iGEM 2021 — Team NYCU-Taipei

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# Team:NYCU-Taipei/Notebook

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:NYCU-Taipei/Engineering

* [![](https://static.igem.org/mediawiki/2021/9/9d/T--NYCU-Taipei--logo.svg)](https://2021.igem.org/Team%3ANYCU-Taipei)
* [MEDAL CRITERIA](https://2021.igem.org/Team%3ANYCU-Taipei/Medal%20Criteria)
* NOTEBOOK
  + [experiments](https://2021.igem.org/Team%3ANYCU-Taipei/Experiments)
  + [progress](https://2021.igem.org/Team%3ANYCU-Taipei/Progress)
* HUMAN PRACTICES
  + [integrated HP](https://2021.igem.org/Team%3ANYCU-Taipei/ihp)
  + [human practices](https://2021.igem.org/Team%3ANYCU-Taipei/Human_Practices)
  + [collaborations](https://2021.igem.org/Team%3ANYCU-Taipei/Collaborations)
  + [education & communication](https://2021.igem.org/Team%3ANYCU-Taipei/Communication)
* [SAFETY](https://2021.igem.org/Team%3ANYCU-Taipei/Safety)
* [PARTS](https://2021.igem.org/Team%3ANYCU-Taipei/Parts)
* PROJECT
  + [description](https://2021.igem.org/Team%3ANYCU-Taipei/Description)
  + [design](https://2021.igem.org/Team%3ANYCU-Taipei/Design)
  + [device](https://2021.igem.org/Team%3ANYCU-Taipei/Device)
  + [model](https://2021.igem.org/Team%3ANYCU-Taipei/Model)
  + [software](https://2021.igem.org/Team%3ANYCU-Taipei/Software)
  + [results](https://2021.igem.org/Team%3ANYCU-Taipei/Results)
  + [implementation](https://2021.igem.org/Team%3ANYCU-Taipei/Implementation)
  + [engineering
    success](https://2021.igem.org/Team%3ANYCU-Taipei/Engineering)
* TEAM
  + [members](https://2021.igem.org/Team%3ANYCU-Taipei/Members)
  + [attributions](https://2021.igem.org/Team%3ANYCU-Taipei/Attributions)

Engineering

Engineering

![](https://static.igem.org/mediawiki/2021/4/4b/T--NYCU-Taipei--banner_engine.svg)

Engineering Success of Kill Switch

Design
With respect to biosafety issues, we designed a kill switch system operating in engineered bacteria. In our design, MazE-MazF toxin-antitoxin with TetR gene, TetR repressible promoter, pBad promoter and RNA thermometer are used to control the survival of bacteria. We can switch on the killing system through temperature and chemical inducement. Here is our design:

![](https://static.igem.org/mediawiki/2021/8/88/T--NYCU-Taipei--design_kill_1.png)

Fig .1 Our kill switch design 1.

Build
To check if the functions of each component in the design can regulate the expression of MazE as we expected, we built three constructs to evaluate the function. With Construct 2 & 3, the expression of MazE and TetR can be evaluated by testing the RFP intensity under different L-arabinose concentrations and temperatures. With the data collected from Construct 2 & 3, we can speculate the MazE expression in Construct 1.

![](https://static.igem.org/mediawiki/2021/5/5e/T--NYCU-Taipei--engineering_2%28new%29.png)

Fig. 2 Schematics of our biobrick construction

The MazE and MAzF have been amplified by PCR from E. coli MG1655 genome DNA.

![](https://static.igem.org/mediawiki/2021/2/2b/T--NYCU-Taipei--engineering_success_fig.3.png)

Fig. 3 Agarose electrophoresis in order to verify MazE and MazF genes. The purified DNA fragments had the expected length and were subsequently used for cloning.
Lane 1, 100bp ladder.
Lane 2, MazE PCR product.
Lane 3 and 4, MazF PCR product.
Land 5, 100bp ladder.

We successfully built our Construct 1 & 2 & 3. For more information, please visit our project  [Results](https://2021.igem.org/Team%3ANYCU-Taipei/Results) page.

![](https://static.igem.org/mediawiki/2021/e/e3/T--NYCU-Taipei--engineering_3.png)

Fig .4 Agarose electrophoresis in order to verify Construct 1 and Construct 2.
Lane 1, 100bp ladder. Lane 1’, partial of Life 1 kb Plus ladder.
Lane 2 and 3, Construct 1 digested with BamHI.
Lane 4, Construct 2 digested with KpnI and NdeI.

Test
To test how our kill switch design works, the expression of MazE and TetR can be evaluated by testing the RFP intensity under different L-arabinose concentrations and temperatures from Construct 2 & 3. We initially expected the bacteria with Construct 2 would show fluorescent red in LB medium (without L-arabinose). However, there was no red color in the colony of the bacteria, which means the RFP gene didn’t express. After using a photometer, the value of RFP intensity (RFP/OD600) is almost zero according to Fig.5, which is out of our expectation.

![](https://static.igem.org/mediawiki/2021/7/77/T--NYCU-Taipei--const2.png)

Fig. 5 After 24 hrs 37 celsius degree cultivation, the RFP intensity of construct 2.

![](https://static.igem.org/mediawiki/2021/6/6c/T--NYCU-Taipei--engineering_5.png)

Fig. 6 The Construct 2 state in LB medium (without L-arabinose)

Learn
To figure out why the RFP didn’t express, we tested the function of the pBad promoter from Construct 3, which has a RFP gene regulated by pBad. The result shows that the leakage of pBad is huge according to Fig.7, which means that the TetR in Const 2 would still be expressed when there is no L-arabinose.

![](https://static.igem.org/mediawiki/2021/1/11/T--NYCU-Taipei--engineering_6.png)

Fig. 7 The RFP intensity of construct 3(Pre B3) in different L-arabinose concentrations.

Then, we used modeling to analyze this system and we received the same answer. In an endogenous situation when there is no MazF in the bacteria, the expression of TetR wouldn’t be terminated, so the MazE won’t be expressed. Please see our  [project modeling](https://2021.igem.org/Team%3ANYCU-Taipei/Model) for more information.

![](https://static.igem.org/mediawiki/2021/a/a5/T--NYCU-Taipei--engineering_7.png)

Fig. 8 Modeling result: the concentration of MazE according to time and the amount of MazF change.

Re-Design
So we then try to construct the kill switch Design 2. In Design 2, we removed gene TetR and promoter pTetR and replaced them with a tandem promoter (a pBad with a constitutive promoter) which regulates the expression of MazF.

Here is our design:

![](https://static.igem.org/mediawiki/2021/a/ac/T--NYCU-Taipei--design_kill_2.png)

Fig. 9 Our kill switch design 2.

Re-Build
To check if the function of our tandem promoter in the design can regulate the expression of MazF as we expected, we evaluated our design by testing the RFP intensity. However, we have to choose whether the promoter pBad or the constitutive promoter should be put in the former. Therefore, we built three constructs to evaluate the function. Strategy1 & 2 was designed to find where to put the pBad promoter, while Strategy3 was designed to compare more constitutive promoters in the same family of J23106.

![](https://static.igem.org/mediawiki/2021/7/7c/T--NYCU-Taipei--engineering_9.png)

Fig. 10 Schematics of our biobrick construction

We successfully used Gibson assembly to build Pre C1, Pre C2, and other tandem promoters (pJ102, pJ118, pJ110, pJ116, and pJ113). Please visit our  [Results](https://2021.igem.org/Team%3ANYCU-Taipei/Results) page for more information.

![](https://static.igem.org/mediawiki/2021/b/b7/T--NYCU-Taipei--engineering_10.png)

Fig. 11 Agarose electrophoresis in order to verify the tandem promoters.
Lane 1, 100bp ladder. Lane 2, pJ116 with BamHI and PstI. Lane 3, pJ118 with BamHI and PstI. Lane 4, pJ113 with BamHI and PstI. Lane 5, pJ110 with BamHI and PstI. Lane 6, pJ102 with BamHI and PstI.

Re-Test
**The RFP expression of pBad located ahead (Pre C1) or behind (Pre C2) the constitutive promoter(J32106)**

![](https://static.igem.org/mediawiki/2021/b/ba/T--NYCU-Taipei--measurement_2.png)

Fig. 12 The RFP intensity of Pre C1 and Pre C2 after 24 hr cultivation.

We found that Pre C2 is hardly induced by L-arabinose and the endogenous expression rate of Pre C2 is very low. However, the expression rate of Pre C1 is great when L-arabinose is added. In conclusion, the result showed that if we put pBad in the former, the expression of tandem promoter will be more stable. This experiment is conducted by our collaboration partner, 2021 TAS\_Taipei. For more information about our collaboration, please visit our  [Collaborations](https://2021.igem.org/Team%3ANYCU-Taipei/Collaborations) page.

**The RFP expression of Pre C1 compared with other 5 tandem promoters in the same family**

![](https://static.igem.org/mediawiki/2021/5/5d/T--NYCU-Taipei--measurement_3.png)

Fig. 13 The RFP intensity of tandem promoters.

We found out that when the concentration of L-arabinose is 0 M, the order of the expression rate of these tandem promoters is the same as that of the constitutive promoter individually (BBa\_J23102＞ BBa\_J23118＞ BBa\_J23106＞ BBa\_J23110＞ BBa\_J23116>BBa\_J23113). Besides, when the concentration of L-arabinose rises from 0 M to 0.025 M, the expression of RFP under the regulation of pJ102, pJ118, and pJ110 decreases while others rise. Interestingly, among these six tandem promoters, only the Pre C1 will be induced by L-arabinose significantly.

Learn
First of all, we learned that tandem promoters with strong constitutive promoters like BBa\_J23102, BBa\_J23118 and BBa\_J23110 have a better expression rate when there is no L-arabinose. After doing paper research [1], we found out that the mechanism of L-arabinose regulating pBad is that L-arabinose will bind and change the pBad inhibitor araC into an inducer. We suggest that the formation of L-arabinose-araC-pBad complex may be a huge barrier for RNA polymerase to transcript. Even though L-arabinose may enhance the expression of pBad, the interference of the transcription by the L-arabinose-araC-pBad complex plays an more important role.

Secondly, although the constitutive promoter J23106 in Pre C1 is from the same constitutive promoter family as others, they have quite different RFP expression characteristics. Besides, the strength of J23106 ranks only in the middle among others. The only difference is that Pre C1 is constructed in pSB1A2, while the others are constructed in pSB1C3. We suggest it is the difference of plasmid backbones that lead to different results. Hence, we are going to transfer these tandem promoters into pSB1A2 to see if these tandem promoters will have different results.

In conclusion, from Design 1 to Design 2, we should choose the promoter before MazE wisely. There are some pBad tandem promoters whose RFP expression ability will drop as the concentration of L-arabinose increases such as pJ102, pJ118, pJ110. Here we take pJ102 for example. We can construct pJ102 before MazE while Pre C1 before MazF. Under this design, in general, the expression of MazE will be higher than MazF, since when the concentration of L-arabinose is zero, the RFP expression of pJ102 is better than that of Pre C1. When the L-arabinose concentration increases, the expression rate of pJ102 will drop while that of Pre C1 will increase simultaneously. As the MazF surpasses MazE, the killing system will turn on and achieve the kill switch.

![](https://static.igem.org/mediawiki/2021/8/83/T--NYCU-Taipei--measurement_4.png)

Fig. 14 Our final kill switch design

References

[1] Öztürk, S., Ergün, B. G., & Çalık, P. (2017). Double promoter expression systems for recombinant protein production by industrial microorganisms. Applied microbiology and biotechnology, 101(20), 7459–7475.

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# Team:NYCU-Taipei/Experiments

* [![](https://static.igem.org/mediawiki/2021/9/9d/T--NYCU-Taipei--logo.svg)](https://2021.igem.org/Team%3ANYCU-Taipei)
* [MEDAL CRITERIA](https://2021.igem.org/Team%3ANYCU-Taipei/Medal%20Criteria)
* NOTEBOOK
  + [experiments](https://2021.igem.org/Team%3ANYCU-Taipei/Experiments)
  + [progress](https://2021.igem.org/Team%3ANYCU-Taipei/Progress)
* HUMAN PRACTICES
  + [integrated HP](https://2021.igem.org/Team%3ANYCU-Taipei/ihp)
  + [human practices](https://2021.igem.org/Team%3ANYCU-Taipei/Human_Practices)
  + [collaborations](https://2021.igem.org/Team%3ANYCU-Taipei/Collaborations)
  + [education & communication](https://2021.igem.org/Team%3ANYCU-Taipei/Communication)
* [SAFETY](https://2021.igem.org/Team%3ANYCU-Taipei/Safety)
* [PARTS](https://2021.igem.org/Team%3ANYCU-Taipei/Parts)
* PROJECT
  + [description](https://2021.igem.org/Team%3ANYCU-Taipei/Description)
  + [design](https://2021.igem.org/Team%3ANYCU-Taipei/Design)
  + [device](https://2021.igem.org/Team%3ANYCU-Taipei/Device)
  + [model](https://2021.igem.org/Team%3ANYCU-Taipei/Model)
  + [software](https://2021.igem.org/Team%3ANYCU-Taipei/Software)
  + [results](https://2021.igem.org/Team%3ANYCU-Taipei/Results)
  + [implementation](https://2021.igem.org/Team%3ANYCU-Taipei/Implementation)
  + [engineering
    success](https://2021.igem.org/Team%3ANYCU-Taipei/Engineering)
* TEAM
  + [members](https://2021.igem.org/Team%3ANYCU-Taipei/Members)
  + [attributions](https://2021.igem.org/Team%3ANYCU-Taipei/Attributions)

experiments

Experiments

![](https://static.igem.org/mediawiki/2021/6/62/T--NYCU-Taipei--banner_exp.svg)

Check out our experiments and protocols!

Plasmaid Extraction

Preparation

pET-21a in DH5-alpha liquid culture harvest for plasmid extraction

1.  LB: 5ml

2.  Amp(100 mg/ml): 2.5ul

3.  Incubator for 18-19 hour

4.  To pick a single colony, you need to put it on a plate as well. You can also use a tip to do it. Burn the tip a little bit in the alcohol lamp. Use a tip and streak out on the plate, after throw the entire tip into the 15cc tube.

Steps

1.  Harvest

A.  Transfer 1.5ml of cultured bacteria cells to a 1.5 ml Eppendorf.

B.  Centrifuge 14000-16000 x g for 1 min to form cell pellet.

C.  Discard the supernatant.

D.  Repeat the harvest steps using the same Eppendorf. (We use 1 ml one time and repeat 4 times)

2.  Resuspension

A.  Add 200 ul of PD1 buffer(make sure Rnase A was added) to the Eppendorf.

B.  Resuspend the cell pellet completely by vortex or pipette until all traces of the cell pellet have been dissolved. (PD1 is in the 4 degree fridge)

3.  Cell lysis

A.  Add 200 ul of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA.

B.  Let stand at room temperature for at least 2 min. Do not exceed 5 mins.

4.  Neutralization

A.  Add 300 ul of PD3 buffer then mix immediately by inverting the tubes 10 times ( invert it very slowly!).

B.  Centrifuge at 14000-16000 xg for 3 min at room temperature. If using over 5 ml bacteria cells, centrifuge at 16000-20000 xg for 5-8 min.

C.  While waiting, place a PDH Column in a 2 ml collection tube.

5.  DNA binding

A.  Transfer all the supernatant to the PDH column.

B.  Centrifuge at 14000-16000 x g for 30 sec at room temperature then discard the flow-through.

C.  Place the PDH column back in the 2 ml collection tube.

6.  Wash

A.  Add 400 ul of W1 buffer into the PDH column. Centrifuge at 14000-16000 x g for 30 sec.

B.  Discard the flow-through then place the PDH column back in the 2 ml Collection tube.

C.  Add 600 ul of Wash buffer into the PDH column.

D.  Centrifuge at 14000-16000 x g for 30 sec at room temp.

E.  Discard the flow-through then place the PDH column back in the 2ml collection tube.

F.  Centrifuge at 14-16000 x g for 3 min at room temp. to dry the column matrix.

G.  Transfer the dried PDH column to a new 1.5 ml Eppendorf.

7.  Elution

A.  Add 50ul (40 ul for higher concentration) of Elution buffer into the Center of the matrix.

B.  Let stand for at least 2 min to allow Elution Buffer to be completely absorbed.

C.  Centrifuge at 14000-16000 x g 2min at room temp to elute the purified DNA.

Enzyme Digestion

Preparation

Total Volume(20ul)

| components | amount per reaction (ul) |
| --- | --- |
| DNA(1 ug) |  |
| 10 x buffer | 2 |
| Enzyme | 0.8-1 |
| Add ddH2O up to 20 uL | |

| components | amount per reaction (ul) |
| --- | --- |
| DNA(1 ug) |  |
| 10 x buffer | 2 |
| Enzyme1 | 0.8 |
| Enzyme2 | 0.8 |
| Add ddH2O up to 20 uL | |

Steps

1.  Prepare a clean PCR tube to undergo this experiment

2.  Add the components in following order: ddH20, DNA(4°C), 10x buffer(-20°C), enzyme(-20°C)

3.  After adding, pipetting the tube to mix it well

4.  Put the tube into the incubator or water bath(37°C) for 1 hr

Electrophoresis

Preparation

1.  Agarose

A.  Big: 50 ml TAE+ 0.5g agarose(1%) + 1g agarose(2%)

B.  Small: 30 ml TAE+ 0.3g agarose(1%) + 0.6g agarose(2%)

2.  Safety dye: 0.5ul/ 10ml
3.  DNA marker: 5-8 marker

A.  100 bp marker

B.  1 kb marker

4.  Loading dye(6x): ⅕ volume of the sample

Steps

1.  Dissolve the agarose (heat by microwave oven)

2.  Wait to cool a little (you could touch it with your hand to feel the temperature) and add safety dye into it

3.  Pour agarose onto the gel tray

4.  Load sample first, then the marker

5.  Run 70 V, at least 30 min

Gel Extraction

Steps

1.  Gel dissociation

A.  Prepare a 55-60°C water bath. At the same time preheat Elution buffer in the Dry bath(69°C)

B.  Transfer the gel slice to a 1.5 ml Eppendorf

C.  Add 500 ul of gel buffer to the sample then mix by vortex.

D.  Incubate at 55-60°C for 10-15 min to ensure the gel slice is completely dissolved.

E.  During incubation, invert the tube every 2-3 min.

2.  DNA binding

A.  Place a DFH Column in a 2 ml Collection tube.

B.  (Wait to cool down to room temp), spin down and transfer 800ul of the sample mixture to the DFH column.

C.  Centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and put the DFH column back in the 2 ml collection tube.
(If the sample mixture is more than 800 ul, repeat the DNA binding step)

3.  Wash

A.  Add 400 ul of W1 buffer into the DFH column.

B.  Centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and place the DFH column back to the collection tube.

C.  Add 600 ul of Wash buffer(make sure ethanol was added) into the DFH column.

D.  Let stand for 1 min at room temp. Centrifuge at 14-16000 x g for 30 sec then discard the flow-through.

E.  Place the DFH column back in the 2 ml collection tube.

F.  Centrifuge at 14-16000 x g for 3 min to dry the matrix.

4.  Elution

A.  Transfer the dried DFH column to a new 1.5 ml Eppendorf.

B.  Add 20-50 ul(25 ul) of pre-heated Elution buffer into the center of the column.

C.  Let stand for at least 2 min (put 5 min).

D.  Centrifuge at 14-16000 x g for 2 min.

Ligation

Preparation

Total Volume(20ul)

| components | amount per reaction (ul) |
| --- | --- |
| T4 DNA ligase buffer | 2 |
| Vector DNA (? bp) |  |
| Insert DNA (? bp) | 2 |
| T4 DNA ligase | 1 |
| Add ddH2O up to 20 uL | |

Calculation

1.  Usually, 1 : 1 or 1 : 3 molar ratio of vector : insert works

Steps

1.  Add the components in following order: water, DNA, T4 DNA ligase buffer, T4 DNA ligase

2.  Use an Eppendorf or PCR tube

3.  Let it stand over 1 hour to let it ligase. (put it in 4 degree if you want to put it overnight)

4.  The next time if you want to transform this product, remember to inactivate (goal : inactivate the ligase, 65 degree 10 min)

5.  Cool down then transform

Transformation

Steps

1.  Prepare 42°C water bath

2.  Prepare the ice with water. Put the sterile Eppendorf on tubes on ice.

3.  Make sure the vector and insert DNA fragment are ready.

4.  Take competent cells out of -80°C and thaw on ice immediately.

5.  Mix 1-5 ul of DNA into 30-35 ul of competent cells. Gently mix by flicking the bottom of the tube.(Do not vortex!)

6.  Incubate the competent cell and DNA mixture on ice for 20-30 mins

7.  Heat shock 42°C water bath for 60 sec.

8.  Put the tubes back on ice for 2 min.

9.  Add 250-1000ul LB(without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min

10.  Place the transformation onto LB agar plate with selective marker. (We add 100 ul in every plate)

11.  Put the plate for 14~20 hr in the incubator.

Fibrin Plate

Preparation

(2 petri dishes : total volume of 16ml solution)

1.  Tris-HCl: 20 mmol/L Tris-HCl buffer (pH 8.0)

2.  Agar solution: 0.128g agar in 10 ml Tris-HCl, 50°C

3.  Fibrinogen: 0.032g fibrinogen in 6 ml 0.9% NaCl, 37°C(note: the final concentration comes to 0.8% agar solution, 0.2% fibrinogen)

4.  Thrombin (58.8U/ml): 55ul of thrombin solution in 500 ul of 0.9% NaCl

5.  Filter paper: 《ADVANTEC》NO.1 Qualitative Filter Paper, NO.1

6.  Petri dish: 5.5 cm in diameter

Steps

1.  Prepare a 50°C and 37°C water bath

2.  Prepare the fibrinogen solution and put it in the 37°C water bath for 15 min to dissolve fibrinogen

3.  Dissolve agar and let it cool down to 50°C

4.  Mix both solution and pour into 2 petri dishes (5.5 cm in diameter)

5.  Let stand for 30 min

6.  Add thrombin solution and put a filter paper on top

7.  Let stand for another 30 min to form fibrin clot

8.  Discard the filter paper and punch holes (3 mm of diameter)

9.  Add 10 ul of sample and put it in 37°C incubator for 24 hours to see the lytic circle

Freeze Drying

Preparation

1.  Protective medium: Sucrose (10%), trehalose (5%), Skimmed milk (10%), Ascorbic acid (0.1~0.15%) in PBS solution

2.  0.9% NaCl

3.  PBS: 1x PBS (pH =7.4)

Freeze-dryer

1.  BENCHTOP 2K , VIRTIS

Steps (before freeze)

1.  Bacteria harvest

A.  5 ml LB medium in one 15 ml tube, put it in the incubator to shake for 16 hours

2.  Label 1.5ml Eppendorf

3.  Form cell pellet and load with desired medium

A.  Load 500 ul of bacteria medium in each Eppendorf

B.  Centrifuge for 16000 x g for 1 min to form cell pellet

C.  Discard the supernatant

D.  Load 500 ul in each Eppendorf with desired medium (protective medium or 0.9% NaCl)

4.  Freeze in -80 degree

A.  Put the Eppendorf in -80 degree for at least 5 hours

5.  Transfer to the freeze-dryer

A.  Take out from the fridge and change the lid with holes on to

B.  After changing the lid, put it into the freeze-dryer as fast as possible. (Do not let it melt.)

C.  Freeze-drying condition : 129mTorr (=17.2 Pa), -85.5 degree, time: 24 hours

6.  Count fresh cells before freeze

A.  Label 5 Eppendorf, and put 900 ul PBS solution in each Eppendorf

B.  Take 100 ul of the sample solution and dilute to 10-1~10-5

C.  Use the plate counting method to calculate viable cells

D.  Add 2.5 ul of each diluted sample and spread on the plate

E.  Put in the 37°C incubator and count them after 24 hours

Steps (after freeze)

1.  Take out from the freeze-dryer

A.  Change the lid and use parafilm for longer storage

B.  Put the samples at desired condition and time

C.  Remember to wash the lid with holes and let it dry to reuse.

2.  Rehydration

A.  Add 500 ul of PBS solution to rehydrate the sample

3.  Count fresh cells after freeze

A.  Label 5 Eppendorf, and put 900 ul PBS solution in each Eppendorf

B.  Take 100 ul of the sample solution and dilute 10-1~10-5

C.  Use the plate counting method to calculate viable cells

D.  Add 2.5 ul of each diluted sample and spread on the plate

E.  Put in the 37°C incubator and count them after 24 hours

Competent Cell

DAY-1 Buffer preparation & bottle sterilization

1.  SOB medium (for 250 mL SOB prepared in flask or Duran bottle)

| components | amount per reaction (gram) |
| --- | --- |
| Yeast extract | 1.25 |
| Tryptone | 5 |
| NaCl | 0.15 |
| KCl | 0.05 |
| MgSO4 | 0.6 |
| Add ddH2O up to 250 mL | |

> Autoclave 121°C, 20 min
> Store at 4°C

2.  CCMB80 transformation buffer (for 1L CCMB80 in flask Duran bottle)

| components | amount per reaction (gram) |
| --- | --- |
| KOAc | 0.98 (10mM) |
| CaCl2 • 2H2O | 11.8 (80mM) |
| MnCl2 • 4H2O | 4 (20mM) |
| MgCl2 • 6H2O | 2 (10mM) |
| Glycerol | 100 (10%) |
| Add ddH2O up to 1L | |

> Adjust pH to 6.4 with HCl
> Sterile filtered
> Store at 4°C

DAY-2 Preculture

Pick single colony to 3 mL SOB broth, incubate under 37°C, 200 rpm, overnight.

DAY-3

1.  Transfer 100X preculture to new SOB broth inside a flask (5X larger than SOB volume),
culture under 37°C, 200 rpm to OD600 = 0.35~0.4

2.  Prepare

A.  A full bucket of ice.

B.  CCMB80 buffer under 4°C.

C.  (optional) Liquid nitrogen in ice bucket.

Every step should be done ON ICE

3.  Following step 1, transfer the cell into 50 mL PP tube. (No more than 25 mL cell in
each.) Incubate on ice (covered by ice) for 10 min.

4.  Centrifuge 1600~2500xg, 10 min. under 4°C.

5.  Decant supernatant gently, and stand the tubes in an inverted position for 1 min. to allow
the last traces of medium to drain away.

6.  (Per 25 mL cell) resuspend with 10 mL chilled CCMB80 by vortex.

7.  Centrifuge 1600~2500xg, 10 min. under 4°C.

8.  (Per 25 mL cell) resuspend with 1 mL chilled CCMB80 by vortex.

9.  Dispend per 100 μL of cell into a new microcentrifuge tube, and then put it immediately
back to ice or liquid nitrogen.

10.  Freeze at -80°C.

Transformation of Competent Cells

1.  Pipet DNA (1 μL of miniprep-grade plasmid DNA, or up to 9 μL of ligation reaction)
into an Eppendorf tube and place this on ice. Place an aliquot of competent cells on ice
and leave until thawed (~10 minutes).

2.  Add 100 μL of competent cells to the DNA, and incubate for 20-30 minutes on ice.

3.  Heat shock in a water bath at 42°C for 30~60 seconds (if unsure, use 60 seconds) and
immediately place on ice.

4.  Add 1 ml of LB medium and shake at 37°C for 30-60 minutes. If you transform
miniprep DNA, plate out 100 μL of the cells onto a suitable selective plate. If you
transform ligation reactions, spin the cells at 3000 rpm for 5 min. Pour off most of the
medium, leaving 100-200 μL in the tube. Resuspend the cells in this remaining medium
and plate everything onto suitable selective plates.

PCR

Preparation

Total Volume(20ul)

| components | Sample (ul) | Control (ul) |
| --- | --- | --- |
| 10X KOD buffer | 2.5 | 2.5 |
| Mg2+ | 1 | 1 |
| DNA template | 50 ng (if using gDNA template, increase the amount of the template) | |
| dNTP (2mM) | 2.5 | 2.5 |
| primer pair | 1.5 | 1.5 |
| KOD polymerase | 0.5 | 0.5 |
| Add ddH2O up to 25 uL | | |

set up PCR machine

1.  HOLD: 94°C, 1 min.
2.  Cycling (\*30)
3.  Denaturing - 94°C, 20 sec
4.  Annealing - 55°C, 30 sec. (or other appropriate annealing temperature)
5.  Extending - 68°C, 1 min. (adjust the time length by the size of the predicted
product)
6.  HOLD: 68°C, 10 min.
7.  HOLD: 20°C, 15 min.
8.  (if we need touchdown, touchdown 10 cycle, then choose an appropriate
annealing temperature for 25 cycle)

RFP intensity measurement

Steps

1.  Making L-arabinose solution

A.  Weigh 6 g L-arabinose and add ddH2O to form a 20 ml solution(2M).
B.  Draw the L-arabinose solution into a 20cc (or 50 cc) syringe.
C.  Place a 0.22um filter onto the syringe. Force the solution through the syringe into a sterile 50 ml centrifuge tube.

2.  Inoculate a liquid culture of the provided bacteria

A.  Prepare a sterile culture tube. Label properly.
B.  Put an inoculating loop into the flame until it is red hot, and wait for a few seconds to allow it to cool.
C.  Use the sterile inoculating loop to pick a bit of the bacteria on the plate and stir into the culture tube.
D.  Recap the tube and incubate it in a shaking incubator overnight.

3.  Test the concentration of cells by ODE testing. Record the OD600 value and the estimated concentration of cells

See Appendix 1.

4.  Inoculate liquid cultures with different concentrations of L-arabinose

A.  Take five 15 ml centrifuge tubes or culture tubes, and label them as 0.2M, 0.1M, 0.05M, 0.025M, 0M(negative control), respectively.
B.  Transfer 5 ml sterile LB medium into each tube.
C.  Add 2 μl of 1000x (100 mg/ml) ampicillin stock solution into each tube. Mix thoroughly. (Final concentration: 100 μg/ml).
D.  Add 222 ul 2M L-arabinose solution into the 0.2M centrifuge centrifuge tube.
E.  Add 105 ul 2M L-arabinose solution into the 0.1M centrifuge centrifuge tube.
F.  Add 50 ul 2M L-arabinose solution into the 0.05M centrifuge centrifuge tube.
G.  Add 25 ul 2M L-arabinose solution into the 0.025M centrifuge centrifuge tube.
H.  Add 5 ul of the bacterial liquid culture into each tube. Mix thoroughly.
I.  Test the concentration of cells of each culture by ODE testing according to the Appendix 1.
J.  Measure RFP intensity of each culture according to Appendix 2.
K.  Put the tubes into a shaking incubator , temperature at 37 °C and shaking speed at 200 RPM.
L.  Test for the concentration of cells and RFP intensity for each tubes according to Appendices 1 & 2 after 12, 24,36 ,and 48hr of incubation.

5.  Retrieve the data from the computer, correct for the measurements for the OD600 and subtract the autofluorescence from the control sample of all the other samples

Appendices

1.  Testing the concentration of cells by ODE testing

A.  setting parameter in the plate reader

i.  Measurement: Absorbance
ii.  Set Temperature: 37°C
iii.  Shake: Medium, 0:30 (MM:SS)
iv.  Multiple Reader per well: 3x3(square)

A.  Set the absorption wavelength to 600 nm and make sure the measuring unit is set to Abs (or A).
B.  Transfer 40ul LB blank and 160 ul PBS buffer into 96 well plate(with clear bottom and flat white sides).

C.  Set the absorption wavelength to 600 nm and make sure the measuring unit is set to Abs (or A).
D.  Transfer 40ul LB blank and 160 ul PBS buffer into 96 well plate(with clear bottom and flat white sides).

E.  Set the absorption wavelength to 600 nm and make sure the measuring unit is set to Abs (or A).

2.  Test the RFP intensity of each culture

A.  Load 160 µl PBS buffer in one well of the 96-well plate (with clear bottom and black sides) and add 40 µl of LB blank .

B.  Load 160 µl PBS buffer in the wells of the 96-well plate (with clear bottom and black sides) and add 40 µl of bacteria culture .
C.  setting parameter in the plate reader

i.  Measurement:Fluorescence Intensity
ii.  Set Temperature: 37°C
iii.  Shake: Medium, 6:00 (MM:SS)
iv.  Excitation: 555,Emission: 585
v.  Optics: Bottom,Gain: 100
vi.  Multiple Reader per well: 3x3(square)
vii.  Read Speed: Normal, Delay: 100 msec, Measurements/Data Point: 10

D.  Start the plate reader machine.

6x Histag purification

Growth of expression cultures

1.  Inoculate 10 ml of LB medium containing the appropriate antibiotics with a fresh bacterial colony harboring the expression plasmid. Grow at 37°C overnight.
2.  Dilute the non-induced overnight culture 1:50 (e.g., inoculate 100 ml LB medium with 2 ml overnight culture) with fresh LB medium containing the appropriate antibiotics. Grow at 37°C with vigorous shaking until the OD600 reaches 0.6~0.8.
3.  Add IPTG to a final concentration of 1 mM and grow the culture at 37°C with vigorous shaking for 4 hours. (In Nattokinase expression, we use 20°C for 16 hr). IPTG (Isopropyl β-d-1-thiogalactopyranoside): MW 238.30.
4.  Harvest the cells by centrifugation at 8000 rpm for 15 min.

Preparation

(for native condition purification)
1.  Lysis Buffer (NPI-10): 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0.
2.  Wash Buffer (NPI-20): 50 mM NaH2PO4, 300 mM NaCl, 20 mM imidazole, pH 8.0.
3.  Elute Buffer (NPI-500): 50 mM NaH2PO4, 300 mM NaCl, 500 mM imidazole, pH 8.0.
4.  Benzonase® Endonuclease 25 U/μl.
5.  Lysozyme stock solution 10 mg/ml in water. Sterilize by filtration and store in aliquots at –20°C.

Purification

(native condition)
1.  Resuspend a pellet derived from 5 ml cell culture volume in 630 μl Lysis Buffer (NPI-10). Add 70 μl Lysozyme Stock Solution (10 mg/ml) and add 3 Units/ml culture volume Benzonase® Nuclease.

A.  If the tagged protein does not bind under these conditions, the amount of imidazole should be reduced to 1-5 mM.

2.  Incubate on ice for 15–30 min.
3.  Centrifuge lysate at 12,000 x g for 15–30 min at 4°C. Collect supernatant.

A.  Save 20 ul of the cleared lysate for SDS-PAGE analysis.

4.  Equilibrate the Ni-NTA spin column with 600 ul Buffer NPI-10. Centrifuge for 2 min at 890 x g.

A.  The spin column should be centrifuged with an open lid to ensure that the centrifugation step is completed after 2 min.

5.  Load up to 600 ul of the cleared lysate containing 6xHis-tagged protein onto the pre-equilibrated Ni-NTA column. Centrifuge for 5 min at 270 x g.

A.  To ensure efficient binding it is important not to exceed 270 x g with centrifugation.
B.  For very concentrated cell lysate, it may be necessary to extend the centrifugation time to 5 or 10 min at 270 x g.
C.  Save the flow-through for analysis by SDS-PAGE to check binding efficiency.

6.  Wash the Ni-NTA spin column twice with 600 ul Buffer NPI-20. Centrifuge for 2 min at 890 x g.

A.  For very low expression levels or highly concentrated lysates, 3 wash steps may be required to achieve high purity.
B.  Save the flow-through (wash fractions) for analysis by SDS-PAGE to check the stringency of the wash conditions.

7.  Elute the protein twice with 300 ul Buffer NPI-500. Centrifuge for 2 min at 890 x g, and collect the elute.

A.  If higher protein concentrations are desired, do not combine the eluates or, alternatively, elute 100-200 ul aliquots.

SDS page for protein expression

Sampling

1.  For liquid culture: incubate bacteria in LB with corresponding antibiotic to OD600 = 0.4~0.6. Take 1 mL to microcentrifuge tube and centrifuge for 16,000 g for 1 minute.
2.  For protein induction after liquid culture: Take 1 mL of inducted bacterial medium and measure OD600. Centrifuge for 16,000 g for 1 minute in microcentrifuge tube.
3.  To make the band in SDS page clear, samples have to be dilution. For example,

if OD600 = 1.537, add 153.7 λ of ddH2O for dilution.
Diluted sample 30 λ + 10 λ 4X dye, cook 100°C for 10 minutes

SDS-PAGE

1.  Prepare gel

A.  Set up the glass plates and put them onto the green clamp.

i.  Nudge the set to the end of the loading stand and fix it with the clamp above.

B.  Loading with ddH2O first for 10 secs to check if the gel stands have been firmly set. Pour out ddH2O and dry up glass plates with paper towel.
C.  Prepare separating gel in a tube.
D.  Prepare separating gel in a tube.

| SDS-Page Separating Gel (for 1mm page) | | | | | | | |
| --- | --- | --- | --- | --- | --- | --- | --- |
|  | 6% | 8% | 10% | 12% | 15% | 18% | 20% |
| ddH2O (ml) | 2.6 | 2.3 | 1.9 | 1.6 | 1.1 | 0.6 | 0.7 |
| 1.5M Tris pH8.8 (ml) | 1.3 | | | | | | |
| 10% SDS (ml) | 0.05 | | | | | | |
| 10% APS (ml) | 0.05 | | | | | | |
| 30% Acrylamide 29:1 (ml) | 1 | 1.3 | 1.7 | 2 | 2.5 | 3 | 3.33 |
| TEMED (ml) | 0.002 | | | | | | |
| Total volume (ml) | 5.02 | | | | | | |

E.  Pipetting and stir with p1000, suck up about 4.5 ml solution and load between the glasses to 2 cm below the green stick.

i.  Load 70% EtOH between the glasses to full.
ii.  Wait for 20 mins for separating gel to dried up.
iii.  \* Notice that APS and TEMED will solidify the gel. Add them the LAST.

F.  Prepare 5% stacking gel in a tube.

| SDS-Page Stacking Gel (5%) | | |
| --- | --- | --- |
|  | 2ml | 4ml |
| ddH2O (ml) | 1.4 | 2.8 |
| 1.5M Tris pH6.8 (ml) | 0.25 | 0.5 |
| 10% SDS (ml) | 0.02 | 0.04 |
| 10% APS (ml) | 0.02 | 0.04 |
| 30% Acrylamide 29:1 (ml) | 0.33 | 0.66 |
| TEMED (ml) | 0.002 | 0.004 |
| Total volume (ml) | 2.022 | 4.044 |

G.  Pipetting and stir with p1000, suck up about 2 ml solution and load between the glasses to full.

i.  Put on the comb (Don’t make bubbles!)
ii.  Wait for 20 mins.

H.  Unload the glasses with gel (comb remained), store in water inside a box under 4°C if needed.

2.  Running SDS page

A.  Prepare 10X tris-Glycine SDS running buffer (700 ml).

i.  70 ml SDS stock buffer + 630 ml ddH₂0, mix well.

B.  Set up the SDS box (with glasses on both sides).

i.  Small glass facing inside.
ii.  Plug into the bottom with a tilt.
iii.  Push the glasses upwards to the block.
iiii.  Push the handles inwards to fix the glasses.

C.  Load samples: prestained protein marker 3~5 λ, samples 5~10 λ.
D.  Running: stacking gel 70~90V for 30 minutes (standard), separating gel 120V 1.5 for hours.

3.  Staining and destaining of SDS page

A.  Remove the glass plates from the gel carefully in the water.
B.  Stain the gel with coomassie blue for 20 minutes on the shaker (60 rpm).
C.  Destain with destaining solution (50% ddH₂0, 40% Methanol, 10% Acetic acid) or in water overnight on the shaker (60 rpm).

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:NYCU-Taipei/Results

* [![](https://static.igem.org/mediawiki/2021/9/9d/T--NYCU-Taipei--logo.svg)](https://2021.igem.org/Team%3ANYCU-Taipei)
* [MEDAL CRITERIA](https://2021.igem.org/Team%3ANYCU-Taipei/Medal%20Criteria)
* NOTEBOOK
  + [experiments](https://2021.igem.org/Team%3ANYCU-Taipei/Experiments)
  + [progress](https://2021.igem.org/Team%3ANYCU-Taipei/Progress)
* HUMAN PRACTICES
  + [integrated HP](https://2021.igem.org/Team%3ANYCU-Taipei/ihp)
  + [human practices](https://2021.igem.org/Team%3ANYCU-Taipei/Human_Practices)
  + [collaborations](https://2021.igem.org/Team%3ANYCU-Taipei/Collaborations)
  + [education & communication](https://2021.igem.org/Team%3ANYCU-Taipei/Communication)
* [SAFETY](https://2021.igem.org/Team%3ANYCU-Taipei/Safety)
* [PARTS](https://2021.igem.org/Team%3ANYCU-Taipei/Parts)
* PROJECT
  + [description](https://2021.igem.org/Team%3ANYCU-Taipei/Description)
  + [design](https://2021.igem.org/Team%3ANYCU-Taipei/Design)
  + [device](https://2021.igem.org/Team%3ANYCU-Taipei/Device)
  + [model](https://2021.igem.org/Team%3ANYCU-Taipei/Model)
  + [software](https://2021.igem.org/Team%3ANYCU-Taipei/Software)
  + [results](https://2021.igem.org/Team%3ANYCU-Taipei/Results)
  + [implementation](https://2021.igem.org/Team%3ANYCU-Taipei/Implementation)
  + [engineering
    success](https://2021.igem.org/Team%3ANYCU-Taipei/Engineering)
* TEAM
  + [members](https://2021.igem.org/Team%3ANYCU-Taipei/Members)
  + [attributions](https://2021.igem.org/Team%3ANYCU-Taipei/Attributions)

results

Results

![](https://static.igem.org/mediawiki/2021/8/88/T--NYCU-Taipei--banner_results.svg)

Nattokinase Production

Construct Plasmid for Nattokinase Expression
First of all, we extracted the genomic DNA from *Bacillus subtilis*  natto HS01(a generous gift from Dr. Hsueh).

![](https://static.igem.org/mediawiki/2021/4/4d/T--NYCU-Taipei--results_NK_1.png)

Fig 1. The result of *Bacillus subtilis* genomic DNA extraction.

Then, we successfully cloned *aprN*  from gDNA of *Bacillus subtilis*  natto HS01 by PCR. At the same time, we added XbaI and XhoI cutting site in front of *aprN*  and behind *aprN* , respectively.

![](https://static.igem.org/mediawiki/2021/f/f5/T--NYCU-Taipei--results_NK_2.png)

Fig 2. The result of nattokinase cloning.

After ligating *aprN*  PCR product to pET-21a(+), we used NotI/ PstI enzyme digestion to test our construction because there is a NotI cutting site in *aprN* .

![](https://static.igem.org/mediawiki/2021/0/05/T--NYCU-Taipei--results_NK_3.png)

Fig 3. Prediction of plasmid-NK digestion(NotI/PstI).

![](https://static.igem.org/mediawiki/2021/e/e8/T--NYCU-Taipei--results_NK_4.png)

Fig 4. The result of plasmid-NK confirmation.

Nattokinase Induction and Test
To know whether *E. coli*  can produce Nattokinase, we transformed the successfully made plasmid-NK construct into *E. coli*  BL21 cell and treated them with 0.7 mM of IPTG after OD600 of the liquid culture reached 0.6, and measured OD600 every 4 hr throughout the 16 hr induction procedure at 20°C.[1, 2] Next, we checked Nattokinase expression by running SDS-PAGE. We can see there are bands presenting between 25~35 kDa after IPTG induction, and Nattokinase should be around 28 kDa. Thus, there are chances that Nattokinase is successfully expressed.

![](https://static.igem.org/mediawiki/2021/2/24/T--NYCU-Taipei--SDS_PAGE_correct_result-1.png)

Fig 5. The SDS-PAGE result of Nattokinase induction.

However, in our fibrin plate assay, no lytic circle was observed after dropping 10 ul medium onto it, which means no Nattokinase activity was detected. This may be due to the expression level in medium was too low to be detected, or our Nattokinase lost its activity somehow. Also, our SDS-PAGE results of Nattokinase purification were not as expected, too.

![](https://static.igem.org/mediawiki/2021/d/d2/T--NYCU-Taipei--results_NK_6.png)

Fig 6. The result of fibrin plate test.

Due to the limitation of our time, we are still in the troubleshooting process. Here are some experiments we plan to do in the near future:

**Gene sequencing**
 Confirm the sequence of *aprN*  sequence and 6xHis-tag are as expected. Make sure our Nattokinase will be produced with 6xHis-tag at the C-terminal.

**Western blot**
 Check the presence of Nattokinase with 6xHis-tag by 6xHis-tag antibody.

**Adjust purification protocol and expression condition**
 In the reference, when E. coli expresses Nattokinase at higher temperature (e.g 37°C, 30°C), Nattokinase will prefer to present at about 40 kDa. On the other hand, while expressing at lower temperature (e.g. 15°C, 20°C), it will present at about 28 kDa (mature protein) instead.[2] So, we are going to check the optimized temperature for our Nattokinase secretion. Also, we may try to use different concentrations of IPTG to induce the expression, add PMSF to avoid unwanted protein degradation (however, this way will permanently inactivate Nattokinase), and so on.

**Retest activity**
 If our Nattokinase is secreted into medium successfully, we can measure its activity by fibrin plate assay and spectrophotometric method to know how our Nattokinase expression level and activity is.

References

[1] Liang X, Jia S, Sun Y, Chen M, Chen X, Zhong J, Huan L. Secretory expression of nattokinase from Bacillus subtilis YF38 in Escherichia coli. Mol Biotechnol. 2007 Nov;37(3):187-94. doi: 10.1007/s12033-007-0060-y. Epub 2007 Jul 17. PMID: 17952663.

[2] Jeong SJ, Cho KM, Lee CK, Kim GM, Shin JH, Kim JS, Kim JH. Overexpression of aprE2, a fibrinolytic enzyme gene from Bacillus subtilis CH3-5, in Escherichia coli and the properties of AprE2. J Microbiol Biotechnol. 2014 Jul;24(7):969-78. doi: 10.4014/jmb.1401.01034. PMID: 24743573.

Kill switch result

For more information about our kill switch part, please visit our [Parts](https://2021.igem.org/Team%3ANYCU-Taipei/Parts) page.

Result of Design 1

**Analysis and Validation of the constructed strains**
The RFP expression of Construct 3
We used RFP(E1010) to monitor the expression of TetR, so that we can figure out how L-arabinose will influence the expression of TetR in our Design 1.

![](https://static.igem.org/mediawiki/2021/f/fb/T--NYCU-Taipei--kilksw_result_1.png)

Fig 1. The RFP intensity of construct 3 (Pre B3) from 0 hr~ 24 hrs in different L-arabinose concentrations.

![](https://static.igem.org/mediawiki/2021/3/34/T--NYCU-Taipei--kilksw_result_2.png)

Fig 2. After 24 hrs cultivation, the RFP intensity of construct 3 in different L-arabinose concentrations

The RFP expression of Construct 2

![](https://static.igem.org/mediawiki/2021/b/be/T--NYCU-Taipei--kilksw_result_3.png)

Fig 3. The RFP intensity of construct 2 in different L-arabinose concentrations and temperatures.

![](https://static.igem.org/mediawiki/2021/8/8b/T--NYCU-Taipei--kilksw_result_4.png)

Fig 4. After 24 hrs cultivation, the RFP intensity of construct 2 in different temperatures.

The comparison between Construct 2 and Construct 3

![](https://static.igem.org/mediawiki/2021/d/de/T--NYCU-Taipei--kilksw_result_5.png)

Fig 5. The RFP intensity of Construct 2 and Construct 3

**Discussion**
1. The RFP wasn’t expressed under 23°C:
According to Fig.3 & 4, the expression of RFP in Construct 2 is nearly zero at 23°C. According to the mechanism of thermometer RBS, the structure of the thermometer RBS will change under 23°C so the RFP gene behind it couldn’t express[1]. That is to say, the MazE won’t be expressed in Construct 1. Hence, we suppose that our kill switch design will work when the LBP escapes from the human body, achieving safety in the environment.

2. The mechanism of pBad promoter interacting with L-arabinose is much more complicated than what we thought:
According to the RFP expression of Construct 3, the relation between pBad and L-arabinose is complicated. First of all, the RFP intensity is still high when the concentration of L-arabinose is zero. We suggested that it’s because the leakage rate of pBad may be too high. Yet, this is out of our expectation since we initially hope the pBad won’t express when the L-arabinose concentration is low. Secondly, L-arabinose would enhance the expression of pBad when the concentration is not too high. However, once the L-arabinose concentration surpasses 0.025, it inhibits the expression of pBad. After doing some paper research, we suggested that too much L-arabinose interacting with pBad would become a huge barrier for the RNA polymerase to transcript the gene.

3. The result of RFP intensity of Construct 2 is beyond our expectation:
To our expectation, we hope that construct 2 will show fluorescent red. If so, the expression of MazE in construct 1 would be stable. Besides, construct 2 shows fluorescent red when the concentration of L-arabinose rises, but drops again when the concentration of L-arabinose rises to 0.05M. What’s more, the RFP increases sharply as the concentration of L-arabinose surpasses 0.05M.

According to our design, the RFP in construct 2 was directly influenced by the expression of TetR. The RFP expression in construct 3 may stand for the expression of TetR in construct 2. According to Fig 5, we can see that the expression rate of construct 2 is almost zero, but construct 3 has about 1000 RFP intensity. On the other hand, as the expression in construct 3 drops, the expression in construct 2 will rise simultaneously. Hence, it means that the expression ability of pBad in construct 3 would significantly affect our kill switch design 1.

4. Conclusion: The pBad leakage may make our Design 1 unusable
Since the pBad expression didn’t function as we expected, we considered the Design 1 inappropriate to serve as our kill switch design. In the meantime, we analyzed the Design 1 system through modeling, which turned out to be the same results. Please see our project [Model](https://2021.igem.org/Team%3ANYCU-Taipei/Model) model page for more information. Therefore, we constructed Design 2 for our new design.

Molecular engineering for Kill Switch Design 2

**Analysis and Validation of the constructed strains**
The RFP expression of Pre C1 and Pre C2

![](https://static.igem.org/mediawiki/2021/8/8a/T--NYCU-Taipei--kilksw_result_6.png)

Fig 6. The RFP intensity of Pre C1 and Pre C2 after 24 hr cultivation.

According to the figure, we found that Pre C2 is hardly induced by L-arabinose and the endogenous expression rate of Pre C2 is very low. On the other hand, the expression rate of Pre C1 is great when L-arabinose is added. This experiment is conducted by our collaboration partner, 2021 TAS\_Taipei. For more information, please visit our [Collaborations](https://2021.igem.org/Team%3ANYCU-Taipei/Collaborations) page.

The RFP expression of Pre C1 compared with other 5 tandem promoters constructed by Strategy 3

![](https://static.igem.org/mediawiki/2021/6/6e/T--NYCU-Taipei--kilksw_result_7.png)

Fig 7. The RFP intensity of tandem promoters.

According to the figures, when the concentration of L-arabinose is 0 M, the order of the expression rate of these tandem promoters is the same as that of the constitutive promoter individually. Besides, when the concentration of L-arabinose rises from 0 M to 0.025 M, the expression of RFP under the regulation of pJ102, pJ118, and pJ110 decreases while others rise. Interestingly, among these six tandem promoters, only the Pre C1 will be induced by L-arabinose significantly.

**Discussion**
1. The expression of Pre C1 and Pre C2 is significantly different:
The mechanism of L-arabinose regulating pBad is that L-arabinose will bind and change the pBad inhibitor araC into an inducer [2]. We suggest that the formation of L-arabinose-araC-pBad may be a huge barrier for RNA polymerase to transcribe the gene. Hence, when the pBad is constructed behind the constitutive promoter J23106, the RNA polymerase will be hard to function J23106 because there is a L-arabinose-araC-pBad complex located behind it.

2. The tandem promoter with relatively strong constitutive promoter will be inhibited as the L-arabinose increases:
Those tandem promoters with strong constitutive promoters like pJ102, pJ118 and pJ110 have a better expression rate when there is no L-arabinose. We suggest that it’s still the L-arabinose-araC-pBad complex which serves as a barrier. Even though L-arabinose may enhance the expression of pBad, the interference of the transcription by the L-arabinose-araC-pBad complex plays an more important role.

3. Pre C1 and the other tandem promoters constructed through strategy 3 have similar components but have quite different RFP expression characteristics:
The constitutive promoter J23106 in Pre C1 is from the same constitutive promoter family as others. Also, the strength of J23106 ranks in the middle among others. Besides, the structure of sequence behind these different tandem promoters is identical. The only difference is that Pre C1 is constructed in pSB1A2, while the others are constructed in pSB1C3. We suggest it is the difference of plasmid backbones that lead to different results. Hence, we are going to transfer these tandem promoters into pSB1A2 to see if these tandem promoters will have different results.

4. To implement kill switch design 2
To make design 2 come true, we should choose the promoter before MazE wisely. According to the result of the RFP expression of each tandem promoter, there are some pBad tandem promoters whose RFP expression ability will drop as the concentration of L-arabinose increases such as pJ102, pJ118, pJ110, which are all with strong constitutive promoters. Here we take pJ102 for example. We can construct pJ102 before MazE while Pre C1 before MazF. Under this design, in general, the expression of MazE will be higher than MazF, since when the concentration of L-arabinose is zero, the RFP expression of pJ102 is better than that of Pre C1. When the L-arabinose concentration increases, the expression rate of pJ102 will drop while that of Pre C1 will increase simultaneously. As the MazF surpasses MazE, the killing system will turn on and achieve the kill switch.

![](https://static.igem.org/mediawiki/2021/2/27/T--NYCU-Taipei--kilksw_result_8.png)

Fig 8. Our final kill switch design

References

[1] Narberhaus, F., Waldminghaus, T., & Chowdhury, S. (2006). RNA thermometers. FEMS microbiology reviews, 30(1), 3–16. https://doi.org/10.1111/j.1574-6976.2005.004.x

[2] Lee, N. L., Gielow, W. O., & Wallace, R. G. (1981). Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 78(2), 752–756.

Oral Delivery system

Freeze-drying (lyophilization)
**Results**
From Fig1, results showed that freeze-dried EcN in the presence of our protective medium can survive from harsh conditions during freeze-drying; whereas the freezing in the presence of 0.9% NaCl resulted in no viable EcN after freeze. Besides, the survival rate remained stable after 48 hours and 4 days of storage at 4°C, with the rate of 58.5% and 41.68% respectively. From Fig2, we compared the viability of bacteria storage at 4°C and 27°C. Both conditions show similar results. After 4 days of storage at 27°C, freeze-dried EcN still showed the survival rate of 43.3%. Freeze-drying in the presence of a protective medium ensures that our bacteria are still viable after lyophilization for storage at either 4°C or 27°C. The survival rate was calculated as the percentage ratio of viable cells after freeze-drying to viable cells before freeze-drying.

![](https://static.igem.org/mediawiki/2021/f/f9/T--NYCU-Taipei--results_oral_1.png)

![](https://static.igem.org/mediawiki/2021/0/0d/T--NYCU-Taipei--results_oral_2.png)

**Statistical analysis and conclusion**
All experiments were carried out in triplicate. The difference between two means before and after freeze-drying was calculated using the t-test.

In conclusion, we evaluated the impact of freeze drying on the bacterial viability using 0.9% NaCl as a control. We suppose that freeze drying (lyophilization) is harmful to EcN. Although we didn’t reach the expected viability as the references, using the protective medium as cryoprotectants is still a good alternative to protect *E. coli*  Nissle 1917 during freeze drying. We realized that conditions for freeze drying and freeze dryers’ differences are also factors resulting in different viability after lyophilization. We hope that our engineered bacteria which can secrete Nattokinase can be freeze-dried for storage and then loaded into capsules to become commercial dietary supplements in the future.

![](https://static.igem.org/mediawiki/2021/4/4f/T--NYCU-Taipei--results_oral_3-2.png)

**Capsule option: BioVXR® Acid-Resistant Capsule**
According to Fig4 provided by DFC, after 120 minutes of the pH1.2 environment, the dissolution rate of BioVXR® is lower than 10%, which has protection for ingredients. After 45 minutes of the pH6.8 environment, the dissolution rate of BioVXR® reached 89.18%. BioVXR® shows good acid resistance ability and dissolutes quickly at pH 6.8.BioVXR® Acid-Resistant Capsules can protect acid-vulnerable ingredients from gastric acid and release in the intestinal tract, which is an ideal choice for the delivery of our EcN to the small intestine. We are grateful and honored to be sponsored by the most leading and finest capsule manufacturing company, Dah Feng Capsule (DFC), in Taiwan. For more information, please visit: [Dah Feng Capsule-DFC](https://www.dfc.com.tw/)

![](https://static.igem.org/mediawiki/2021/a/a2/T--NYCU-Taipei--results_oral_4.png)

![](https://static.igem.org/mediawiki/2021/5/50/T--NYCU-Taipei--results_oral_5.png)

**Note:**
1. The graphs are provided by Dah Feng Capsule (DFC)
2. BioVXR® Acid Resistant Capsules are sponsored by Dah Feng Capsule (DFC)

**References**

[1] Mawad, A., Helmy, Y.A., Shalkami, AG. et al. E. coli Nissle microencapsulation in alginate-chitosan nanoparticles and its effect on Campylobacter jejuni in vitro. Appl Microbiol Biotechnol 102, 10675–10690 (2018)

[2] Dah Feng Capsule (DFC) website: https://www.dfc.com.tw/

[3] Bellali S, Bou Khalil J, Fontanini A, Raoult D, Lagier JC. A new protectant medium preserving bacterial viability after freeze drying. Microbiol Res. (2020)

Remote control

E.coli MG1655 gDNA extraction and LexA cloning
We successfully extracted the genomic DNA of E.coli MG1655 and then used it as template for LexA cloning.

![](https://static.igem.org/mediawiki/2021/8/8e/T--NYCU-Taipei--optoresultMG1655.png)

Fig 1. E.coli K12 MG1655 genomic DNA extraction (M: lamba HindIII; 1: gDNA sample 1; 2: 0.5xgDNA sample-1; 3: gDNA sample-2; 4: 0.5xgDNA sample 2)

![](https://static.igem.org/mediawiki/2021/9/9a/T--NYCU-Taipei--optoresultLexA.png)

Fig 2. LexA cloning (M: 100bp marker; 1-6: LexA cloning sample 1-6 ; the lightest bands of the cloning samples cooperate to size of 600bp approximately.)

BphP1 and QPAS1 cloning
We successfully cloned BphP1 and QPAS1 from template pKA-207I10 ordered from Addgene.

![](https://static.igem.org/mediawiki/2021/5/5f/T--NYCU-Taipei--optoresultQPAS1.png)

Fig 3. QPAS1 cloning (M: 100bp marker; 1-10: QPAS1 cloning sample 1-10 ; the lightest bands of the cloning samples cooperate to size of 400bp-500bp approximately.)

![](https://static.igem.org/mediawiki/2021/d/dd/T--NYCU-Taipei--optoresultBphP1.png)

Fig 4. BphP1 cloning (M: 1000bp marker; 1 and 2: Bphp1 cloning sample 1 and 2 ; the lightest bands of the cloning samples cooperate to size of 2000bp approximately.)

Plasmid construction and functional test
Currently, we successfully constructed most of the plasmid we design for the optogenetics system. However, due to time limitations, we did not finish the functional tests of these plasmids we constructed.

Adhesive protein

Overview (Experimental Design)
OmpA-FimH fusion protein connected by GS flexible linker was designed hopefully to bring FimH, the adhesive protein, to the outer membrane of our *E. coli*  host with OmpA transmembrane protein. (See link for our design)

 The adhesive protein part consists of two major experiments.
- FimH expression test under body temperature
- OmpA fusion protein mobility test.

![](https://static.igem.org/mediawiki/2021/2/2d/T--NYCU-Taipei--results_adhesive_protein_F0.png)

Experiment results & proof of concept
**Cloning PCR of targeted DNA and restriction site addition**

![](https://static.igem.org/mediawiki/2021/c/c1/T--NYCU-Taipei--results_adhesive_protein_F1.png)

Fig 1. Cloning PCR of our targeted DNA.

1. Cloning PCR of FimH aa 22~300 from *E. coli*  BL21 genomic DNA

![](https://static.igem.org/mediawiki/2021/4/41/T--NYCU-Taipei--results_adhesive_protein_F2.png)

Fig 2. DNA electrophoresis result of PCR of FimH aa 22~300 from *E. coli*  gDNA. NdeI and XhoI cutting sites were added from primer to 5’ and 3’ end respectively.

2. Cloning PCR of OmpA from *E. coli*  BL21 genomic DNA

![](https://static.igem.org/mediawiki/2021/7/77/T--NYCU-Taipei--results_adhesive_protein_F3.png)

Fig 3. DNA electrophoresis result of PCR of OmpA from *E. coli*  gDNA.NdeI and XbaI cutting sites were added from primer to 5’ and 3’ end respectively.

3. Cloning PCR of GFP from iGEM part BBa\_E0040

![](https://static.igem.org/mediawiki/2021/2/28/T--NYCU-Taipei--results_adhesive_protein_F4.png)

Fig 4. DNA electrophoresis result of PCR of GFP from BBa\_E0040. XbaI and XhoI cutting sites were added from primer to 5’ and 3’ end respectively.

**Enzyme digestion and ligation of targeting constructs**
Enzyme digestions were carried out on gel extracted linear PCR products to their corresponding digestion sites shown in the following graph.

![](https://static.igem.org/mediawiki/2021/8/8f/T--NYCU-Taipei--results_adhesive_protein_F5.png)

Fig 5. Cloning of our targeted constructs.

Ligation of the inserts to pET21a+ by T4 ligase finally succeeded after several attempts. See notebook and protocolfor detailed description.

![](https://static.igem.org/mediawiki/2021/5/5a/T--NYCU-Taipei--results_adhesive_protein_F6.png)

Fig 6. DNA electrophoresis result of FimH-pET21a ligation product and NdeI / XhoI enzyme digestion check. A respected 952 bps releasement were observed.

**FimH protein induction test**
To make sure FimH protein can be induced under body temperature, we transformed the successfully made FimH-pET21a+ construct into *E. coli*  BL21 cell and treated them with 0.25, 0.5, 1.0, 2.0 mM of IPTG after OD600 of the liquid culture reached 0.4, and sample every one hour through the 4 hours induction procedure of FimH protein at 37°C.

![](https://static.igem.org/mediawiki/2021/3/3e/T--NYCU-Taipei--results_adhesive_protein_F7.png)

Fig 7. SDS page result of FimH protein induction.

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