRBS1, TP24-rrnD-fadRBS2, TP24-rrnD-fadRBS3 after 16 hours of induction under 5mM fatty acid (n=3)

Replacement of pFadBA promoter with pFadD promoter, Lac, and additional FadR binding sites

We can see clearly that pFadD\_Lac ([BBa\_K3040014](http://parts.igem.org/Part%3ABBa_K3040014)), pFadD ([BBa\_K3040013](http://parts.igem.org/Part%3ABBa_K3040013)) promoter with an additional lac binding site, has relatively low leakage and has 2-3 fold increase in expression as the fatty acid concentration rises. Though compared to the original promoter pFadBA, both pFadD and pFadD\_FadR ([BBa\_K3040015](http://parts.igem.org/Part%3ABBa_K3040015)) also has reduction in leakage, we assumed them not functioning since they show no changes in expression as the concentration of fatty acid rise.

![](https://static.igem.org/mediawiki/2019/a/a6/T--NTHU_Taiwan--result12.png)

Figure 12. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under 5mM fatty acid (n=3).

![](https://static.igem.org/mediawiki/2019/5/54/T--NTHU_Taiwan--result13.png)

Figure 13. Protein expression of fatty acid promoter pFadD, pFadD-FadR, pFadD–lac after 16 hours of induction under different fatty acid concentration (n=3).

Combination of gene of TesA protein with pFadBA promoter

The fold change of fluorescence after 16 hours 5mM oleic acid induction can come up to 9-fold. This has greatly improved the native strength of the promoter since it can only increase to about 2-fold. This modification helped us to control the strength of promoter more precisely compared to the native pfadBA since the induced-transcription range of the promoter has been broaden. (TesA\_FadBA is [BBa\_K3040007](http://parts.igem.org/Part%3ABBa_K3040007))

![](https://static.igem.org/mediawiki/2019/4/4e/T--NTHU_Taiwan--result14.png)

Figure 14. Protein expression of fatty acid promoter TesA-FadBA after 16 hours of induction under different fatty acid concentration (n=3).

We have successfully proposed five new functional biobricks ([BBa\_K3040005](http://parts.igem.org/Part%3ABBa_K3040005), [BBa\_K3040006](http://parts.igem.org/Part%3ABBa_K3040006), [BBa\_K3040007](http://parts.igem.org/Part%3ABBa_K3040007), [BBa\_K3040011](http://parts.igem.org/Part%3ABBa_K3040011), [BBa\_K3040014](http://parts.igem.org/Part%3ABBa_K3040014)) with improvements in the original pFadBA promoter in leakage reduction and strength enhancement.

# Conclusion

We demonstrated that our cold-adapted lipase and fatty acid promoter could be used to construct the Tunable-Temperature-Sensitive-System (TTSS). We proved that the catalytic activity changes over temperature and that the new promoters we designed had better expression.

#### **FOLLOW US**

[![](https://static.igem.org/mediawiki/2019/9/93/T--NTHU_Taiwan--Facebook.png)](https://www.facebook.com/NTHU.iGEM/)
[![](https://static.igem.org/mediawiki/2019/f/fe/T--NTHU_Taiwan--Instagram.png)](https://www.instagram.com/igem_tw_nthu/?fbclid=IwAR1DB5OWM9-rSEoxWU95uJwIUaos3lcTa07citdUi1U3kJuYHInHiW3IwtE)

#### **CONTACT US**

![](https://static.igem.org/mediawiki/2019/3/3e/T--NTHU_Taiwan--email.png)

nthuxigem@gmail.com