iGEM 2022 — Team nycu-taipei

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Notebook

[Reference](#reference)

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---

#### Reference

1. The 1st citation.
2. The 2nd citation.
3. The 3rd citation.
4. The 4th citation.
5. The 5th citation.
6. The 6th citation.
7. The 7th citation.
8. The 8th citation.
9. The 9th citation.
10. The 10th citation.

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Engineering

[First cycle](#1)
[Second cycle](#2)
[Third cycle](#3)

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## The First Cycle

---

#### Design

For identifying the *E. coli* growth status, we combined a phase-dependent promoter with a fluorescent protein to
specifically express distinct color in each phase. In our design, early log phase promoter, mid-log phase
promoter, late log phase promoter, and stationary phase promoter are combined with AmilCP, mCherry, yPet,
mCerulean respectively, so the early log phase promoter will express color blue, mid-log phase will express
color red, late log phase will express color yellow, and stationary phase will express color green. Note that
the chromoprotein, AmilCP, which has slightly longer maturation time is used since the *dusB* expression will
begin at lag phase. Here is our design.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/design0-12.png)

#### Build

To measure the characteristics of the potential phase specific promoters, we combined the promoters with
RBS(B0034), reporter gene(YPet, mCerulean, mCherry, AmilCP) and double terminator(B0015). Since the AmilCP
expression is rather hard to measure, the early log phase promoter is combined with the mCerulean for
measurement.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering0-1.png)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering0-2.png)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering0-3.png)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering0-4.png)

The early log phase promoter, *dusB* promoter and *cyoA* promoter, the mid-log phase promoter, *nirBDC* promoter and
*glpABC* promoter, the late log phase promoter, *ansB* promoter, and the stationary phase promoter, *hchA* promoter,
*rmf* promoter, and *slp* promoter have been cloned by PCR from *E. coli* MG1655 genomic DNA. For more information
regarding how we construct the plasmid, please visit the [Result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering0-5.jpg)

The *glpABC* promoter, *dusB* promoter, and *hchA* promoter are the first three constructs to be successfully built.
The other construct will be discussed in the next cycle for the reason mentioned in the learn part. The
validation is performed by restriction enzyme digestion method with BamHI and XhoI. For more information,
please visit our project [Result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering1.png)

Fig. 1) The expected result for enzyme digestion confirmation for *dusB*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering2.jpg)

Fig. 2) The result for enzyme digestion confirmation of *dusB*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering3.png)

Fig. 3) The expected result for enzyme digestion confirmation for *glpABC*-mCherry

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering4.jpg)

Fig. 4) The result for enzyme digestion confirmation of *glpABC*-mCherry

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering5.png)

Fig. 5) The expected result for enzyme digestion confirmation for *hchA*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering6.jpg)

Fig. 6) The result for enzyme digestion confirmation of *hchA*-mCerulean

#### Test

To evaluate if the promoter can specifically express in the desired phase, the fluorescent intensity is
tested, as well as the OD value.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering7.png)

Fig. 7) The fluorescent intensity - time graph and OD600 value - time graph for *dusB*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering8.png)

Fig. 8) The fluorescent intensity - time graph and OD600 value - time graph for *glpABC*-mCherry

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering9.png)

Fig. 9) The fluorescent intensity - time graph and OD600 value - time graph for *hchA*-mCerulean

#### Learn

First of all, we found that despite the promoter express phase-specific rise of gene expression, the
fluorescent protein won't degrade on time. Based on the observation, the data suggests two possibilities. One
is that the promoter we use isn't phased specifically but expresses from specific phases. The other is that
the *E. coli* DH5α has a poor ability to degrade mCerulean and mCherry.

Secondly, we found that fluorescent protein is difficult to be observed by naked eye in the early log phase.
It might be due to the relatively low amount of bacteria in those phases or the weak expression of the
fluorescent protein.

Thirdly, the promoter's function did not work as we expected.

1. The *dusB* promoter expression is completely different from our expectations. It shows little expression
   during the early log phase but has a sharp increase when entering the late log phase. Since the *dusB*
   promoter function is quite different, we could not contribute it to any possible reason. In the end, we
   decided to remove the *dusB* promoter from our project.
2. The *hchA* promoter expressed before entering the stationary phase. The result is predictable for that the
   *hchA* mRNA expression shows the uprise from late log phase and reaches its peak at stationary phase. The
   point we are missing is the late log expression might be too strong so that it suppress our late log phase
   indicator.
3. The *glpABC* promoter expressed more like the late log phase promoter. The result is also predictable
   since the *glpABC* mRNA expression reaches its peak in the middle of the mid log phase. The late log phase
   expression might be due to the time required for protein synthesize or the time required for fluorescent
   protein maturation.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering10.png)

Fig. 10) mRNA expression of glpA, glpB, glpC under the *glpABC* promoter

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering11.png)

Fig. 11) mRNA expression of *hchA* under the *hchA* promoter

## The Second Cycle

---

#### Design

Since the early exponential phase is too difficult to be observed, and the *dusB* promoter fails to express the
desired phase, we remove the early exponential phase indicator from our project. Besides, we combine the *hchA*
promoter with the AmilCP to delay the late log phase expression, serving as the timer. Lastly, the color of
mid-log phase indicator and late log phase indicator are switched since the *glpABC* promoter with mCherry
expresses more like the late log phase promoter, and the other mid log phase, *nirBDC*-mCerulean, are
constructed.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering12.jpg)

Fig. 12) The construct for *nirBDC*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering13.jpg)

Fig. 13) The construct for *hchA*-AmilCP

#### Build

The mid-log phase indicator, *nirBDC*-mCerulean and the stationary phase indicator, *hchA*-AmilCP are successfully
built. The validation is also performed by restriction enzyme digestion method with BamHI and XhoI. For more
information, please visit our project [Result](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering14.png)

Fig. 14) The expected result for enzyme digestion confirmation for *nirBDC*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering15.jpg)

Fig. 15) The result for enzyme digestion confirmation of *nirBDC*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering16.png)

Fig. 16) The expected result for enzyme digestion confirmation for *hchA*-AmilCP

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering17.jpg)

Fig. 17) The result for enzyme digestion confirmation of *hchA*-AmilCP

#### Test

To evaluate if the promoter can specifically express in the desired phase, the fluorescent intensity is
tested, and as well as the OD value. For *hchA*-AmilCP, the photos are taken since it could not emit the
fluorescence.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering18.png)

Fig. 18) The fluorescent intensity and OD600 - time graph for *nirBDC*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering19.png)

Fig. 19) OD600 value - time graph for *hchA*-AmilCP

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering20-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering20-2.jpg)

Fig. 20) The photo of 9hr to 10hr for Fig. 19

#### Learn

The expression of the *nirBDC* promoter with mCerulean and the *hchA* promoter with AmilCP have accorded to our
expectation. Therefore, the remaining question is the lasting fluorescent intensity.

## The Third Cycle

---

#### Design

Two hypotheses were proposed for the cause of lasting fluorescence:

1. Continuous production of the fluorescent protein.
2. Slow degradation of fluorescent protein by *E. coli* DH5α

To determine which hypothesis is correct, we append the fast degradation mCherry (
[BBa\_K4461013](http://parts.igem.org/Part%3ABBa_K4461013)) to *glpABC* promoter for
testing.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering25.png)

#### Build

We successfully built our construct, and the validation is performed by BamHI and HindIII. For more
information, please visit our project [Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D) page

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering21.jpg)

Fig. 21) The result for enzyme digestion confirmation of *glpABC*-mCherry with tag

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering22.png)

Fig. 22) The expected result for enzyme digestion confirmation of *glpABC*-mCherry with tag

#### Test

Both fluorescent intensity and OD of *glpABC*-mCherry and *glpABC*-mCherry-tag are tested, and the data are
compared to determine which hypothesis is correct

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering23.png)

#### Learn

Since we could not see the reduction of fluorescent intensity after the late-log phase, our promoter is more
likely to continuously express from a specific phase. For future improvement, the protease-degradation tag
system is designed to enable the fast elimination of fluorescent protein in the next phase.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/engineering24.png)

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Experiments

[LB Preparation](#LB)
[Plasmid Extraction](#plasmid)
[PCR](#pcr)
[Enzyme Digestion](#enzyme-digestion)
[Electrophoresis](#electrophoresis)
[Gel extraction](#gel-extraction)
[Gibson assembly](#gibson)
[Ligation](#ligation)
[Transformation](#transformation)
[Competent cell preparation](#competent-cell)
[FI & OD measurement](#FI-OD)

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## Here are our experiments & protocols

---

#### LB Preparation

* Liquid Medium with Chloramphenicol (CM) Preparation
  1. 8g of LB broth (Bioshop LBL407.1) are added with 400ml ddH2O
  2. the solution is then sterilized
  3. after the cooldown, 200 μl CM is added
* Solid Medium with CM Preparation
  1. 8g of LB broth (Bioshop LBL407.1) are added with 400ml ddH2O
  2. add 6g agar to the solution
  3. the solution is then sterilized
  4. after the cooldown, 200 μl CM is added
  5. pour the solution into the petri dish

---

#### Plasmid Extraction

* Preparation
  1. single colony is picked on the LB plate with CM
  2. the colony is cultured in 5ml LB+CM medium for 16-18hr
* Steps
  + Harvest
    1. transfer 1ml of cultured bacterial cells to a 1.5 ml microcentrifuge tube
    2. centrifuge at 14-16000 x g for 1 minute at room temperature
    3. discard the supernatant completely
    4. repeating the harvesting step in the same tube until 3 ml sample are collected
  + Resuspend
    1. add 200 μl of PD1 buffer (make sure Rnase A was added) to the Eppendorf
    2. resuspend the pellet by vortex or pipette until the cell pellet is completely dissolved
  + Cell Lysis
    1. add 200 μl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. (do not vortex, it will cause the shearing of the genomic DNA)
    2. stand the sample at room temperature for 2 to 5 minutes. Do not exceed 5 minutes
  + Neutralization
    1. add 300 μl of PD3 Buffer to the sample then mix immediately by inverting the tube 10 times
    2. centrifuge at 14-16000 x g for 3 minutes at room temperature
    3. during the centrifugation, place a PDH column in a 2 ml collection tube
  + DNA Binding
    1. transfer all of the supernatants to the PDH column
    2. centrifuge at 14-16000 x g for 30 seconds at room temperature
    3. the DNA binding step can be repeated for better yield
    4. discard the flow-through and place the PDH column back in the 2 ml Collection Tube
  + Wash
    1. add 400 μl W1 buffer to PDH column
    2. centrifuge at 14-16000 x g for 30 sec
    3. discard the flow-through and place the PDH column back into the collection tube
    4. add 600 μl of Wash buffer into the PDH column
    5. stand for 1 min at room temp, then centrifuge at 14-16000 x g for 30 sec
    6. discard the flow-through and place the PDH column back in the 2 ml collection tube
    7. centrifuge at 14-16000 x g for 3 min to dry the matrix
  + Elution
    1. add 400 μl of W1 buffer into the PDH column
    2. centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and place the PDH column back into the collection tube
    3. add 600 μl of Wash buffer(make sure ethanol was added) into the PDH column
    4. let stand for 1 min at room temp. Centrifuge at 14-16000 x g for 30 sec then discard the flow-through
    5. place the PDH column back in the 2 ml collection tube
    6. centrifuge at 14-16000 x g for 3 min to dry the matrix

---

#### PCR

* Preparation
  + | Component | Sample | Control |
    | --- | --- | --- |
    | 10X KOD buffer | 2 | 2 |
    | MgSO4 | 0.8 | 0.8 |
    | DNA template | 60-80 ng (if using gDNA template, increase the amount of the template) | 0 |
    | dNTP (2mM) | 0.5 | 0.5 |
    | primer pair | 1 | 1 |
    | KOD plus enzyme | 0.2-0.4 | 0.2-0.4 |
    | add ddH2O to 20 μl | | |
  + | Component | Sample | Control |
    | --- | --- | --- |
    | 2X dreamtaq green master mix | 10 μl | 10 μl |
    | DNA template | 60-80 ng (if using gDNA template, increase the amount of the template) | 0 |
    | primer pair | 1 | 1 |
    | add ddH2O to 20 μl | | |
* Machine set up
  + | Step | Temp(°C) | Time |
    | --- | --- | --- |
    | 1 | 94 | 5 min |
    | 2 | 94 | 30 sec |
    | 3 | 57-59 | 30 sec |
    | 4 | 68-72 | 1 min for each kb |
    | 5 | 68-72 | 7 min |
    | repeat step 3 to step 5 for 30-35 cycle | | |
    | 6 | 20 | 10 min |

---

#### Enzyme Digestion

* Preparation
  + | component | amount per reaction |
    | --- | --- |
    | DNA | 1000 ng |
    | rCutsmart buffer | 2 μl |
    | restriction enzyme | 0.5 μl |
    | add sterilized ddH2O to 20 μl | |
  + | component | amount per reaction |
    | --- | --- |
    | DNA | 1000 ng |
    | rCutsmart buffer | 2 μl |
    | restriction enzyme 1 | 0.5 μl |
    | restriction enzyme 2 | 0.5 μl |
    | add sterilized ddH2O to 20 μl | |
* Steps
  1. prepare the sterilized eppendorf
  2. add the component above in the following order. ddH2O, rCutsmart buffer, DNA, restriction enzyme
  3. mix the component by pipetting
  4. put it in the 37°C water bath for an hour

---

#### Electrophoresis

* Preparation
  + Large : 50ml TAE buffer + 0.5 - 1 g agarose (1-2%)
  + Small : 30 ml TAE buffer + 0.3 - 0.6 g agarose (1-2%)
* Steps
  1. add the component with protocol above
  2. heat the liquid by microwave oven to dissolve the agarose
  3. wait for it cool down until you can touch it with your hand and still feeling temperature
  4. pour agarose onto the gel tray
  5. add 4 μl loading dye to 20 μl sample for big well or add 2 μl loading dye to 10 μl sample for small well
  6. load sample and marker
  7. run 70 V, at least 30 min

---

#### Gel extraction

* Gel Dissociation
  1. prepare a 55-60°C dry bath
  2. transfer the gel slice to the eppendorf
  3. add 500 μl of gel buffer to the sample then mix by vortex (if two slice are used, added 600 μl instead)
  4. incubate at 55-60°C for 10-15 min to ensure the gel slice is completely dissolved
  5. during incubation, invert the tube every 2-3 min
* DNA binding
  1. place a DFH Column in a 2 ml Collection tube
  2. wait for the medium cool down to the room temperature
  3. transfer 800 μl of the sample to 2 ml Collection tube
  4. centrifuge at 14-16000 x g for 30 sec
  5. discard the flow-through and put the DFH column back in the 2 ml collection tube
* Wash
  1. add 400 μl W1 buffer to DFH column
  2. centrifuge at 14-16000 x g for 30 sec
  3. discard the flow-through and place the DFH column back into the collection tube
  4. add 600 μl of Wash buffer into the DFH column
  5. stand for 1 min at room temp, then centrifuge at 14-16000 x g for 30 sec
  6. discard the flow-through and place the DFH column back in the 2 ml collection tube
  7. centrifuge at 14-16000 x g for 3 min to dry the matrix
* Elution
  1. add 400 μl of W1 buffer into the DFH column
  2. centrifuge at 14-16000 x g for 30 sec. Discard the flow-through and place the DFH column back into the collection tube
  3. add 600 μl of Wash buffer(make sure ethanol was added) into the DFH column
  4. let stand for 1 min at room temp. Centrifuge at 14-16000 x g for 30 sec then discard the flow-through
  5. place the DFH column back in the 2 ml collection tube
  6. centrifuge at 14-16000 x g for 3 min to dry the matrix

---

#### Gibson assembly

* Preparation
  + Component

    |  |  |
    | --- | --- |
    | fragment 1 (? bp) |  |
    | fragment 2 (? bp) |  |
    | fragment 3 (? bp) |  |
    | 2X Gibson Assembly master mix | 9 |
    | add ddH2O to 18 μl | |
  + PCR machine set up 50°C for 15 min
  + prepare a 42°C dry bath
  + prepare ice with water
* Calculation
  + Optimized cloning efficiency is 50-100 ng of vector with 2-3 fold molar excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp
* Steps
  1. add the components in following order: ddH2O, 2X Gibson Assembly master mix, DNA fragments
  2. use PCR machine to incubate for 50°C 15 min
  3. perform the transformation immediately

---

#### Ligation

* Preparation

  | Aomponent | Amount per Reaction |
  | --- | --- |
  | vector DNA | 0.02 pmol |
  | insert DNA-1 | 0.06 pmol |
  | insert DNA-2 | 0.06 pmol |
  | T4 ligase buffer | 2 μl |
  | T4 ligase | 1 μl |
  | add ddH2O to 20 μl | |
* Steps
  1. add the components in the following order: ddH2O, DNA, T4 DNA ligase buffer, T4 DNA ligase to the eppendorf
  2. stand over 2-4 hour at room temperature
  3. stand overnight at 4°C

---

#### Transformation

1. prepare 42°C dry bath, and the ice with water
2. take competent cells out of -80°C and thaw on ice immediately
3. add 2 μl of plasmid and 30 μl competent cell into eppendorf
4. stand on ice for 30 min
5. heat shock for 1 min
6. put the tubes back on ice for 2 min
7. add 1000 μl LB to the eppendorf and incubate at 37°C for 45 min
8. centrifuge at 14-16000 x g for 1 min
9. remove 800 μl of supernatant then resuspend
10. place the transformation onto LB agar plate with selective marker
11. culture for 16-18 hr in the incubator

---

#### Competent cell preparation

* DAY 1 - Buffer preparation & bacteria cultivation
  + 0.1M MgCl2 preparation
  + 0.1M CaCl2 preparation
  + 85% 100 mM CaCl2 & 15% glycerol mixture preparation
  + bacteria culture
    - pick a single colony to the 50 ml LB and incubate under 37°C, 200 rpm for 16-20hr
    - two flasks of 125 ml LB are pre-warm under 37°C
* DAY 2
  1. after the 16-20hr cultured, transfer 12.5 ml medium to 125 ml pre-warm LB
  2. the bacteria are cultured to OD value 0.2 (approximately 5hr-6hr)
  3. added 75 ml of pre-warm 37°C LB medium, and culture for another 30 minutes
  **Every step should be done on ice**4. centrifuge 150 ml of the medium at 5000 rpm 10 minutes in 4 °C
  5. discard the supernatant, and resuspend with 37.5 ml MgCl2
  6. stand for 5 minutes then centrifuge 4000rpm, 10 min. under 4°C
  7. discard the supernatant, and resuspend with 7.5 ml CaCl2
  8. stand for 20 minutes then centrifuge 4000rpm, 10 min. under 4°C
  9. iscard the supernatant, and resuspend with 1.5 ml CaCl2+glycerol

---

#### Fluorescent intensity and OD value measurement

1. pick a single colony to 5ml LB+CM medium, and culture for 17 hr
2. transfer the cultured medium to LB+CM for final OD value 0.05 and volume 50 ml
3. transfer 100 μl medium to transparent 96 well microplate for OD measurement
4. transfer 100 μl medium to black 96 well microplate for fluorescent intensity measurement
5. repeat step 3 and step 4 hourly until entering the stationary phase

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Results

[Construct Plasmid](#construct)
[Promoter Functional Test](#func-test)
[Ligation](#ligation)
[Validation](#validation)
[Measurement for Cultivate Condition](#condition)
[Native Color Indicator](#native)
[Cell Division Indicator](#cell-division)

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## Construct Plasmid for Growth Phase Indicator

---

We applied a series of existing plasmid constructs which were kindly provided to us by our supervisor for the
construction of our growth phase indicator product. Each plasmid consists of a constitutive promoter
([BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)), a color indicator gene, and
double terminators ([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)). First of
all, pSB1C3 plasmids
containing our selected reporter gene (mCerulean, mCherry, yPet, AmilCP) were confirmed by restriction enzyme
digestion (**Fig. 1**). In the following steps, these plasmids will be used to construct our
newly
assembled
plasmid, which replaces the constitutive promoter with our phase-dependent promoter.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result1-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result1-2.jpg)

Fig. 1) The result of enzyme digestion confirmation of YPet, mCerulean, mCherry, AmilCP

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result2.png)

Fig. 2) The expected result of enzyme digestion confirmation

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result3-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result3-2.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result3-3.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result3-4.jpg)

Fig. 3) The construct of YPet, mCerulean, mCherry, AmilCP

Then, we extracted the genomic DNA from *E. coli* K-12 MG1655

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result4.jpg)

Fig. 4) the result of *E. coli* K12 Mg1655 gDNA extraction

The potential phase-dependent promoters, *dusB, nirBDC, glpABC, ansB, rmf* and *hchA*
promoter, were cloned from gDNA of *E. coli* K-12 MG1655. Note that the length of the sequence that is
cloned is
larger than the promoter we used. This is to prevent the amplification of unintended bands. After the cloning
from gDNA of *E. coli* K-12 MG1655, overlapping sequences were added by PCR on both sides of the promoter
sequence, which allows the performance of Gibson assembly.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result5.jpg)

Fig. 5) The result of PCR cloning of each promoter from gDNA

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result6.jpg)

Fig. 6) The result of PCR cloning to append overlapping site to promoter

pSB1C3 plasmids containing the fluorescent-terminator sequence were cloned into two pieces by PCR and are
added with overlapping sequences. This will prevent the situation that the construct size is too similar to
the original plasmid and causes low yield after Gibson transformation

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result7.png)

Fig. 7) The construct for pSB1C3 plasmids containing the constitutive promoter (J23100), RBS (BBa\_B0034),
and fluorescent-terminator sequence

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result8.png)

Fig. 8) The sequence for vector 2, which contains RBS, reporter gene and the double terminator

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result9-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result9-2.jpg)

Fig. 9) The result of PCR cloning for each vector 2, containing a different reporter gene. The expected
result for YPet is 1072 bp, for mCerulean is 1078 bp, for AmilCP is 1027 bp, for mCherry is 1040 bp

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result10.png)

Fig. 10) The sequence for vector 1, which is the pSB1C3 backbone

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result11.jpg)

Fig. 11) The result of PCR cloning for vector 1. The expected result is 1874 bp

#### Early Log Phase

The potential early log phase promoter, *dusB* promoter, was combined with RBS-mCerulean-double
terminator sequence and pSB1C3 backbone sequence by Gibson assembly.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result12.png)

Fig. 12) The construct for *dusB*-mCerulean

The plasmid was then transformed into *E. coli* DH5α competent cells, and the successful colonies
were checked by enzyme digestion.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result13.png)

Fig. 13) The expected result for enzyme digestion confirmation for dusB-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result14.jpg)

Fig. 14) The result for enzyme digestion confirmation of *dusB*-mCerulean

#### Mid Log Phase

The potential mid log phase promoter, *nirBDC* promoter, was combined with RBS-mCerulean-double
terminator sequence and pSB1C3 backbone sequence by Gibson assembly

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result15.jpg)

Fig. 15) The construct for *nirBDC*-mCerulean

The plasmid was then transformed into *E. coli* DH5α competent cells, and the successful colonies
were checked by enzyme digestion.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result16.png)

Fig.16) The expected result for enzyme digestion confirmation for *nirBDC*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result17.jpg)

Fig. 17) The result for enzyme digestion confirmation of *nirBDC*-mCerulean

The potential mid log phase promoter, *glpABC* promoter, was combined with RBS-mCherry-double terminator
sequence and pSB1C3 backbone sequence by Gibson assembly. Note that it turns out that *glpABC* promoter
expression more likely occurs during the late log phase.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result18.jpg)

Fig. 18) The construct for *glpABC*-mCherry

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result19.png)

Fig.19) The expected result for enzyme digestion confirmation for *glpABC*-mCherry

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result20.jpg)

Fig. 20) The result for enzyme digestion confirmation of *glpABC*-mCherry

#### Late Log Phase

The potential late log phase promoter, *ansB* promoter, was only cloned from the gDNA since the
*glpABC*
expression represents the pattern of a late log phase promoter.

#### Stationary Phase

The potential stationary phase promoter, *hchA* promoter, was combined with either RBS-mCerulean-double
terminator sequence or RBS-AmilCP-double terminator sequence and pSB1C3 backbone sequence by Gibson assembly.
The two different colors were both constructed to give a more observable option.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result21-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result21-2.jpg)

Fig. 21) The construct for *hchA*-mCerulean and *hchA*-AmilC

The plasmid was then transformed into *E. coli* DH5α competent cells, and the successful
colonies were checked by enzyme digestion.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result22.png)

Fig. 22) The expected result for enzyme digestion confirmation for *hchA*-AmilCP

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result23.jpg)

Fig. 23) The result for enzyme digestion confirmation of *hchA*-AmilCP

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result24.png)

Fig. 24) The expected result for enzyme digestion confirmation for *hchA*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result25.jpg)

Fig. 25) The result for enzyme digestion confirmation of *hchA*-mCerulean

## Functional Test for Promoter and the Validation

---

The fluorescence intensity and OD value were both tested to determine whether our promoter expressed in each
specific phase.

#### *dusB*

Although literature evidence strongly supports the phase-dependent expression of the *dusB-fis* promoter
during
early-exponential phase, the measured fluorescent intensity data demonstrates a pattern that closely resembles
a constitutive promoter.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result26.png)

Fig. 26) The fluorescent intensity - time graph and OD600 value - time graph for
*dusB*-mCerulean

#### *nirBDC*

The expression curve of the *nirBDC* promoter occurs during the mid-exponential phase, which was the same
as we expected.

#### *glpABC*

The *glpABC* promoter did not express from the mid-exponential phase as we expected. However, its
expression
perfectly matches the late-exponential phase. Therefore, the *glpABC* promoter was used to indicate the
late-exponential phase.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result27.png)

Fig. 27) The fluorescent intensity and OD600 - time graph for *nirBDC*-mCerulean and
*glpABC*-mCherry

#### *hchA*

The *hchA* promoter is continuously expressed, and shows the rather fast incline from the
late-exponential
phase. We combined it with AmilCP, which has a slightly longer maturation time (1h), to delay its expression
and enable the *hchA* promoter to indicate stationary phase. Although this is not the optimal method for
indicating stationary phase, our project is still workable with this part. Despite the fact that we could not
find the stationary phase-dependent promoter ourselves, the MIT 2006 iGEM has developed a stationary phase
promoter osmY which has been recorded on the iGEM Parts of Registry.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result28.png)

Fig. 28) The fluorescent intensity - time graph and OD600 value - time graph for
*hchA*-mCerulean

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result29.png)

Fig. 29) OD600 value - time graph for *hchA*-AmilCP

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result30-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result30-2.jpg)

Fig. 30) The photo of 9hr to 10hr for Fig. 29

Since the fluorescence will remain after the desired phase, ssrA degradation tags were attached to the
fluorescent protein to increase its degradation rate in *E. coli*. This allowed us to test whether the
lasting
fluorescence is caused by the continuous expression of the fluorescent protein or the slow degradation rate of
fluorescent protein by *E. coli*. The possible results are shown in **(Fig. 31)**.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result31.png)

Fig. 31) The possible result when the tag is attached to fluorescent protein

In order to attach the degradation tag behind the fluorescent protein, point mutation is performed to prevent
the effect of the stop codon. Confirmation of whether the point mutation has been successfully performed is
done by enzyme digestion since the cutting site, HindIII, is deleted after point mutation.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result32.jpg)

Fig. 32) The result for enzyme digestion confirmation of *glpABC*-mCherry mutation

PCR is performed to append cutting sites, HindIII and EcoRI, between fluorescent protein and terminator. The
tag with HindIII and EcoRI cutting sites is synthesized due to its short length. The ligation is then
performed. The confirmation is performed by restriction enzyme digestion method with BamHI and EcoRI.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result33.jpg)

Fig. 33) The result for enzyme digestion confirmation of *glpABC*-mCherry with tag

The result suggests that our promoter is specific express from desired phases. Therefore, the degradation
system is designed to prevent the lasting fluorescent intensity. However, due to the time limit, we are unable
to finish it.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result34.png)

Fig. 34) The result for fluorescent intensity and OD measurement of *glpABC*-mCherry with tag

## Ligation of Each Phase to Form the Three-Phases Indicator

---

The mid-exponential phase, late-exponential phase, and stationary phase indicator sequence are cut with
NotI/XhoI, NotI/PstI and PstI/XhoI respectively. The ligation products are confirmed by PCR with primers
located at *nirBDC* promoter and *glpABC* promoter.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result35.jpg)

Fig. 35) The result for PCR confirmation of *nirBDC*-mCerulean, *glpABC*-mCherry, and *hchA*-AmilCP
ligation

fluorescence intensity are tested and pictures are taken as well

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result36.png)

Fig. 36) The result for fluorescent intensity and OD measurement of ligation

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result37-1.jpg)
![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result37-2.jpg)

Fig. 37) The photo of 10 hr to 11 hr from Fig. 36 to show AmilCP expression

## Validation of Our Growth Phase Indicator

---

For validation, we use the ribosome dynamic as the indicator for the growth phase since it shows the growth
phase dependent pattern. MCrg, the generous gift from Dr. Nikolay, are used as the biomarker for ribosome
dynamics by expressing fluorescent-labeled ribosomal proteins L19-GFP and S2-mCherry. We made it as the
competent cell to check whether the color change of our indicator could correspond to the growth phase
indicated by ribosome dynamics. However, due to the time limit, the successful colonies are not yet to be
found.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result38.jpg)

Fig. 38) the MCrg competent cell are culture on LB plate to make sure it is not dead during the competent
cell preparation

## Measurement Data for Different Cultivate Condition

---

The measurements start with OD600=0.05 and total LB+CM volume of 50ml, the OD600 are
recorded by a microplate
reader. The sample is the *E. coli* DH5α with pSB1C3 plasmid serving as the CM resistance. The loading
volume is
100 μl, and the cultivating environment is 200rpm and 37°C

#### Different Quantity of Bacteria That are Cultured

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result39.png)

Fig. 39) The result for OD measurement with a different initial quantity of bacteria. The meaning of the
data can be seen in the [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page

#### Different Status of Bacteria That are Cultured

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result40.png)

Fig. 40) The result for OD measurement when regrowing bacteria from different growth phases. The meaning of
the data can be seen in the [Modeling](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) page.

## Native Color Indicator

---

First, the part K1789003 is confirmed using Colony PCR. It was run with primer VF2/VR and confirmed with 1 %
Agar gel electrophoresis for 45 min, the result **(Fig. 41)** expected to be 800 bp.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result41.jpg)

Fig. 41)

The fluorescent intensity of Part K1789003 is observed using Tecan Elisa Reader and its data is collected as
shown below (Fig. 42). We then narrowed down the Excitation/Emission range of Part K1789003 between
470nm/510~530nm and reobserved. Its data is collected as shown below (Fig. 43). In both data, in order to
identify the background value, LB+CM is used as the source of A3 well.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result42.jpg)

Fig. 42)

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result43.jpg)

Fig. 43)

We later concluded that the liquid culture of DH5α bacteria strain would be a better source for the
background value of original cell culture than LB+CM so
fluorescent intensity of Part K1789003 is reobserved with K1789003/DH5α/LB+CM, thus our observation data
is made more complete.

After confirmation, we planned of using K608008 (J23104 + RBS)
as template and RF cloning method, K1789003(sGFP1) would be inserted to replace the original GFP.

## Cell Division Indicator

---

To indicate cell division, we clone both of the ftsZ and ftsQ promoters from *E. coli* DH5α gDNA since
they show oscillation during the cell cycle.

![](https://static.igem.wiki/teams/4461/wiki/wiki-resources/pictures/result44.jpg)

Fig. 41) The result of PCR cloning of ftsQ promoter and ftsZ promoter

#### Plasmid Construction and Functional Test

Currently, we successfully constructed both of the potential promoters for our cell division indicator system.
However, due to time limitations, we have not combined it with the reporter gene. The functional tests of
these plasmids had not been performed yet.

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