iGEM 2023 — Team mit-mahe

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Growth Curve

WEEK 1

×

## **Week 1**

---

---

## Experiment 1: Revival of *Pseudomonas putida* culture

18/07/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani**

---

#### ***Pseudomonas putida* culture:**

The MTCC (Microbial Type Culture Collection and Gene Bank) number of the culture: 1072

* A lyophilised culture that was stored in a glass ampoule was transferred into Nutrient broth media,
  distributed into test tubes, and incubated for further use at the required optimum temperature and time
  period as mentioned by the culture suppliers (for *Pseudomonas putida*, the incubation was done at 30°C
  for 24 hours).
* The cultures were monitored to observe growth and detect signs of contamination.

---

---

## Experiment 2: Inoculation and streak plating of the culture

09/08/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani, Akanksha Kelkar**

---

#### ***Pseudomonas putida* culture:**

* 1 ml of culture from one test tube was transferred into an Eppendorf tube to prevent contamination.
* A heat-sterilized inoculation loop was dipped into the Eppendorf tube containing the culture. A Petri dish
  containing nutrient broth agar was taken and quadrant streaking was done.

#### **Result:**

**The streaked plate was observed:**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-petri-dish-streaked-with-pseudomonas-putida.jpg)
*(Figure: A petri dish streaked with Pseudomonas putida.)*

---

---

## Experiment 3: Confirmatory tests for *Pseudomonas putida*

09/08/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani, Akanksha Kelkar**

---

#### **Confirmatory tests for *Pseudomonas putida***

Confirmatory tests were performed to confirm the characteristics of the bacteria. These tests included a
catalase test, a gram staining test, an indole spot test, and a MR-VP test.

1. **Catalase test-** *Pseudomonas putida* has the ability to produce catalase, and a positive
   catalase test indicates that the organism possesses this characteristic. When performed, the following
   result was obtained.
![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/catalase-positive-reaction-p-putida.jpg)
*(Figure: Catalase positive reaction: Evident by immediate effervescence (bubble formation)*
2. **Gram staining**- A Gram staining test was performed on the culture. The cells appeared pink when viewed
   under the microscope, confirming that they are Gram-negative (as expected, the shape was bacillus).
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/microscopic-view-p-putida.jpg)
   *(Figure: Microscopic view of a bacterial culture Gram stain - the cells appear as dark pink dots
   on a lighter pink background with a magnification of 100x)*

   | Test | Expected Result | Observations | Results |
   | --- | --- | --- | --- |
   | Gram staining | Gram-negative | Cells appeared pink when viewed under a microscope | Cells were concluded to be Gram-negative |
   | Catalase Test | Evidence of catalase production | Effervescence was observed | Cells were concluded to be producing catalase |

---

---

## Experiment 4: Primary inoculation for confirmatory tests for *Pseudomonas putida*

11/08/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani, Akanksha Kelkar**

---

***Pseudomonas putida* culture**

*Pseudomonas putida* was inoculated in a test tube with LB broth and left overnight to be used for the
indole spot confirmatory test. One of the prepared plates was used for subculturing.

---

---

## Experiment 5: Primary inoculation for confirmatory tests for *Pseudomonas putida*

12/08/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica, Nidhi Mallya, Pooja Agarwal**

---

***Pseudomonas putida culture***

The culture was observed to have been contaminated and was unsuitable for use in the indole spot confirmatory
test. Another sample was incubated at 30°C, 180 rpm overnight.

---

---

## Experiment 6: Confirmatory tests for *P. putida* (Indole Test)

11/08/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani, Akanksha Kelkar**

---

#### ***Pseudomonas putida culture***

**Confirmatory Test for *Pseudomonas putida* (Indole Test):**

1. **Indole test**- The Indole spot test was performed with the inoculated culture. A piece of Whatman
   filter paper was placed in a petri dish cover and saturated with Kovács reagent. The paper was then smeared
   with cell paste from the culture. There was no visible colour change, indicating a negative result. The
   organism was confirmed to lack the ability to produce tryptophanase.
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/indole-negative-test-of-pseudomonas-putida.png)
   *(Figure: Indole negative test of Pseudomonas putida)*
2. **MRVP test preparation**- To prepare for the Methyl Red and Voges-Proskauer test, MR-VP broth medium was
   prepared in three test tubes and autoclaved. *Pseudomonas putida* was inoculated into the appropriate
   tube, with the other tube serving as blank. All the test tubes were incubated at 37°C for 24 hours.

   | Test | Expected Result | Observations | Results |
   | --- | --- | --- | --- |
3. **Confirmatory test for *Pseudomonas putida(MRVP)*:**

   **Methyl red test**- A Methyl Red Test was performed, and a negative result was obtained. This
   indicates that it does not produce organic acid end products following the metabolism of glucose, or acid
   end products are enzymatically converted into non-acidic compounds.This indicates that it does not produce
   organic acid end products, or acid end products are enzymatically converted into non-acidic compounds.
   Thus proving that *Pseudomonas putida* does not ferment glucose.

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/negative-methyl-red-test-p-putida.jpg)
   *(Figure: Negative Methyl Red test)*
4. **Voges- Proskauer test**- Voges- Proskauer test was performed and a negative result was obtained. This
   indicates the absence of non-acidic and neutral compounds being formed as end products following the
   metabolism of glucose.
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/negative-voges-proskauer-test-p-putida.jpg)
   *(Figure: Negative Voges- Proskauer test)*

| Tests | Expected Results | Observation | Conclusion |
| --- | --- | --- | --- |
| Methyl Red test | Negative result (Culture should remain yellow) | Negative results were observed | Cells produce stable non-acidic end products |
| Voges- Proskauer test | Negative result (No colour change in culture) | Negative results were observed | There is an absence of non-acidic and neutral compounds being formed as end products |

---

---

## Experiment 7: Growth curve for *P. putida* in presence of Acetonitrile

13/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **Growth curve of *Pseudomonas putida* in the presence of Acetonitrile**

This growth curve was necessary since, the growth curve in presence of TCC included addition of acetonitrile
for dissolving, hence we needed to ensure that there is not any effect of acetonitrile on the growth.

* Growth curves for *Pseudomonas putida* when in the presence of acetonitrile were prepared.
* Three conical flasks were taken, and 25 ml LB broth was prepared in each and labelled Blank, Control and
  Culture + Acetonitrile, respectively and autoclaved.
* Observation/OD at 600nm was measured at different time intervals as mentioned in the table below.

  | Time (hours) | Control | Acetonitrile |
  | --- | --- | --- |
  | 0 | 0.1 | 0.2 |
  | 2 | 0.9 | 0.9 |
  | 4 | 2.5 | 2.7 |
  | 6 (diluted) | 5 (0.5) | 6 (0.6) |
  | 8 (diluted) | 9 (0.9) | 9 (0.9) |
  | 18 (diluted) | 9 (0.9) | 9 (0.9) |

  (dilution- 1/10th)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-acetonitrile.png)
**Result:**It was found that the presence of acetonitrile doesn't affect the growth of *Pseudomonas
putida*.

WEEK 2

×

## Week 2

## Experiment 8: Growth curve for *P. putida* in presence of Acetonitrile

13/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **Growth curve for growth of *Pseudomonas putida* in the presence of acetonitrile**

This growth curve was necessary since, the growth curve in presence of TCC included addition of acetonitrile
for dissolving, hence we needed to ensure that there is not any effect of acetonitrile on the growth.

* Three test tubes, one blank, one a control, and one containing a mix of culture and 1% acetonitrile
  solution.
* Observation/OD at 600nm was measured at different time intervals as mentioned in the table below.

  | Time (hours) | Control | Acetonitrile |
  | --- | --- | --- |
  | 0 | 0 | 0 |
  | 2 | 0 | 0 |
  | 4 | 0 | 0 |
  | 6 | 0.2 | 0.2 |
  | 8 | 1.9 | 1.8 |
  | 10 (diluted) | 6(0.6) | 6(0.6) |
  | 12 (diluted) | 7(0.6) | 8(0.7) |
  | 14 (diluted) | 9(0.9) | 10(1.0) |
  | 24 (diluted) | 7(0.7) | 7(0.7) |

  (dilution- 1/10th)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-acetronitrile-2.png)

**Result:** It was found that the presence of acetonitrile doesn't affect the growth of *Pseudomonas
putida*.

---

---

## Experiment 9: Growth curve for *P. putida* in presence of Acetonitrile

13/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **Growth curves for *Pseudomonas putida* in the presence of acetonitrile**

This growth curve was necessary since, the growth curve in presence of TCC included addition of acetonitrile
for dissolving, hence we needed to ensure that there is not any effect of acetonitrile on the growth.

* Three test tubes, one blank, one a control, and one containing a mix of culture and 1% acetonitrile
  solution, were taken and observed.
* Observation/OD was measured at different time intervals as mentioned in the table below.

  | Time (hours) | Control | Acetonitrile |
  | --- | --- | --- |
  | 0 | 0 | 0 |
  | 2 | 0.03 | 0.023 |
  | 3 | 0.1 | 0.1 |
  | 4 | 0.3 | 0.3 |
  | 5 | 0.8 | 0.8 |
  | 6 | 0.2 | 0.2 |
  | 7 (diluted) | 3(0.3) | 3(0.3) |
  | 8 (diluted) | 4(0.404) | 4(0.4) |
  | 9 (diluted) | 4.3(0.423) | 4.3(0.430) |
  | 10 (diluted) | 4.1(0.412) | 4.4(0.441) |
  | 12 (diluted) | 5.91(0.591) | 6.19(0.619) |

  (dilution- 1/10th)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-acetonitrile-3.png)

**Result:** It was confirmed that the presence of acetonitrile does not affect the growth of
*Pseudomonas putida*. The sudden increase in the growth curve may be attributed to either contamination
or the utilization of an alternative carbon source(Diauxic growth).

---

---

## Experiment 10: Growth curve for *P. putida* at pH 8.0

16/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **Growth curve for *Pseudomonas putida* at pH 8.0**

* Growth curves were prepared for *Pseudomonas putida*.
* Four conical flasks were taken and labelled Flask 1, Flask 2, Flask 3, and Blank. The pH was adjusted
  using 1N NaOH and all flasks were autoclaved.
* The flasks were observed for a time period of 12 hours.

| Time (hours) | Flask 1 | Flask 2 | Flask 3 |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 3 | 0.017 | 0.00 | 0.031 |
| 4 | 0.032 | 0.00 | 0.038 |
| 6.5 | 0.592 | 0.074 | 0.684 |
| 7.25 | 0.929 | 0.135 | 1.117 |
| 8.25 | 1.833 | 0.414 | 1.957 |
| 9.25 (diluted) | 2.26 (0.026) | 1.02 (0.102) | 2.6 (0.26) |
| 10.25 (diluted) | 3.48(0.348) | 2.23(0.223) | 3.49(0.349) |
| 11.25 (diluted) | 3.91(0.391) | 2.46(0.246) | 4.05(0.405) |
| 12 (diluted) | 3.7(0.37) | 2.5(0.25) | 4.71(0.471) |

(dilution- 1/10th)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-ph8.png)

---

---

## Experiment 11: Growth curve for *P. putida* at pH 6.0 and 7.0

18/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **Growth curve for *Pseudomonas putida* at pH 6 and 7**

* Growth curves were prepared for *Pseudomonas putida.*
* Three conical flasks were taken and labelled Flask 1, Flask 2, and Blank. The pH was adjusted using 1N HCl
  and all flasks were autoclaved.
* The flasks were observed over a time period of 12 hours.

#### **At pH 6:**

| Time (hours) | Flask 1 | Flask 2 | Average |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 2 | 0.057 | 0.003 | 0.030 |
| 3 | 0.048 | 0.011 | 0.0295 |
| 4 | 0.108 | 0.057 | 0.0825 |
| 5 | 0.313 | 0.174 | 0.2435 |
| 6.5 | 1.034 | 0.66 | 0.847 |
| 8.25 (diluted) | 2.16(0.216) | 1.74(0.174) | 1.95 |
| 9 (diluted) | 2.88(0.288) | 2.45(0.245) | 2.665 |
| 10 (diluted) | 3.83(0.383) | 3.11(0.311) | 3.47 |
| 11 (diluted) | 4.63(0.463) | 3.83(0.383) | 4.23 |
| 12 (diluted) | 5.39(0.539) | 4.76(0.476) | 5.075 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-ph6.jpg)

#### **At pH 7:**

| Time (hours) | Flask 1 | Flask 2 | Average |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 2 | 0.027 | 0.074 | 0.0505 |
| 3 | 0.013 | 0.074 | 0.0435 |
| 4 | 0.099 | 0.131 | 0.115 |
| 5 | 0.373 | 0.437 | 0.405 |
| 6.5 | 1.127 | 1.344 | 1.2355 |
| 8.25 (diluted) | 2.13(0.213) | 2.88(0.288) | 2.505(0.2505) |
| 9 (diluted) | 2.83(0.283) | 3.23(0.323) | 3.03(0.303) |
| 10 (diluted) | 3.3(0.33) | 3.38(0.338) | 3.34(0.34) |
| 11 (diluted) | 4.62(0.462) | 5.1(0.510) | 4.86(0.486) |
| 12 (diluted) | 4.93(0.493) | 5.32(0.532) | 5.125(0.5125) |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-ph7.png)

#### **Comparisons were drawn between growth curves at different pH levels to determine the optimal pH for growth.**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-ph-comparison.png)

**Result:**The optimal pH for growth of *Pseudomonas putida* was observed to be pH 7.5/7.7.

---

---

WEEK 3

×

## Week 3

## Experiment 12: Growth curve for *P. putida* at 24 and 37 degrees Celsius

21/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### ****The effect of temperature on the growth of *Pseudomonas putida* was studied:****

1. 24°C

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 1 | 0 | 0 |
   | 2 | 0.9 | 0.9 |
   | 3 | 0.306 | 0.082 |
   | 4 | 0.493 | 0.165 |
   | 5 | 1.331 | 0.409 |
   | 6 | 2.291 | 0.939 |
   | 7 | 2.75 | 1.5 |
   | 8 | 3.33 | 2.42 |
   | 11 | 10.42 | 9.76 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-at-24.png)
2. 37°C

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 1 | 0 | 0.001 |
   | 2 | 0 | 0.019 |
   | 3 | 0 | 0.025 |
   | 4 | 0 | 0.028 |
   | 5 | 0.008 | 0.028 |
   | 6 | 0.024 | 0.034 |
   | 7 | 0 | 0.08 |
   | 8 | 0.06 | 0.15 |
   | 11 | 3.39 | 0.18 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-at-37.png)

#### **Comparisons were drawn between growth curves at different temperatures to determine the optimal temperature for growth.**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-temp-comparison.png)

**Result:**The abrupt surge in growth at 9th hour in flask 2 is likely due to contamination. It is
observed that *Pseudomonas putida* exhibits superior growth at 24 degrees Celsius, whereas its growth is
adversely affected at 37 degrees Celsius.

---

---

## Experiment 13: Growth curve for *P. putida* at different concentrations of TCC

22/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **Growth curves for *Pseudomonas putida* at different concentrations of TCC**

1. 2000ng/L

   | Time (hours) | Control | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 | 0 |
   | 1 | 0.074 | 0.005 | 0.026 | 0.0155 |
   | 2 | 0.225 | 0.025 | 0.045 | 0.035 |
   | 3 | 0.891 | 0.127 | 0.339 | 0.233 |
   | 4 | 1.384 | 0.323 | 0.558 | 0.4405 |
   | 6 | 2.22 | 1.817 | 2.41 | 2.1135 |
   | 7 | 2.82 | 2.51 | 3.06 | 2.785 |
   | 8 | 3.334 | 2.83 | 4.11 | 3.47 |
   | 9 | contaminated | 4.7 | 4.55 | 4.625 |
   | 10 | contaminated | 4.13 | 4.78 | 4.455 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-2000ngl-tcc.png)
2. 3000 ng/L

   | Time (hours) | Control | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 | 0 |
   | 1 | 0.074 | 0.011 | 0.26 | 0.0185 |
   | 2 | 0.225 | 0.037 | 0.045 | 0.0405 |
   | 3 | 0.891 | 0.178 | 0.207 | 0.1925 |
   | 4 | 1.384 | 0.416 | 0.455 | 0.4355 |
   | 6 | 2.22 | 1.997 | 2.04 | 2.0185 |
   | 7 | 2.82 | 2.68 | 2.76 | 2.72 |
   | 8 | 3.334 | 3.12 | 2.97 | 3.045 |
   | 9 | contaminated | 3.6 | 3.23 | 3.415 |
   | 10 | contaminated | 3.68 | 3.77 | 3.725 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-3000ngl-tcc.png)
3. 4000 ng/L

   | Time (hours) | Control | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 | 0 |
   | 1 | 0.074 | 0.005 | 0.012 | 0.0085 |
   | 2 | 0.225 | 0.032 | 0.033 | 0.0325 |
   | 3 | 0.891 | 0.162 | 0.203 | 0.1825 |
   | 4 | 1.384 | 0.361 | 0.328 | 0.3445 |
   | 6 | 2.22 | 2.007 | 1.921 | 1.964 |
   | 7 | 2.82 | 2.69 | 2.7 | 2.695 |
   | 8 | 3.334 | 3.37 | 2.71 | 3.04 |
   | 9 | contaminated | 3.54 | 3.88 | 3.71 |
   | 10 | contaminated | 3.77 | 3.95 | 3.86 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-4000ngl-tcc.png)

**Result:** It was observed that *Pseudomonas putida* had a slower growth in the presence of TCC when
compared to the control.

---

---

## Experiment 14: Reviving of culture for *P. fluorescens*

23/08/2023

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

Another chassis, *Pseudomonas fluorescens*, was considered for our project. The culture was revived, and
growth curves were prepared for it.

The MTCC (Microbial Type Culture Collection and Gene Bank) number of the culture: 6627

#### *****Pseudomonas fluorescens culture:*****

* A lyophilised culture that was stored in a glass ampoule was transferred into growth media, distributed
  into test tubes, and incubated for further use in nutrient broth at the required optimum temperature and
  time period as mentioned by the culture suppliers (for *Pseudomonas fluorescens*, the incubation was
  done at 30°C for 24 hours).
* The cultures were monitored to observe growth and detect signs of contamination.

---

---

## Experiment 15: Inoculation and streak plating of the culture

24/08/2023

**Project: CarbanEl**

**Authors: Akanksha Kelkar, Vidhula Jessica**

---

#### ***Pseudomonas fluorescens* culture**

* 1 ml of culture from one test tube was transferred into an Eppendorf tube for testing.
* A heat-sterilized inoculation loop was dipped into the Eppendorf tube containing the culture. A Petri dish
  containing nutrient broth agar was taken and quadrant streaking was done.

---

---

## Experiment 16: Confirmatory tests for *P. fluorescens*

25/08/2023

**Project: CarbanEl**

**Authors: Nidhi Mallya, Vidhula Jessica**

---

#### **Confirmatory tests for *Pseudomonas fluorescens***

1. **Gram stain test**- A Gram staining test was performed on the culture. The cells appeared pink when
   viewed under the microscope, confirming that they are Gram-negative.

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/microscopic-view-p-fluorescens.jpg)

   *(Figure: Microscopic view of a bacterial culture - the cells appear as dark pink dots on a lighter
   pink background at a magnification of 100x)*
2. **Indole spot test**- The Indole spot test was performed with the inoculated culture. A piece of
   Whatman filter paper was placed in a petri dish cover and saturated with Kovács reagent. The paper was
   then smeared with cell paste from the culture.There was no visible colour change, indicating a negative
   result. The organism was confirmed to lack the ability to produce tryptophanase.

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/indole-negative-test-p-fluorescens.png)

   *(Figure: Indole negative test of Pseudomonas fluorescens)*
3. **Voges- Proskauer test**- This test was performed and a negative result was obtained. This indicates
   the absence of non-acidic and neutral compounds being formed as end products following metabolism of
   glucose.
4. **Methyl red test**- A Methyl Red Test was performed, and a negative result was obtained. This
   indicates that it does not produce organic acid end products, or acid end products are enzymatically
   converted into non-acidic compounds. This indicates that it does not produce organic acid end products, or
   acid end products are enzymatically converted into non-acidic compounds. Thus proving that *Pseudomonas
   fluorescens* does not ferment glucose.

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/negative-mrvp-test-p-fluorescens.png)

   *(Figure: Two test tubes held up. The first one, labelled VP, has dark yellow liquid in it,
   indicating a negative result for the Voges Proskauer test. The second, labelled MR, has a light yellow
   liquid, indicating a negative result for the Methyl Red test)*
5. **Catalase Test**- *Pseudomonas fluorescens* has the ability to produce catalase, and a positive
   catalase test indicates that the organism possesses this characteristic. When performed, we obtained
   positive results.

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/catalase-positive-test-p-fluorescens.jpg)

   *(Figure: A slide held up to show the presence of effervescence, confirming the production of
   catalase.)*

| Test | Expected Result | Observations | Results |
| --- | --- | --- | --- |
| Gram staining | Gram-negative | Cells appeared pink when viewed under a microscope | Cells were concluded to be Gram-negative |
| Indole test | Negative result (No visible colour change) | Negative results were observed | Cells lack the ability to produce tryptophanase |
| Methyl Red test | Negative result (Culture should remain yellow) | Negative results were observed | Cells produce stable non-acidic end products following metabolism of glucose |
| Voges-Proskauer test | Negative result (No colour change in culture) | Negative results were observed | There is an absence of non-acidic and neutral compounds being formed as end products |
| Catalase Test | Evidence of catalase production | Effervescence was observed | Cells were concluded to be producing catalase |

---

---

## Experiment 17: Growth curve of *Pseudomonas fluorescens* with Acetonitrile

26/08/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **Growth curve for growth of *Pseudomonas fluorescens* in the presence of Acetonitrile**

This growth curve was necessary since, the growth curve in presence of TCC included addition of acetonitrile
for dissolving, hence we needed to ensure that there is not any effect of acetonitrile on the growth.

* Three test tubes, one blank, one a control, and one containing a mix of culture and 1% acetonitrile
  solution, were taken and observed.
* Observation/OD was measured at different time intervals as mentioned in the table below.

| Time (hours) | Acetonitrile-Flask 1 | Acetonitrile-Flask 2 |
| --- | --- | --- |
| 0 | 0 | 0 |
| 1 | 0.006 | 0.008 |
| 2 | 0.051 | 0.055 |
| 3 | 0.154 | 0.055 |
| 4 | 0.561 | 0.156 |
| 5 | 0.753 | 0.261 |
| 6 | 1.373 | 0.53 |
| 7 | 1.951 | 0.82 |
| 8 | 2.42 | 0.909 |
| 9 | 3.2 | 2.03 |
| 10 | 4.07 | 3.28 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluorescens-acetonitrile.png)

It was found that the presence of acetonitrile does not appreciably affect the growth of *Pseudomonas
fluorescens*.

---

---

WEEK 4

×

## Week 4

## Experiment 18: Growth curve of *P. fluorescens* at 24 and 30 degrees Celsius

27/08/2023

**Project: CarbanEl**

**Authors: Wet Lab team**

---

#### **The effect of temperature on the growth of *Pseudomonas fluorescens* was studied:**

1. 24°C

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0 | 0 | 0 |
   | 1 | 0 | 0.001 |
   | 2 | 0.006 | 0.003 |
   | 3 | 0.01 | 0.012 |
   | 4 | 0.033 | 0.015 |
   | 5 | 0.036 | 0.02 |
   | 6 | 0.049 | 0.067 |
   | 7 | 0.087 | 0.095 |
   | 8 | 0.161 | 0.107 |
   | 9 | 0.21 | 0.17 |
   | 10 | 0.237 | 0.285 |
   | 11 | 0.391 | 0.401 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluorescens-at-24c.png)
2. 30°C

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0 | 0 | 0.001 |
   | 1 | 0.001 | 0.002 |
   | 2 | 0.009 | 0.002 |
   | 3 | 0.013 | 0.042 |
   | 4 | 0.038 | 0.049 |
   | 5 | 0.049 | 0.061 |
   | 7 | 0.056 | 0.066 |
   | 8 | 0.102 | 0.093 |
   | 9 | 0.162 | 0.22 |
   | 10 | 1.51 | 1.557 |
   | 11 | 2.223 | 2.29 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluorescens-at-30c.png)

#### **Comparisons were drawn between growth curves at different temperatures to determine the optimal temperature for growth.**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-temp-comparison.png)

**Result:**It was observed that *Pseudomonas fluorescens* exhibits better growth performance at 30
degrees Celsius.

---

---

## Experiment 19: Growth curve of *P. fluorescens* at ph 6.0, 7.0 and 8.0

29/08/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **The effect of pH on the growth of *Pseudomonas fluorescens* was studied:**

* Growth curves were prepared for *Pseudomonas fluorescens*.
* Three conical flasks were taken and labelled Flask 1, Flask 2, and Blank.
* The pH was adjusted using 1N NaOH and all flasks were autoclaved.
* The pH was adjusted using 1N NaOH and all flasks were autoclaved.

**At pH 6:**

| Time (hours) | Flask 1 | Flask 2 | Average |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 2 | 0 | 0.002 | 0.001 |
| 5 | 0.002 | 0.005 | 0.0035 |
| 6 | 0.047 | 0.016 | 0.0315 |
| 7 | 0.072 | 0.043 | 0.0575 |
| 10 | 0.086 | 0.059 | 0.0725 |
| 12 | 0.095 | 0.071 | 0.083 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-at-ph-6.png)

**At pH 7:**

| Time (hours) | Flask 1 | Flask 2 | Average |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 2 | 0.083 | 0.002 | 0.0425 |
| 5 | 0.081 | 0.02 | 0.0505 |
| 6 | 0.061 | 0.011 | 0.036 |
| 7 | 0.14 | 0.066 | 0.103 |
| 10 | 0.408 | 0.415 | 0.4115 |
| 12 | 1.288 | 1.322 | 1.305 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-at-ph-7.png)

**At pH 8:**

| Time (hours) | Flask 1 | Flask 2 | Average |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 2 | 0 | 0 | 0 |
| 5 | 0 | 0.041 | 0.0205 |
| 6 | 0.004 | 0.07 | 0.05 |
| 7 | 0.008 | 0.139 | 0.0735 |
| 10 | 0.488 | 0.88 | 0.684 |
| 12 | 1.361 | 2.062 | 1.7115 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-at-ph-8.png)

#### **Comparisons were drawn between growth curves at different pH levels to determine the optimal pH for growth**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-ph-comparison.png)

**Result:** The optimal pH for growth of *Pseudomonas fluorescens* was observed to be pH 8.

---

---

## Experiment 20: Growth curve of *P. fluorescens* for determining lag phase

30/08/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **Determination of the lag phase of *Pseudomonas fluorescens* with two duplicates:**

* Growth curves were prepared for *Pseudomonas fluorescens*.
* Three conical flasks were taken and labelled Flask 1, Flask 2, and Blank.
* The flasks were observed over a time period of 10 hours.

| Time (hours) | Flask 1 | Flask 2 |
| --- | --- | --- |
| 0 | 0 | 0 |
| 1 | 0.006 | 0.008 |
| 2 | 0.051 | 0.055 |
| 3 | 0.154 | 0.055 |
| 4 | 0.561 | 0.156 |
| 5 | 0.753 | 0.261 |
| 6 | 1.373 | 0.53 |
| 7 | 1.951 | 0.82 |
| 8 | 2.42 | 0.909 |
| 9 | 3.2 | 2.03 |
| 10 | 4.07 | 3.28 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluorescens-lag-phase.png)

**Result:** It was observed that the lag phase for *Pseudomonas fluorescens* extended for a duration
of 5 hours before entering the exponential growth phase.

---

---

## Experiment 21: Growth curve of *P. putida* at different concentrations of TCC

31/08/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **The effect of various concentrations of TCC on the growth of *Pseudomonas putida* was observed**

1. 13000ng/ml

   | Time (hours) | Control | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 | 0 |
   | 2 | 0.074 | 0 | 0.012 | 0.006 |
   | 3 | 0.225 | 0 | 0.011 | 0.006 |
   | 4 | 0.891 | 0 | 0.032 | 0.016 |
   | 4 | 1.384 | 0.361 | 0.328 | 0.3445 |
   | 5 | 1.384 | 0.019 | 0.115 | 0.067 |
   | 6 | 2.22 | 0.045 | 0.207 | 0.126 |
   | 7 | 2.282 | 0.088 | 0.417 | 0.253 |
   | 8 | 3.334 | 0.266 | 0.713 | 0.49 |
   | 9 | 3.5 | 0.645 | 1.342 | 0.994 |
   | 10 | 3.7 | 2.178 | 2.581 | 2.38 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-13000ngml-tcc.png)
2. 17000ng/ml

   | Time (hours) | Control | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 | 0 |
   | 2 | 0.074 | 0.001 | 0 | 0 |
   | 3 | 0.225 | 0.051 | 0 | 0.026 |
   | 4 | 0.891 | 0.133 | 0.004 | 0.069 |
   | 5 | 1.384 | 0.265 | 0.003 | 0.134 |
   | 6 | 2.22 | 0.445 | 0.047 | 0.246 |
   | 7 | 2.282 | 0.813 | 0.131 | 0.472 |
   | 8 | 3.334 | 1.296 | 0.283 | 0.664 |
   | 9 | 3.5 | 1.917 | 1.2 | 1.559 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-17000ngml-tcc.png)
3. 21000ng/ml

   | Time (hours) | Control | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 | 0 |
   | 2 | 0.074 | 0.002 | 0 | 0.001 |
   | 3 | 0.225 | 0.023 | 0.067 | 0.045 |
   | 4 | 0.891 | 0.034 | 0.078 | 0.056 |
   | 5 | 1.384 | 0.094 | 0.254 | 0.174 |
   | 6 | 2.22 | 0.19 | 0.444 | 0.317 |
   | 7 | 2.282 | 0.374 | 0.696 | 0.535 |
   | 8 | 3.334 | 0.645 | 1.198 | 0.922 |
   | 9 | 3.5 | 1.094 | 1.672 | 1.383 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-21000ngml-tcc.png)

**Result:** It was observed that the growth of *Pseudomonas putida* was affected in the presence of
different TCC concentrations and was stunted as compared to control.

---

---

## Experiment 22: Growth curve of *P. fluorescens* at different concentrations of TCC

01/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **The effect of various concentrations of TCC on the growth of *Pseudomonas fluorescens* was observed**

1. 13000ng/ml

   | Time (hours) | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 |
   | 1 | 0 | 0 | 0. |
   | 2 | 0.034 | 0.014 | 0.024 |
   | 4 | 0 | 0.032 | 0.016 |
   | 4 | 0.1 | 0.022 | 0.061 |
   | 5 | 0.125 | 0.062 | 0.0935 |
   | 6 | 0.215 | 0.115 | 0.165 |
   | 7 | 0.426 | 0.26 | 0.343 |
   | 8 | 0.654 | 0.406 | 0.53 |
   | 9 | 1.024 | 0.761 | 0.8925 |
   | 10 | 1.373 | 1.1 | 1.2365 |
   | 21.5 | 6.45 | 4.22 | 5.335 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-13000ngml-tcc.png)
2. 17000ng/ml

   | Time (hours) | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 |
   | 1 | 0 | 0 | 0. |
   | 2 | 0.047 | 0.017 | 0.032 |
   | 4 | 0.104 | 0.078 | 0.091 |
   | 5 | 0.212 | 0.136 | 0.174 |
   | 6 | 0.306 | 0.191 | 0.2485 |
   | 7 | 0.563 | 0.438 | 0.5005 |
   | 8 | 0.839 | 0.66 | 0.7495 |
   | 9 | 1.293 | 1.024 | 1.1585 |
   | 10 | 1.744 | 1.503 | 1.6235 |
   | 21.5 | 4.99 | 5.33 | 5.16 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-17000ngml-tcc.png)
3. 21000ng/ml

   | Time (hours) | Flask 1 | Flask 2 | Average |
   | --- | --- | --- | --- |
   | 0 | 0.007 | 0.019 | 0.013 |
   | 1 | 0.02 | 0.023 | 0.0215 |
   | 2 | 0.021 | 0.024 | 0.0225 |
   | 4 | 0.049 | 0.043 | 0.046 |
   | 5 | 0.094 | 0.186 | 0.14 |
   | 6 | 0.138 | 0.281 | 0.2095 |
   | 7 | 0.322 | 0.519 | 0.4205 |
   | 8 | 0.528 | 0.834 | 0.681 |
   | 9 | 0.844 | 1.189 | 1.0165 |
   | 10 | 1.138 | 1.492 | 1.315 |
   | 21.5 | 5.05 | 6.96 | 6.005 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-21000ngml-tcc.png)

**Result:**It was observed that TCC did not deter the growth of *Pseudomonas fluorescens*.

---

---

WEEK 5

×

## Week 5

---

## Experiment 23: Revival of *Acinetobacter baylyi* culture

10/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### ***Acinetobacter baylyi* culture:**

* A culture that was stored in a glass ampoule was transferred into growth media, distributed into test
  tubes, and incubated for further use at the required optimum temperature and time period as mentioned by the
  culture suppliers. (30 °C for 24 hours).
* The cultures were monitored to observe growth and detect signs of contamination.
* 1 ml of culture from one test tube was transferred into an Eppendorf tube for testing.
* A heat-sterilized inoculation loop was dipped into the Eppendorf tube containing the culture. A Petri dish
  containing nutrient broth agar was taken and quadrant streaking was done.

---

---

## Experiment 24: Confirmatory Test for *A. baylyi*

10/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **Confirmatory tests for *Acinetobacter baylyi*:**

Confirmatory tests were performed to confirm the characteristics of the bacteria. These tests included a gram
staining test, an indole spot test, and a MR-VP test.

1. **Gram staining**- A Gram staining test was performed on the culture. The cells appeared pink and the
   morphology was observed as coccobacillary when viewed under the microscope, confirming that they are
   Gram-negative.
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/microscopic-view-a-baylyi.png)

   *(Figure: Microscopic view of a bacterial culture - the cells appear as dark pink dots on a lighter
   pink background at a magnification of 100x.)*
2. **Indole spot test**- The Indole spot test was performed with the inoculated culture. A piece of Whatman
   filter paper was placed in a petri dish cover and saturated with Kovács reagent. The paper was then smeared
   with cell paste from the culture.There was no visible colour change, indicating a negative result. The
   organism was confirmed to lack the ability to produce tryptophanase.
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/indole-negative-test-a-baylyi.png)

   *(Figure: Indole negative test of Acinetobacter baylyi)*
3. **Methyl Red Voges Proskauer test preparation**- The broth for the test was prepared and inoculated. It
   was kept in the incubator shaker for 24 hours at 37°C.

   After 24 hours

   1. **For Methyl Red Test:** Methyl Red Reagent was added.
   2. **For Voges Proskauer Test:** Barritt Reagent A and Barritt Reagent B were added.

   And a negative result was obtained. This indicates that it does not produce organic acid end products, or
   acid end products are enzymatically converted into non-acidic compounds. Thus proving that
   *Acinetobacter baylyi* does not ferment glucose.

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/mrvp-a-baylyi.jpg)

   *(Figure: Two test tubes held up. The first one, labelled MR, light yellow liquid, indicating a
   negative result for the Methyl Red test. The second, labelled VP, has dark yellow liquid in it,
   indicating a negative result for the Voges Proskauer test.)*

| Test | Expected Result | Observations | Results |
| --- | --- | --- | --- |
| Gram staining | Gram-negative | Cells appeared pink when viewed under a microscope | Cells were concluded to be Gram-negative |
| Indole test | Negative result (No visible colour change) | Negative results were observed | Cells lack the ability to produce tryptophanase |
| Methyl Red test | Negative result (Culture should remain yellow) | Negative results were observed | Cells produce stable non-acidic end products following metabolism of glucose |
| Voges-Proskauer test | Negative result (No colour change in culture) | Negative results were observed | There is an absence of non-acidic and neutral compounds being formed as end products |
| Catalase Test | Evidence of catalase production | Effervescence was observed | Cells were concluded to be producing catalase |

---

---

## Experiment 25: Growth curve of *A. baylyi* in the presence of acetonitrile

10/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **The effect of acetonitrile on the growth of *Acinetobacter baylyi* was observed**

This growth curve was necessary since, the growth curve in presence of TCC included addition of acetonitrile
for dissolving, hence we needed to ensure that there is not any effect of acetonitrile on the growth.

| Time (hours) | Control | Flask 1 | Flask 2 |
| --- | --- | --- | --- |
| 0 | 0 | 0 | 0 |
| 1 | 0.02 | 0.023 | 0.0215 |
| 3 | 0.034 | 0.025 | 0.053 |
| 4 | 0.417 | 0.03 | 0.157 |
| 5 | 1.148 | 0.192 | 0.367 |
| 6 | 2.001 | 0.438 | 1.023 |
| 7 | 3.4 | 0.681 | 1.245 |
| 8 | 4.32 | 1.091 | 1.818 |
| 9 | 4.63 | 1.53 | 2.109 |
| 10 | 4.65 | 1.91 | 3.09 |
| 21.5 | 4.66 | 4.2 | 4.03 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-acetonitrile.png)

**Result:** It is observed that Acetonitrile affects the growth of *Acinetobacter baylyi* and acts as
an inhibitor.

---

---

## Experiment 26: Growth curve of *A. baylyi* at pH 6.5, 7.5, 8.5

14/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **The effect of pH on the growth of *Acinetobacter baylyi* was observed**

* Growth curves were prepared for *Acinetobacter baylyi.*
* Three conical flasks were taken and labelled Flask 1, Flask 2, and Blank.
* The pH was adjusted using 1N NaOH and all flasks were autoclaved.
* The flasks were observed over a time period of 12 hours.

#### ****At pH 6.5:****

| Time (hours) | Flask 1 | Flask 2 |
| --- | --- | --- |
| 0 | 0 | 0 |
| 1 | 0 | 0 |
| 2 | 0.079 | 0.056 |
| 5 | 0.83 | 0.776 |
| 7 | 2.08 | 1.579 |
| 9 | 3.2 | 2.7 |
| 11 | 3.63 | 3.5 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-ph-6-5.png)

#### **At pH 7.5:**

| Time (hours) | Flask 1 | Flask 2 |
| --- | --- | --- |
| 0 | 0 | 0 |
| 1 | 0.032 | 0.03 |
| 2 | 0.063 | 0.068 |
| 5 | 0.777 | 0.618 |
| 7 | 2.18 | 2.267 |
| 9 | 3.78 | 3.92 |
| 11 | 4.32 | 4.02 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-ph-7-5.png)

#### **At pH 8.5:**

| Time (hours) | Flask 1 | Flask 2 |
| --- | --- | --- |
| 0 | 0 | 0 |
| 1 | 0 | 0 |
| 2 | 0.131 | 0.142 |
| 5 | 0.5 | 0.709 |
| 7 | 2.018 | 2.276 |
| 9 | 2.5 | 2.6 |
| 11 | 3.03 | 3.55 |
![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-ph-8-5.png)

#### **Comparisons were drawn between growth curves at different pH levels to determine the optimal pH for growth**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/ph-comparison-ab.png)

**Result:** It was observed that the growth of *Acinetobacter baylyi* was not significantly
influenced by variation in the pH. Thus we infer that it would thrive within this pH range typically found
in wastewater treatment plants.

---

---

WEEK 6

×

## Week 6

---

## Experiment 27: Growth curve of *A. baylyi* at different concentrations of TCC

19/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

#### **The effect of different TCC concentrations on the growth of *Acinetobacter baylyi* was observed.**

1. 13000ng/ml

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0 | 0 | 0 |
   | 1 | 0 | 0 |
   | 2 | 0.069 | 0.055 |
   | 3 | 0.139 | 0.058 |
   | 4 | 0.143 | 0.159 |
   | 5 | 0.201 | 0.34 |
   | 6 | 0.587 | 0.6 |
   | 7 | 1.015 | 1.088 |
   | 8 | 1.377 | 1.46 |
   | 9 | 1.68 | 1.878 |
   | 10 | 2.115 | 2.056 |
   | 11 (diluted) | 2.79 (0.279) | 3.18 (0.318) |
   | 12 (diluted) | 2.93 (0.293) | 2.89 (0.289) |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-13000ngml.png)
2. 15000ng/ml

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0. | 0.024 | 0.059 |
   | 1 | 0.037 | 0.048 |
   | 2 | 0.119 | 0.049 |
   | 3 | 0.13 | 0.05 |
   | 4 | 0.135 | 0.042 |
   | 5 | 0.26 | 0.26 |
   | 6 | 0.415 | 0.48 |
   | 7 | 0.716 | 0.893 |
   | 8 | 1.215 | 1.189 |
   | 9 | 1.58 | 1.658 |
   | 10 | 2.032 | 1.992 |
   | 11 (diluted) | 3.06 | 2.51 |
   | 12 (diluted) | 3.36 | 3.67 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-15000ngml-tcc.png)
3. 19000ng/ml

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0. | 0.013 | 0.027 |
   | 1 | 0.004 | 0.039 |
   | 2 | 0.045 | 0 |
   | 4 | 0.139 | 0.153 |
   | 5 | 0.091 | 0.271 |
   | 6 | 0.334 | 0.346 |
   | 7 | 0.868 | 0.993 |
   | 8 | 1.223 | 1.428 |
   | 9 | 1.749 | 1.814 |
   | 10 | 2.101 | 2.089 |
   | 11 (diluted) | 2.3 (0.23) | 2.66 (0.266) |
   | 12 (diluted) | 4.21(0.421) | 3.67(0.367) |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-19000ngml-tcc.png)
4. 21000 (ng/ml)

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0. | 0 | 0.048 |
   | 1 | 0 | 0.089 |
   | 2 | 0.066 | 0.099 |
   | 3 | 0.069 | 0.107 |
   | 4 | 0.208 | 0.163 |
   | 5 | 0.273 | 0.283 |
   | 6 | 0.355 | 0.452 |
   | 7 | 0.782 | 0.69 |
   | 8 | 1.084 | 1.005 |
   | 9 | 1.636 | 1.008 |
   | 10 | 2.02 | 1.598 |
   | 11 (diluted) | 2.75(0.275) | 3.53(0.353) |
   | 12 (diluted) | 3.11(0.311) | 3.98(0.398) |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-21000ngml-tcc.png)

**Results:** It is observed that *Acinetobacter baylyi* is capable of growing in the presence of TCC.
*Acinetobacter baylyi* and other gram negative bacteria have efflux pumps to remove toxic substances that
allow it to survive[1](#ref1). This might explain why *A.baylyi* grows in a TCC
containing environment.

---

---

## **Comparison of the growth of *Pseudomonas putida*, *Pseudomonas fluorescens* and *Acinetobacter baylyi* at different TCC concentrations**

---

1. 13000ng/ml

   | Time (hours) | *P. putida* | *P. fluorescens* | *A. baylyi* |
   | --- | --- | --- | --- |
   | 0 | 0 | 0 | 0 |
   | 1 | 0.002 | 0 | 0 |
   | 2 | 0.006 | 0.024 | 0.069 |
   | 3 | 0.006 | 0.03 | 0.139 |
   | 4 | 0.016 | 0.061 | 0.143 |
   | 5 | 0.067 | 0.0935 | 0.201 |
   | 6 | 0.126 | 0.165 | 0.587 |
   | 7 | 0.253 | 0.343 | 1.015 |
   | 8 | 0.49 | 0.53 | 1.377 |
   | 9 | 0.994 | 0.8925 | 1.68 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/comparison-of-three-cultures-at-13000-ngml.png)

   **Results:** It was observed that at a TCC concentration of 13,000ng/ml,*Acinetobacter baylyi*
   exhibited a better growth pattern that *Pseudomonas putida*and *Pseudomonas fluorescens*.
2. 21000ng/ml

   | Time (hours) | *P. putida* | *P. fluorescens* | *A. baylyi* |
   | --- | --- | --- | --- |
   | 0 | 0 | 0.013 | 0 |
   | 1 | 0 | 0.0215 | 0 |
   | 2 | 0.001 | 0.0225 | 0.066 |
   | 3 | 0.045 | 0.03 | 0.069 |
   | 4 | 0.056 | 0.046 | 0.208 |
   | 5 | 0.174 | 0.14 | 0.273 |
   | 6 | 0.317 | 0.2095 | 0.355 |
   | 7 | 0.535 | 0.4205 | 0.782 |
   | 8 | 0.922 | 0.681 | 1.084 |
   | 9 | 1.383 | 1.0165 | 1.636 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/comparison-of-three-cultures-at-21000ngml.png)

   **Results:** It was observed that at a TCC concentration of 21,000ng/ml, *Acinetobacter baylyi*
   showed higher growth followed by *Pseudomonas putida* and *Pseudomonas fluorescens* showed the
   least growth.

---

---

WEEK 7

×

## Week 7

---

## Experiment 28: Growth curve of *E. coli* BL21 with transformed tccA + pET22b+

27/09/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

| Time (hours) | BL21 | BL21 Transformed |
| --- | --- | --- |
| 1 | 0.104 | 0.027 |
| 2 | 0.189 | 0.05 |
| 3 | 0.898 | 0.089 |
| 4 | 0.208 | 0.163 |
| 5 | 1.723 | 0.102 |
| 6 | 2.535 | 0.831 |
| 7 | 3.15 | 2.72 |
| 8 | 3.17 | 2.54 |
| 9 | 3.29 | 2.92 |
| 10 | 3.31 | 2.95 |
| 11 | 3.69 | 36.56 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/e-coli-bl21-growth-curve.png)

| Time (hours) | BL21 | BL21 Transformed |
| --- | --- | --- |
| 1 | 0.071 | 0.023 |
| 2 | 0.132 | 0 |
| 3 | 0.397 | 0 |
| 4 | 1.271 | 0 |
| 5 | 1.723 | 0.102 |
| 6 | 2.056 | 0.002 |
| 7 | 2.54 | 0.078 |
| 8 | 2.3 | 0.137 |
| 9 | 3.23 | 0.496 |
| 10 | 2.63 | 0.909 |
| 11 | 2.72 | 1.011 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/e-coli-bl21-tcc.png)

**Result**: Although the transformed BL21 did not grow as much as the non- transformed, TCC content in the
supernatant was measured and it showed less concentration of TCC as compared to the initial concentration in
which it was inoculated. The inoculated TCC concentration was observed to be 0.030 OD in LB broth and the
final OD was 0.001. However, more experimentation is required to clearly conclude this result.

---

---

WEEK 8

×

## Week 8

---

---

## Experiment 29: Growth curve of *A. baylyi* at 30 and 37 degree celsius

03/10/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

**The effect of temperature on the growth of *Acinetobacter baylyi* was studied:**1. 30 degrees Celsius

   | Time (hours) | Flask 1 | Flask 2 |
   | --- | --- | --- |
   | 0 | 0 | 0 |
   | 1 | 0 | 0 |
   | 2 | 0 | 0 |
   | 4.5 | 0.042 | 0.04 |
   | 5.5 | 0.043 | 0.055 |
   | 6.5 | 0.123 | 0.181 |
   | 7.5 | 0.44 | 0.64 |
   | 9 | 1.251 | 1.522 |
   | 11 | 2.7 | 3.05 |
   | 12 | 3.01 | 3.19 |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-at-30c.png)
2. 37 degrees Celsius

| Time (hours) | Flask 1 | Flask 2 |
| --- | --- | --- |
| 0 | 0.115 | 0 |
| 3 | 0.093 | 0.015 |
| 4 | 0.001 | 0 |
| 5 | 0.018 | 0.038 |
| 6 | 0.472 | 0.341 |
| 7 | 1.02 | 0.94 |
| 8 | 1.584 | 1.535 |
| 9 | 2.016 | 1.989 |
| 10 | 3.1 | 2.42 |
| 11 | 3.05 | 3.11 |
| 12 | 3.11 | 3.63 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-at-37c.png)

**Comparisons were drawn between growth curves at different temperatures to determine the optimal
temperature for growth.**

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-temp-comparison.png)

**Result**: It was observed that the growth of *Acinetobacter baylyi* was not deterred by the change
in temperature.

---

---

## Experiment 30: Growth curve of *Pseudomonas putida, Pseudomonas fluorescens, Acinetobacter baylyi* for calibrating wet weight

09/10/2023

**Project: CarbanEl**

**Authors: Wet Lab Team**

---

A secondary inoculation was done for all three cultures and OD and weight was calculated every 30 minutes
till the culture reached stationary state.

For ***Pseudomonas putida***

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/putida-wet-weight.jpg)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-putida-wet-weight.png)

For ***Pseudomonas fluorescens***

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/fluoro-wet-weight.jpg)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/p-fluoro-wet-weight.png)

For ***Acinetobacter baylyi***

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/baylyi-wet-weight.jpg)

![](https://static.igem.wiki/teams/4641/wiki/notebook-growth-curves/notebook-growth-curves/a-baylyi-wet-weight.png)

## **References:**

1. Kumar, A. (2021, June 17). Update on Multidrug Resistance Efflux Pumps in Acinetobacter spp.
   NCBI. Retrieved October 4, 2023, from <https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218648/>

Cloning and Expression

WEEK 1

×

---

---

### **Experiment 1: (C&E) Amplification of tccA gene**

**02/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica, Nidhi Mallya**

---

#### **Dissolving lyophilized IDT tccA gene fragment**

1. 100 μL autoclaved Milli-Q water was added to the lyophilized tccA gene.
2. Invert the tube a few times.
3. The sample was then vortexed for 1 sec
4. The sample was centrifuged at 14000 rpm for 30sec and placed in ice immediately
5. OD was measured at 260 nm
6. The mixture was placed on ice
7. Concentration was checked at 260nm (10 μL/ mL) and stored at -20°C.

1. **TccA Primer Synthesis for Amplification of Gene Fragment**
   * Primer Design

     | Primer | Primer sequence (5’ - 3’) | Number of bases | GC content | Tm value(°C) |
     | --- | --- | --- | --- | --- |
     | Forward Primer | CAGCCGGCGATGGCCATGGATATCG | 25 | 64% | 69.5 |
     | Reverse Primer | GTTAGCAGCCGGATCTCAGTGG | 22 | 59.1% | 64 |
   * Primer Report

     | Primer | Primer sequence (5’ - 3’) | Yield (OD) | Volume for 100 pmol/μL |
     | --- | --- | --- | --- |
     | Forward Primer | CAGCCGGCGATGGCCATGGATATCG | 8.4 | 368.9 |
     | Reverse Primer | GTTAGCAGCCGGATCTCAGTGG | 5 | 249.3 |
2. **Dissolving lyophilised tccA primers**
   1. 368.9 μL of autoclaved Milli-Q water was added to Forward Primer, and 249.3 μL of autoclaved
      Milli-Q water was added to tccA Reverse Primer.
   2. The mixture was gently mixed.
   3. The sample was vortexed for 1 sec.
   4. The sample was centrifuged at 14000 rpm for 30sec.
   5. The sample was placed in ice immediately.
   6. 10 μL of sample was taken and dissolved in 90 μL of Milli-Q water.
   7. Pipette mix it GENTLY.
   8. Check OD at 260nm and stored at -20 °C
3. **PCR Reaction Mix Preparation for tccA Gene Amplification**
   * A reaction mixture for 50 μL PCR Reaction was prepared in a 0.2 mL PCR tube
   * Thus, the Total PCR Reaction Mixture = 50 μL
   * The total 50 μL PCR reaction mixture was then divided into two separate 0.2 mL PCR tubes, each
     having 25 μL. The 3 PCR reaction mixtures were divided finally into 6 samples.

   | Component | Volume | Concentration Available | Volume | For 10 Reactions |
   | --- | --- | --- | --- | --- |
   | Q5 High-Fidelity 2X Master Mix | 12.5 ul | 2X | 1.25 ul | 62.5 ul |
   | 10uM Forward Primer | 1.25 ul | 10 mM | 1.25 ul | 6.25 ul |
   | 10uM Reverse Primer | 1.25 ul | 10 mM | 1.25 ul | 6.25 ul |
   | Template DNA | 1 pg - 10ng |  | 10ng (1 full) | 5 ul |
   | Nuclease-Free Water | To make 25 ul |  | To make 25 ul | 45 ul |
   | Total Volume | 25 ul |  | 25 ul |  |

   | Component | Negative-1 | Negative-2 | Positive |
   | --- | --- | --- | --- |
   | Q5 High-Fidelity 2X Master Mix | 12.5 ul |  | 1.25 ul |
   | 10uM Forward Primer | 1.25 ul | 1.25 ul | 1.25 ul |
   | 10uM Reverse Primer | 1.25 ul | 1.25 ul | 1.25 ul |
   | Template DNA |  | 5 ng (0.5 ul of 10ng/ul) | 10ng (1 full) |
   | Nuclease-Free Water | To make 25 ul | To make 25 ul | To make 25 ul |
   | Total Volume | 25 ul | 25 ul | 25 ul |

   For negative control- 1. No template DNA 2. No Q5 For positive control- Mam7 (mam7 was used as it has
   been already used it our lab with consistent Results and standardised protocols)
4. **Thermocycling Conditions for tccA Gene Amplification through PCR**
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/thermocycling-conditions-for-tcca-gene-amplification.png)

   (Thermocycling conditions for tccA gene amplification)

   Annealing Temperature- Gradient PCR from 55 C- 72 C.

   **PROTOCOL FOR GRADIENT PCR:**
   * CalcuLations were made for a master mix containing water, 2X Q5 high-fidelity master mix from NEB,
     and primers. If n reactions are required, prepare a master mix for n+1 reactions.
   * This master mix was prepared in an 0.5 mL Eppendorf tube and aliquoted into 9 separate PCR tubes
     for 8 samples for gradient PCR and sample 11 (negative control with no DNA). Sample 9 was the
     negative control with 10 ng of tccA and water.
   * Sample 10 was a positive control with the MAM7 gene.
5. ****Running of gel electrophoresis for Amplified tccA****

   (Lane 1 of Gel 1 and Gel 2: 1kb DNA ladder
   Lane 2-7 in Gel 1: tccA amplified 55-
   Lane 2-3 in
   Gel 2: tccA amplified)

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/running-of-gel-electrophoresis-for-amplified-tcca-run-1.png)

---

---

### **Experiment 2: (C&E) Amplification of tccA gene**

**04/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica, Nidhi Mallya**

---

1. **PCR Reaction Mix Preparation for tccA Gene Amplification**
   * A Master Mix for 50 μL PCR Reaction Mixture was prepared in a 0.2 mL PCR tube
   * Thus, the Total PCR Reaction Mixture = 50 μL
   * The total 50 μL PCR reaction mixture was then divided into two separate 0.2 mL PCR tubes, each
     having 25 μL. The 3 PCR reaction mixtures were divided finally into 6 samples.

   | Component | Volume | Concentration Available | Volume | For 10 Reactions |
   | --- | --- | --- | --- | --- |
   | Q5 High-Fidelity 2X Master Mix | 12.5 ul | 2X | 1.25 ul | 62.5 ul |
   | 10uM Forward Primer | 1.25 ul | 10 mM | 1.25 ul | 6.25 ul |
   | 10uM Reverse Primer | 1.25 ul | 10 mM | 1.25 ul | 6.25 ul |
   | Template DNA | 1 pg - 10ng |  | 10ng (1 full) | 5 ul |
   | Nuclease-Free Water | To make 25 ul |  | To make 25 ul | 45 ul |
   | Total Volume | 25 ul |  | 25 ul |  |

   | Component | Negative-1 | Negative-2 | Positive |
   | --- | --- | --- | --- |
   | Q5 High-Fidelity 2X Master Mix | 12.5 ul |  | 1.25 ul |
   | 10uM Forward Primer | 1.25 ul | 1.25 ul | 1.25 ul |
   | 10uM Reverse Primer | 1.25 ul | 1.25 ul | 1.25 ul |
   | Template DNA |  | 5 ng (0.5 ul of 10ng/ul) | 10ng (1 full) |
   | Nuclease-Free Water | To make 25 ul | To make 25 ul | To make 25 ul |
   | Total Volume | 25 ul | 25 ul | 25 ul |

   For negative control- 1. No template DNA 2. No Q5 For positive control- Mam7
2. **Thermocycling Conditions for tccA Gene Amplification through PCR**
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/thermocycling-conditions-for-tcca-gene-amplification.png)

   (Thermocycling conditions for tccA gene amplification)

   Annealing Temperature- Gradient PCR from 67C- 69 C.

   #### **PROTOCOL FOR GRADIENT PCR:**

   * CalcuLations were made for a master mix containing water, 2X Q5 high-fidelity master mix, and
     primers. If n reactions are required, prepare a master mix for n+1 reactions.
   * This master mix was prepared in an Eppendorf tube and aliquoted into 9 separate PCR tubes for 8
     samples for gradient PCR and sample 11 (negative control with no DNA). Sample 9 was the negative
     control with 10 ng of tccA and water.
   * Sample 10 was a positive control with the MAM7 gene.
3. **Running of gel electrophoresis for Amplified tccA** (RUN 2)
   ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/amplified-tcca-run-2.png)

---

---

### **Experiment 3: (C&E) Primary Inoculation of DH5alpha and pET22b+ in LB broth**

**05/09/2023**

**Project: CarbanEl**

**Authors: I Vidhula Jessica, Aditi Balasubramani, Manu Upadhye**

---

#### **Primary inoculation of *E.coli* DH5alpha**

1. 200 μL of DH5alpha glycerol stock was inoculated in 5 mL of LB broth
2. A heat fixed loop was used to pick a colony from pET22b+ plate and Inoculated into 5 mL of LB broth
   with 5 μL of Ampicillin

**Result:** No growth was observed in the test tubes inoculated with pET22b+

**Conclusion:**The experiment to be repeated, after streaking new plates of pET22b+ dh5alpha plate.

---

---

### **Experiment 4: (C&E) PCR cleanup**

**06/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, Manu Upadhye**

---

#### **PCR cleanup (QIAGEN):**

**Lanes from the lower range of temperature (65-67) was purified**

1. Each separate 25 μL of Amplified tccA
2. 125 μL of Buffer PB was added to the tccA amplified mix.
3. The mixture was then transferred to a QIAquick column which was placed in the provided 2 mL collection
   tube.
4. The column was centrifuged for 30–60s at 13000rpm, 32°C
5. The flow-through was then discarded, and the QIAquick column was placed back in the same tube.
6. 750 µl Buffer PE was added to the QIAquick column and centrifuged for 30–60s at 13000rpm,32°C for
   washing.
7. The QIAquick column was once again centrifuged for 1min at 13000rpm, 32°C in a new 2 mL collection
   tube that was provided to remove residual wash buffer.
8. The QIAquick column was then placed in an autoclaved 1.5 mL Eppendorf tube.
9. 25 μL of Buffer EB (10 mM Tris·Cl, pH 8.5) was added to the centre of the QIAquick membrane and let to
   stand for 1min
10. The QIAquick column was centrifuged for 1min at 13000rpm, 32°C to elute the DNA.
11. To the same QIAquick membrane, 15 μL of Buffer EB was added to the centre and placed in a new 2 mL
    collection tube that was provided, which was centrifuged for 1min at 13000rpm, 32°C to elute the DNA
    again.
12. The concentration of the gene was determined by a UV spectrophotometer at 260nm.

| Lane | Concentration (ug/mL) |
| --- | --- |
| 1 | 30.7 |
| 2 | 14.2 |
| 3 | 24.7 |
| 4 | 36.6 |

---

---

### **Experiment 5: (C&E)Primary inoculation of pET22b+**

**08/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

1. A heat sterilised loop was used to pick a colony from pET22b+ plate and Inoculated into 5 mL of LB
   broth with 5 μL of Ampicillin
2. The test tube was incubated at 37 °C for 16 hours at 180rpm.

---

---

### **Experiment 6: (C&E)Plasmid isolation of pET22b+**

**09/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

**Plasmid Isolation of pET22b+ (QIAGEN miniprep kit)**

1. The culture samples (1-10) were pelleted by centrifuging at 13000rpm for 10min, 4°C
2. The pelleted bacterial cells were resuspended in 250 μl Buffer P1 and transferred to a microcentrifuge
   tube.
3. 250 μl of Buffer P2 was added and mixed thoroughly by inverting the tube 4–6 times until the solution
   became clear.
4. 350 μl of Buffer N3 was added and mixed immediately and thoroughly by inverting the tube 4–6 times.
5. The mixture was then centrifuged for 10 min at 13,000 rpm (~17,900 x g) in a table-top
   microcentrifuge.
6. 800 μl of the supernatant was added to a QIAprep 2.0 spin column by pipetting.
7. The mixture was then centrifuged for 30–60 s at 13000rpm and the flow-through was discarded
8. The QIAprep 2.0 spin column was washed by adding 0.75 mL Buffer PE. m. The mixture was centrifuged for
   30–60 s for 13000rpm
9. The QIAprep 2.0 spin column was transferred to the collection tube.
10. The tube was then centrifuged for 1 min to remove the residual wash buffer.
11. The QIAprep 2.0 column was placed in a clean 1.5 mL microcentrifuge tube.
12. To elute DNA, 30 μl of Buffer EB (10 mM Tris-HCl, pH 8.5) was added first to the center of the QIAprep
    2.0 spin column, and let stand for 5 minutes, then centrifuged for 1 min at 13000rpm.
13. The elution was again done by adding 15 μl of Buffer EB (10 mM Tris-HCl, pH 8.5) and was let to stand
    for 4min, centrifuged at 13000rpm for 1min.
14. The OD Value of the Isolated plasmid was then measured using a spectrophotometer.

| Plasmid Sample | Concentration (in ug/ mL) |
| --- | --- |
| 1 | 81.7 |

---

---

WEEK 2

×

## Week 2

### **Experiment 7: (C&E)Plasmid isolation of pET22b+**

**10/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

To ensure sufficient stock of the plasmid, we performed plasmid isolation again with the same protocols
as above and measured concentration.

| Plasmid Sample | Concentration (in ug/mL) |
| --- | --- |
| 1 | 120.3 |
| 2 | 53.7 |

---

---

### **Experiment 8: (C&E)Double Digestion of tccA and pET22b+**

**14/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

### **Double digest of amplified tccA and pET22b+ with BamHI and XhoI**

1. Double digestion reaction mix preparation for tccA gene and pET22b+ vector as follows:

   | Component | TccA | pET22b+ |
   | --- | --- | --- |
   | Template DNA | (for 250ng of insert) | (for 250ng of vector) |
   | 10X cut smart buffer | 2.5 | 2.5 |
   | XhoI enzyme | 0.25 | 0.25 |
   | BamHI enzyme | 0.25 | -0.25 |
   | MilliQ water | Make upto 25 μL | Make upto 25 μL |
   | Total Volume | 25 | 25 |
2. Thermocycling conditions for double digest of amplified tccA and pET22b+

   | Step | Temperature (°C) | Time |
   | --- | --- | --- |
   | Incubation | 37 | 3 hours |
   | Enzyme inactivation (BamHI and XhoI) | 65 | 20 minutes |
   | Hold | 4 | Infinite |

   ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/double-digest-tcca-and-pet22b.png)

   (lane 1- 1kb ladder, lane 2,3,4-pET22b+, lane 5,6,7- tccA amplified)

---

---

WEEK 3

×

## Week 3

### **Experiment 9: Primary inoculation for competent cell preparation**

**21/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

### **Primary inoculation for competent cell preparation**

1. 200 μL of E.coli DH5alpha glycerol stock was Inoculated into a test tube with 5 mL of LB broth and
   another test tube was taken and 2 mL of LB broth was added as blank.
2. The test tubes were incubated at 37 °C, 180 rpm for 16 hours.

---

---

### **Experiment 10: (C&E)Gibson Assembly**

**22/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

### **Gibson assembly (NEB E5510S kit was used)**

| Component | Volume(μl) | Concentration Available |
| --- | --- | --- |
| Gibson Assembly Master Mix | 10 | 2X |
| Template DNA | 2 | 50 ng/μl |
| Insert DNA | 0.25 | 38 ng/μl |
| Nuclease-free Water | 7.25 |  |
| Total Volume | 20 |  |

---

---

### **Experiment 11: (C&E)Competent cell preparation and Transformation**

**22/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Transformation of Gibson assembled tccA + pET22b+ to E.coli dh5alpha using CaCl2 method.**

1. #### **Chemical and Media Preparation**

   1. 0.1M CaCl2
      1. 11.1g of anhydrous CaCl2 was weighed out and added to 80 mL of autoclaved distilled water.
      2. The solution was mixed until the CaCl2 was fully dissolved
      3. The solution was then topped up to 100 mL
      4. 10 mL of the above solution was taken and then diluted by adding 90 mL of autoclaved distilled
         water for 1:10 dilution
      5. The solution was then filter sterilized through a PVDF filter of pores size 0.22μm.
   2. LB Ampiclin plates (X10)
      1. 6g of LB agar powder was weighed out and added to 250 mL of distilled water
      2. The LB agar solution was then autoclaved. 0.25 μL of Ampiclin was then dissolved into the LB
         agar solution
      3. 25 mL of LB-ampicillin solution was then poured into each Petri plate.
   3. 100 mL LB broth
      1. 2.5g of LB broth powder was weighed out and added to 100 mL of distilled water
      2. The LB broth solution was then autoclaved.
2. #### **Competent Cell preparation**

   1. E.coli DH5-alpha culture preparation One culture sample were prepared with one blank containing
      just the LB broth
   1. 200 μL of E.coli DH5-alpha from the glycerol stock was Inoculated onto 5 mL of LB
   2. The culture was then incubated at 37°C and 200rpm for 16 hours.
   3. The amount of growth of DH5-alpha culture was measured using a spectrophotometer at absorbance
      A600.2. The sample was transferred to 15 mL falcon tubes that were placed on ice.
   3. The falcon tube was then centrifuged at 4°C at 5600g for 10 minutes for pelleting the DH5-alpha
      culture.
   4. The supernatant was discarded.
   5. Each pellet was then resuspended with 1 mL ice-cold 0.1M CaCl2.
   6. The total 2 mL cell suspension was then incubated on ice for 30 minutes
   7. The suspension was then again centrifuged at 4°C at 5600g for 10 minutes
   8. The supernatant was discarded
   9. Each pellet was then again resuspended in 500 μL of ice-cold 0.1M CaCl2.
   10. Five 100 μL aliquots of competent cells were prepared in ice-cold 1.5 mL eppendorf tube
   11. The prepared competent cells were then stored at -80°C
3. #### **Transformation**

   1. 10ng of plasmid (pET22b+) was added to each aliquot (For required transformant and positive
      control)
   2. The cells were then incubated on ice for 30 minutes
   3. The cells were then placed in a 42°C water bath for exactly 30 seconds
   4. The cells were again placed on ice for 2 minutes
   5. 1 mL of pre-warmed LB media was then added to each aliquot.
   6. The aliquots or cells were then incubated at 37°C, 200rpm for 1-hour.
4. #### **Plating and incubation**

   1. 100 μL of the transformed cells from the aliquots of transformed cells were then spread plated on
      the LB ampicillin plates.
   2. The plates were then incubated at 37°C for 12-16 hours.

**Result: 23/09/2023**

Viewed transformed plates

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/blank.jpg)

Blank (dh5alpha on LB Ampicillin Plate)

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/empty-vector-pet22b.jpg)

(LB Ampicillin plate with empty vector pET22b+)

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/transformed-plate-in-dh5alpha.jpg)

(Transformed plate-contains tccA gibson assembled pET22b+ transformed DH5alpha cells)

---

---

### **Experiment 12: (C&E)Colony Polymerase Chain Reaction**

**23/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### Colony PCR(try 1)

1. PCR reaction Mix preparation for colony PCR of transformed cells
   1. Colony Cell suspension preparation:
      A freshly autoclaved micropipette was touched onto an
      isolated colony and then dipped into the respective Eppendorf tube containing 100 μL of
      Nuclease-free (autoclaved Milli-Q) water.
   2. A colony PCR was done for 10 colonies with 25 μL of the colony PCR mix for each colony
   3. Thus each PCR reaction mix consisted of:

      | Component | Volume (μL) |
      | --- | --- |
      | Q5 Master Mix (2X) (from NEB) | 12.5 |
      | TccA Forward Primer | 1.5 |
      | TccA Reverse Primer | 1.5 |
      | Autoclaved MilliQ Water | 8.5 |
      | Colony Cell Suspension | 1.0 |
      | Total | 25 |
   4. A positive control was prepared of 25 μL.
2. Thermocycling conditions for colony PCR

   | Step | Temperature °C | Time | Cycles |
   | --- | --- | --- | --- |
   | Initial Denaturation and Lysis | 98 | 3 minutes | - |
   | Cycling Denaturation | 98 | 15 seconds | 30 Cycles |
   | Cycling Annealing | 51 | 30 seconds | 30 Cycles |
   | Cycling Extension | 72 | 90 seconds | 30 Cycles |
   | Final Extension | 72 | 10 minutes | - |
   | Hold | - | - | - |
3. Gel electrophoresis for Colony PCR
   1. Preparation of 1% agarose Gel
      1. 0.3 g of agarose was weighed and added to 30 mL of 1X TAE to give 1% agarose gel.
      2. The agarose was boiled till it dissolved completely in a clear solution
      3. 2 μL of SYBR safe dye (invitrogen) was added to the solution
   2. Electrophoresis:
      1. The electrophoresis combs were set to be approximately 2 cm away from the cathode
      2. The agarose solution was poured into the central part of the tank when the temperature reached
         approximately 60 °C.
      3. The gel was kept undisturbed at room temperature fro the agarose to solidify.
      4. 1X TAE buffer was poured into the gel tank till the buffer level stodd at 0.5-0.8cm above the
         gel surface
      5. The combs were gently lifted ensuring the wells remained inatact.
      6. The power cord was connected to the electrophoretic power supply

      | Well | DNA Template | Volume of Reaction Mixture | Volume of Gel Dye |
      | --- | --- | --- | --- |
      | 1 (of both gels) | Ladder (1kb) | 6 μL | - |
      | 2-7 (gel 1) | Colony 1-6 | 25 μL | 4 |
      | 2-5 (gel 2) | Colony 7-10 | 25 μL | 4 |

**Result:**

DNA ladder did not show up in the UV transilluminator, co μLd be due to inappropriate preparation of
ladder,

**Conclusion:** Repeat the experiment after vortexing the DNA ladder before loading.

#### **Inoculated 10 colonies in LB for plasmid isolation**

1. From the cell suspension sample, 50 μL was Inoculated into 5 mL of LB broth containing ampicillin (5
   μL).
2. The test tubes were incubated in 37 °C at 180 rpm.

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/colony-pcr-result-1.png)

(Colony PCR result: Ladder is not visible, the experiment was repeated again)

---

---

### **Experiment 13: (C&E)Plasmid isolation for selected colonies**

**24/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Plasmid isolation (QIAGEN miniprep Kit)**

1. The culture samples (1-10) were pelleted by centrifuging at 13000rpm for 10min, 4°C
2. The pelleted bacterial cells were resuspended in 250 μl Buffer P1 and transferred to a microcentrifuge
   tube.
3. 250 μl of Buffer P2 was added and mixed thoroughly by inverting the tube 4–6 times until the solution
   became clear.
4. 350 μl of Buffer N3 was added and mixed immediately and thoroughly by inverting the tube 4–6 times.
5. The mixture was then centrifuged for 10 min at 13,000 rpm (~17,900 x g) in a table-top
   microcentrifuge.
6. 800 μl of the supernatant was added to a QIAprep 2.0 spin column by pipetting.
7. The mixture was then centrifuged for 30–60 s at 13000rpm and the flow-through was discarded
8. The QIAprep 2.0 spin column was washed by adding 0.75 mL Buffer PE. m. The mixture was centrifuged for
   30–60 s for 13000rpm
9. The QIAprep 2.0 spin column was transferred to the collection tube.
10. The tube was then centrifuged for 1 min to remove the residual wash buffer.
11. The QIAprep 2.0 column was placed in a clean 1.5 mL microcentrifuge tube.
12. To elute DNA, 30 μl of Buffer EB (10 mM Tris-HCl, pH 8.5) was added first to the center of the QIAprep
    2.0 spin column, and let stand for 5 minutes, then centrifuged for 1 min at 13000rpm.
13. The elution was again done by adding 15 μl of Buffer EB (10 mM Tris-HCl, pH 8.5) and was let to stand
    for 4min, centrifuged at 13000rpm for 1min.
14. The OD Value of the Isolated plasmid was then measured using a spectrophotometer at 260nm.

| Colony | Concentration (ug/mL) |
| --- | --- |
| 1 | 60.8 |
| 2 | 38.4 |
| 3 | 39.6 |
| 4 | 43.2 |
| 5 | 24.0 |
| 6 | 32.1 |
| 7 | 46.8 |
| 8 | 35.6 |
| 9 | 36.9 |
| 10 | 54.6 |

---

---

### **Experiment 14: (C&E)Streaking of Master Plate**

**25/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Streaked master plate for the colonies**

An inoculation loop was heat sterilised and plate was streaked for each colony to prepare a master plate

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/the-10-colonies.jpg)

(All 10 colonies from the cell suspension streak plated)

**Result (25/09/2023):**

Viewed master plate after 24 hours

WEEK 4

×

## Week 4

### **Experiment 15: (C&E) Double digestion of isolated Plasmid (tccA+pET22b+)**

**28/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Double digestion of Gibson assembled tccA + pET22b+ cloned in DH5alpha**

This experiment was carried out as an additional step to ensure the presence of the insert in the pET22b+

1. Double digestion reaction mix preparation

   | Component | Volume |
   | --- | --- |
   | template | (500ng of vector) |
   | 10X cutsmart buffer | 5 μL |
   | XhoI enzyme | 0.5 μL |
   | BamHI enzyme | 0.5 μL |
   | Nuclease Free water | Make upto 50 μL |
   | Total | 50 μL |
2. Thermocycling conditions for pET22b+ double digestion

   | Step | Temperature (°C) | Time |
   | --- | --- | --- |
   | Incubation | 37 | 3 hours |
   | Enzyme inactivation (BamHI and XhoI) | 65 | 20 minutes |
   | Hold | 4 | Infinite |

**Result:**

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/double-digestion.png)

(Lane 1 of Gel 1 and 2 – 1kb DNA ladder
Lane 2-7 of gel 1 – digestion of plasmid from colony 1-5 and
8-9
Lane 2 of gel 2 – digestion of plasmid from colony 10
Lane 3 of gel 2 – positive control)

---

---

### **Experiment 16: (C&E)Colony Polymerase Chain Reaction**

**28/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Colony PCR (try 2)**

PCR reaction Mix preparation for colony PCR of transformed cells:

1. Colony Cell suspension preparation:
   A freshly autoclaved toothpick was touched onto a colony and
   then dipped into the respective Eppendorf tube containing 100 μL of Nuclease-free (autoclaved Milli-Q)
   water.
2. A colony PCR was done for 10 colonies with 25 μL of the colony PCR mix for each colony
3. Thus each PCR reaction mix consisted of:

   | Component | Volume (μL) |
   | --- | --- |
   | Q5 Master Mix (2X) (from NEB) | 12.5 |
   | T7 Forward Primer | 1.5 |
   | TccA Reverse Primer | 1.5 |
   | Autoclaved MilliQ Water | 8.5 |
   | Colony Cell Suspension | 1 |
   | Total | 25 |
4. A positive control was prepared of 25 μL.

Thermocycling conditions for colony PCR

| Step | Temperature °C | Time | Cycles |
| --- | --- | --- | --- |
| Initial Denaturation | 98 | 3 minutes | - |
| Cycling Denaturation | 98 | 15 seconds | 30 cycles |
| Cycling Annealing | 51 | 30 seconds | 30 cycles |
| Cycling Extension | 72 | 90 seconds | 30 cycles |
| Final Extension | 72 | 10 minutes | - |
| Hold | 4 | Indefinite | - |

Gel electrophoresis for Colony PCR

1. Preparation of 1% agarose Gel
   1. 0.3 g of agarose was weighed and added to 30 mL of 1X TAE to give 1% agarose gel.
   2. The agarose was boiled till it dissolved completely in a clear solution
   3. 2 μL of SYBR safe dye (Invitrogen) was added to the solution
2. Electrophoresis:
   1. The electrophoresis combs were set to be approximately 2 cm away from the cathode
   2. The agarose solution was poured into the central part of the tank when the temperature reached
      approximately 60 °C.
   3. The gel was kept undisturbed at room temperature fro the agarose to solidify.
   4. 1X TAE buffer was poured into the gel tank till the buffer level stodd at 0.5-0.8cm above the gel
      surface
   5. The combs were gently lifted ensuring the wells remained inatact.
   6. The power cord was connected to the electrophoretic power supply

| Well | DNA template | Volume of reaction mixuture (μL) |
| --- | --- | --- |
| 1 (of both gels) | Ladder (1kb) | 6 | - |
| 2-7 (gel 1) | Colony 1-6 | 25 | 4 |
| 2-5 (gel 1) | Colony 7-10 | 25 | 4 |

**1kb ladder was loaded after vortexing:**

*(Lane 1 of gel 1 and 2- 1kb DNA Ladder
Lane 2-7 of gel 1- Colony PCR of colonies 1-7
Lane 2-4
of gel 2- Colony PCR of colonies 8-10
Lane 5 of gel 2- Positive control
However, we are unsure of
the marks on gel 2, it could be due to unequal mixing of dye during preparation of agarose)*

---

---

### **Experiment 17: (C&E)Primary inoculation for competent cell preparation (BL21)**

**29/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

A heat sterilised loop was used to pick a colony from BL21 plate and Inoculated into 5 mL of LB broth

The test tube was incubated at 37 °C Celsius for 16 hours at 180rpm.

---

---

### **Experiment 18: (C&E)Competent cell preparation and transformation (BL21)**

**30/09/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Transformation of Gibson assembled tccA + pET22b+ to E.coli BL21 using CaCl2 method.**

1. **Chemical and Media Preparation**

   1. 0.1M CaCl2
      1. 11.1g of anhydrous CaCl2 was weighed out and added to 80 mL of autoclaved distilled water.
      2. The solution was mixed until the CaCl2 was fully dissolved
      3. The solution was then topped up to 100 mL
      4. 10 mL of the above solution was taken and then diluted by adding 90 mL of autoclaved distilled
         water for 1:10 dilution
      5. The solution was then filter sterilized through a PVDF filter of pores size 0.22μm.
   2. LB Ampicillin plates (X10)
      1. 6g of LB agar powder was weighed out and added to 250 mL of distilled water
      2. The LB agar solution was then autoclaved ● 0.25 μL of Ampicillin was then dissolved into the
         LB agar solution.
      3. 25 mL of LB-ampicillin solution was then poured into each Petri plate.
   3. 100 mL LB broth

      1. 2.5g of LB broth powder was weighed out and added to 100 mL of distilled water
      2. The LB broth solution was then autoclaved.
2. **Competent Cell Preparation**

   1. *E.coli* BL21 culture preparation One culture sample were prepared with one blank containing
      just the LB broth
      1. 200 μL of *E.coli* BL21 from the glycerol stock was Inoculated onto 5 mL of LB
      2. The culture was then incubated at 37°C and 200rpm for 16 hours.
      3. The amount of growth of BL21 culture was measured using a spectrophotometer at absorbance
         A600.
   2. The sample was transferred to 15 mL falcon tubes that were placed on ice.
   3. The falcon tube was then centrifuged at 4°C at 6000rpm for 10 minutes for pelleting the BL21
      culture.
   4. The supernatant was discarded.
   5. Each pellet was then resuspended with 1 mL ice-cold 0.1M CaCl2.
   6. The total 2 mL cell suspension was then incubated on ice for 30 minutes
   7. The suspension was then again centrifuged at 4°C at 6000rpm for 10 minutes
   8. The supernatant was discarded.
   9. Each pellet was then again resuspended in 500 μL of ice-cold 0.1M CaCl2.
   10. Five 100 μL aliquots of competent cells were prepared in ice-cold 1.5 mL falcon tubes
   11. The prepared competent cells are then stored at -80°C.
3. **Transformation**

   1. 10ng of plasmid was added to each aliquot (For required transformant and positive control)
   2. The cells were then incubated on ice for 30 minutes
   3. The cells were then placed in a 42°C water bath for exactly 30 seconds
   4. The cells were again placed on ice for 2 minutes,
   5. 1 mL of pre-warmed LB media was then added to each aliquot.
   6. The aliquots or cells were then incubated at 37°C, 200rpm for 1-hour.
   7. In order to increase the concentration/ efficiency, centrifuged to pellet down the cells for 4
      minutes at 4000rpm, discarded 700-750 μL of supernatant and resuspended the pellet.
4. **Plating and incubation:**

   1. Rest of the transformed cells from the aliquots of were then spread plated on the LB ampicillin
      plates.
   2. The plates were then incubated at 37°C for 12-16 hours.
      ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/blank-plate.jpg)

      (Blank)

      ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/competent-cells-plated-on-lb-plate-with-ampicillin.jpg)

      Competent cells plated on LB plate with ampicillin
      (This shows that the ampicillin is
      working)

      ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/competent-cells-transformed-with-pet22b-empty-vector.jpg)

      (Competent cells transformed with pET22b+ empty vector)

      ![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/transformed-e-coli-bl21-with-tcca-pet22b.jpg)

      (Transformed E.coli BL21 with tccA+pET22b+)

---

---

### **Experiment 19: (C&E)Expression Studies: inoculation**

**01/10/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

#### **Expression Studies**

1. From the transformed plate, streak plate one single colony and grow it.
2. Inoculate from that plate into 3 mL of LB broth, 37 °C, 180rpm overnight.

---

---

### **Experiment 21: (C&E)Expression Studies**

**04/10/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

After 48 hour incubation, the OD was checked at 600nm.

The pelleted down uninduced cells were resuspended into the broth again and OD was checked.

To get a uniform result, we diluted the higher OD as follows:

---

---

### **Experiment 22: (C&E)Expression Studies: SDS PAGE**

**04/10/2023**

**Project: CarbanEl**

**Authors: Aditi Balasubramani, I Vidhula Jessica**

---

1. Clean the glass plates and spacers of the gel casting unit with deionized water and ethanol.
2. Assembled the plates with the spacers on a stable, even surface.
3. Prepared resolving gel solution using the following volumes for 12.5%:

   | **Component** | **Resolving Gel** |
   | --- | --- |
   | MilliQ water | 1.151 mL |
   | 30.8% Acrylamide | 2.079 mL |
   | 1M Tris: pH 8.8 | 1.663 mL |
   | 10% SDS | 50 microL |
   | 10% APS (freshly prepared) | 50 microL |
   | TEMED | 5 microL |
4. Poured the gel solution in the plates assembled with spacers. To maintain an even and horizontal
   resolving gel surface, overlayed the surface with ethanol
5. The gel was allowed to set for about 20-30 min at room temperature.
6. Prepared stacking gel solution using the following volumes:

   | **Component** | **Stacking Gel** |
   | --- | --- |
   | MilliQ water | 1.752 mL |
   | 30.8% Acrylamide | 0.369 mL |
   | 1M Tris: pH 6.8 | 0.324 mL |
   | 10% SDS | 25 microL |
   | 10% APS (freshly prepared) | 25 microL |
   | TEMED | 3 microL |
7. Discarded the overlayed ethanol on the resolving gel
8. Added the 5% stacking gel solution until it overflows. Inserted the comb immediately ensuring no air
   bubbles were trapped in the gel or near the wells.
9. Allowed the gel to set for about 20-30 min at room temperature.

**Sample Preparation and loading**

1mL of loading buffer was added to each of the samples (Induced and Uninduced) of 1mL each and incubated
at 99 degrees for 20 minutes.

These samples were then loaded onto the gel with two ladders and electrophoresis was carried out at 50V
for 3 hours.

The gel was then carefully removed placed in a plastic container and Coomassie Blue dye was added and
left overnight on the shaker.

Then destaining solution was added and kept for 4 hours then observed under white light.

**Results:**

![](https://static.igem.wiki/teams/4641/wiki/notebook-cloning-and-expression/sds-page.jpg)

(SDS gel image- Lane 1,2,5,6 uninduced cells, Lane 3,4,7,8 induced cells)

**Conclusion:** No distinct clear bands were observed, could be due to extended time for destaining.
The experiment will be repeated.

---

---

Detection

WEEK 1

×

---

---

## **Experiment 1: Finding the best-suited solvent for preparation of standards**

18/07/2023

**Project: CarbanEl
Authors: Manu Upadhye**

---

* Preparation of standards with various solvents for TCC detection: water, acetone, and
  acetonitrile.
* It was found in literature that TCC was insoluble in water, and we concluded in our experiments
  that acetone was too volatile.

**---

---

## Experiment 2: TCC Detection: Preparation of standards (using water as solvent)**

18/07/2023

**Project: CarbanEl

Authors: Aditi Balasubramani Manu Upadhye, Rajat Sreeraj, Vidhula Jessica**

---

* 1.0 mg of TCC was added to 10 ml of water and vortexed to create a solution.
* The solution was then heated.
* The compound did not dissolve in water. This is due to Triclocarban’s low solubility in water
  (approx. 50 ng cm-3)
* Through a literature search, we came to the conclusion that TCC was not soluble in water. Hence,
  we switched to using other solvents.

**---

---

## Experiment 3: Preparation of standards (using acetone as solvent)**

19/07/2023

**Project: CarbanEl

Authors: Aditi Balasubramani, Manu Upadhye, Rajat Sreeraj, Vidhula Jessica**

---

**Preparation of standards (using acetone as solvent)**

* 1.0 mg of TCC was added to 10ml of acetone (100ppm), and serial dilution was performed until 1ppm
  solution was obtained. The solution was then homogenised, and the absorbance and peaks were detected
  using a UV spectrophotometer (Figure 1).
* As the peaks were detected, serial dilution was performed in the range 25ppb-250ppb and absorbance
  and peaks were detected for the same (Figure 2).

**Comments:**

The absorbance values were found to be largely negative. Hence, one wavelength was fixed, and the
absorbance of the same solutions was detected. Multiple readings were taken with different
concentrations of TCC, and the peak was obtained at 260nm and absorbance as 0.025.

**---

---

## Experiment 4: Preparation of standards for detection (with acetone as solvent)**

20/07/2023

**Project: CarbanEl

Authors: Manu Upadhye, Vidhula Jessica**

---

**Attempt 2:**

As the first attempt did not have a positive result, the wavelength was fixed at 260nm, and samples
with different concentrations of TCC, in the range of 25ppb-250ppb, were taken, and absorbance was
measured as in Table 1a and 1b.

Try 1:

| **Concentration of TCC (ppb)** | **Absorbance (260nm)** |
| --- | --- |
| 25 | 0 |
| 50 | 0 |
| 75 | 0 |
| 100 | 0 |
| 150 | 0.018 |
| 200 | 0.008 |
| 250 | 0.009 |

(Table 1a)

Try 2:

| **Concentration of TCC (ppb)** | **Absorbance (260nm)** |
| --- | --- |
| 25 | 0.025 |
| 50 | 0.018 |
| 75 | 0.026 |
| 100 | 0.022 |
| 150 | 0.064 |
| 200 | 0.010 |
| 250 | 0.103 |

(Table 1b)

(Table 1a, 1b: Absorbance values of solutions with different concentrations of TCC ranging from
25-250ppb)

**---

---

## Experiment 5: Preparation of standards for detection of TCC (with acetone as solvent)**

21/07/2023

**Project: CarbanEl

Authors: Manu Upadhye, Vidhula Jessica**

---

**Attempt 3:**
As previous attempts were unsuccessful, another attempt was made to prepare standards using acetone.
Standards were prepared with the concentrations of TCC varying from 1000-4000 ppm. However, positive
results were not obtained.

From this, it was concluded that acetone is a volatile solvent whose concentration can vary
with time; this makes it unsuitable for our experimentation. After a literature survey, an alternative
solvent was decided upon.

**---

---

##**

WEEK 2

×

---

---

Experiment 6: Preparation of standards for TCC detection (with acetonitrile as solvent)

01/08/2023

**Project: CarbanEl

Authors: Manu Upadhye, Rajat Sreeraj**

---

We commenced our attempts to prepare standards using acetonitrile. 1.0 mg of TCC was measured and
dissolved in 10 ml of acetonitrile. The solution was labelled with the concentration (100ppm). This
was further diluted to 1ppm by taking 0.1 ml of the 100 ppm solution and adding it to 9.9ml of
Acetonitrile. Acetonitrile was taken as blank for this experiment.

The preparation of standards was done as follows:

| **TCC Concentration (ppb)** | **Volume of 1 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
| --- | --- | --- |
| 25 | 0.25 | 9.75 |
| 50 | 0.5 | 9.5 |
| 75 | 0.75 | 9.25 |
| 100 | 1.0 | 9.0 |

**Results:**

| **Concentration of TCC (ppb)** | **Absorbance (260nm)** |
| --- | --- |
| 25 | 0.015 |
| 50 | 0.034 |
| 75 | 0.057 |
| 100 | 0.069 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-detection/tcc-and-abs-260.png)
(The above graph depicts the relationship between the concentration of TCC (in ppb) and absorbance at
260 nm)

**---

---

## Experiment 7: Preparation of standards for TCC detection (with acetonitrile as solvent)**

02/08/2023

**Project: CarbanEl

Authors: Manu Upadhye, Vidhula Jessica**

---

1.0 mg of TCC was measured and dissolved in 10 ml acetonitrile. The solution was then labelled (100
ppm). Acetonitrile was taken as blank for this experiment. 1.0 ml of the first solution was pipetted
out and added to another test tube containing 9 ml and was labelled. (10ppm)

The preparation of standards was done as follows:

| **TCC Concentration (ppb)** | **Volume of 10 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
| --- | --- | --- |
| 100 | 0.1 | 9.9 |
| 250 | 0.25 | 9.75 |
| 500 | 0.5 | 9.5 |
| 1000 | 1.0 | 9.0 |

**Results:**

| **TCC Concentration (ppb)** | **Absorbance 1 (260nm)** | **Absorbance 2 (260nm)** | **Absorbance 3 (260nm)** | **Absorbance 4 (260nm)** | **Absorbance 5 (260nm)** | **Averange Absorbance (260nm)** |
| --- | --- | --- | --- | --- | --- | --- |
| 100 | 0.011 | 0.011 | 0.011 | 0.012 | 0.013 | 0.0116 |
| 250 | 0.021 | 0.023 | 0.023 | 0.023 | 0.022 | 0.0224 |
| 500 | 0.045 | 0.047 | 0.047 | 0.048 | 0.048 | 0.047 |
| 1000 | 0.117 | 0.18 | 0.122 | 0.122 | 0.124 | 0.1206 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-detection/tcc-and-acetonitrile.png)
**Figure:** A graph depicting absorbance values for various concentrations of TCC is shown.
Calculation of LOD required a minimum of 6 readings to obtain a good quality curve.

**---

---

## Experiment 8: Detection of TCC in ppb concentrations and plotting calibration curve (with standards prepared with acetonitrile as solvent)**

03/08/2023

**Project: CarbanEl

Authors: Manu Upadhye, Rajat Sreeraj**

---

1.0 mg of TCC was measured and dissolved in 10 ml acetonitrile. Then, the solution was labelled with
the concentration amount (100ppm). Acetonitrile was taken as blank for this experiment.

1.0 ml of the first solution was pipetted out and added to another test tube containing 9 ml after
labelleling. (10ppm)

To prepare the standards:

| **TCC Concentration (ppb)** | **Volume of 10 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
| --- | --- | --- |
| 100 | 0.1 | 9.9 |
| 250 | 0.25 | 9.75 |
| 500 | 0.5 | 9.5 |
| 1000 | 1.0 | 9.0 |
| 2000 | 2.0 | 8.0 |
| 3000 | 3.0 | 7.0 |

**Observation:** Absorbance was detected for the standard samples five times and average absorbance
was considered, as shown in the table below;

| **TCC Concentration (ppb)** | **Absorbance 1 (260nm)** | **Absorbance 2 (260nm)** | **Absorbance 3 (260nm)** | **Absorbance 4 (260nm)** | **Absorbance 5 (260nm)** | **Averange Absorbance (260nm)** |
| --- | --- | --- | --- | --- | --- | --- |
| 100 | 0.017 | 0.018 | 0.018 | 0.018 | 0.019 | 0.018 |
| 250 | 0.035 | 0.035 | 0.036 | 0.036 | 0.036 | 0.0356 |
| 500 | 0.063 | 0.063 | 0.064 | 0.064 | 0.066 | 0.064 |
| 1000 | 0.158 | 0.159 | 0.160 | 0.160 | 0.160 | 0.1594 |
| 2000 | 0.308 | 0.307 | 0.308 | 0.308 | 0.309 | 0.308 |
| 3000 | 0.421 | 0.421 | 0.423 | 0.423 | 0.424 | 0.4224 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-detection/calibration-curve.png)
**Figure:**  Graph depicting absorbance values for various concentrations of TCC.

**---

---

## Experiment 9: Detection of TCC in ppb concentrations and plotting calibration curve (with standards prepared with acetonitrile as solvent)**

04/08/2023

**Project: CarbanEl

Authors: Manu Upadhye, Vidhula Jessica**

---

Standards were then prepared for TCC detection using acetonitrile. The following results were
obtained:

| **TCC Concentration (ppb)** | **Absorbance 1 (260nm)** | **Absorbance 2 (260nm)** | **Absorbance 3 (260nm)** | **Absorbance 4 (260nm)** | **Absorbance 5 (260nm)** | **Averange Absorbance (260nm)** |
| --- | --- | --- | --- | --- | --- | --- |
| 100 | 0.002 | 0.002 | 0.003 | 0.004 | 0.002 | 0.0026 |
| 250 | 0.020 | 0.019 | 0.019 | 0.020 | 0.020 | 0.0196 |
| 500 | 0.027 | 0.026 | 0.027 | 0.027 | 0.028 | 0.027 |
| 1000 | 0.043 | 0.042 | 0.044 | 0.043 | 0.043 | 0.043 |
| 2000 | 0.131 | 0.131 | 0.131 | 0.132 | 0.131 | 0.1312 |
| 3000 | 0.225 | 0.225 | 0.224 | 0.222 | 0.225 | 0.2242 |

![](https://static.igem.wiki/teams/4641/wiki/notebook-detection/average-absorbance.png)
**Figure:**  Graph depicting absorbance values for various concentrations of TCC.

**---

---

##**

WEEK 3

×

---

---

Experiment 10: Preparation of standards for TCC detection (for LCMS)

15/08/2023

**Project: CarbanEl

Authors: Manu Upadhye**

---

A solution of TCC and acetonitrile was prepared with a concentration of 100 ppm. Samples in the range
of 100 ppb – 3000 ppb were prepared as follows:

| **TCC Concentration (ppb)** | **Volume of 10 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
| --- | --- | --- |
| 100 | 0.1 | 9.9 |
| 250 | 0.25 | 9.75 |
| 500 | 0.5 | 9.5 |
| 1000 | 1.0 | 9.0 |
| 2000 | 2.0 | 8.0 |
| 3000 | 3.0 | 7.0 |

These samples are to be shipped off to an external party to be analysed using an LCMS system

**---

---

##**

WEEK 4

×

---

---

Experiment 11: Preparation of standard solutions for the byproduct 4-CA

02/10/2023

**Project: CarbanEl

Authors: Manu Upadhye, Aritro Biswas**

---

To prepare 100ppm solution of 4-CA, which is a degradation product of TCC;
1mg of 4-CA is taken and added to 10ml of Acetonitrile

| **TCC Concentration (ppm)** | **Volume of 100 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
| --- | --- | --- |
| 1 | 0.1 | 9.9 |
| 5 | 0.5 | 9.5 |
| 10 | 1.0 | 9.0 |
| 15 | 1.5 | 8.5 |
| 20 | 2.0 | 8.0 |
| 30 | 3.0 | 7.0 |

Our calibration curve for 4-CA
![](https://static.igem.wiki/teams/4641/wiki/notebook-detection/4ca-calibration-curve.png)
The effect of 4-CA concentration on OD is shown here. (267nm)

A scan was run with the 10ppm solution from 220nm to 300nm to find the wavelength to run the
standard solutions on. The peak we got was 267.

**---

---

## Experiment 12: Preparation of standard solutions for the byproduct 3,4-DCA**

02/10/2023

**Project: CarbanEl

Authors: Manu Upadhye, Aritro Biswas**

---

TTo prepare 100ppm solution of 3,4-DCA, a degradation byproduct of TCC other than 4-CA;
1mg of 3,4-DCA is taken and added to 10ml of Acetonitrile
Further dilution is done to obtain samples of lower concentration.

| **TCC Concentration (ppm)** | **Volume of 100 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
| --- | --- | --- |
| 1 | 0.1 | 9.9 |
| 5 | 0.5 | 9.5 |
| 10 | 1.0 | 9.0 |
| 15 | 1.5 | 8.5 |
| 20 | 2.0 | 8.0 |
| 30 | 3.0 | 7.0 |

Our calibration curve for 3,4-DCA
![](https://static.igem.wiki/teams/4641/wiki/notebook-detection/34dca-calibration-curve.png)
The effect of 3,4-DCA concentration on OD is shown here. (266nm)
A scan was run with the 10ppm solution from 220nm to 300nm to find the wavelength to run the standard
solutions on. The peak we got was 266.

{% endblock %}

{% extends "my-layout.html" %}
{% block sidebar\_content %}

* [Success](#section1)
* [Module 0](#section2)
* [Module 1](#section3)
* [Module 2](#section4)
* [Module 3](#section5)
* [Module 4](#section5)

{% endblock %}
{% block heading %}Engineering success{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4641/wiki/engineering-success-dbtl/mm.png)

MODULE 0

Overview:

The first module of our DBTL cycle shows how our initial project idea shifted on the basis of
iHP and testing.

Design:

The team of MIT-MAHE set out to solve the problem of **triclocarban entering our environment** and causing
major issues. We initially identified a novel TccA amidase enzyme expressed in Ochrobactrum sp. TCC-2. This
amidase had only 27-38% sequence similarity with other known amidases. This enzyme is capable of breaking down
triclocarban (TCC) into 4-chloroaniline and 3,4-dichloroaniline.[1]

We initially wanted to implement this system within the aeration tank of the wastewater treatment plant. The aim
was to increase the enzyme efficiency to function within the hydraulic retention time of the wastewater
treatment plant.

Hydraulic retention time (HRT) of the aeration tank of a wastewater treatment plant is the amount of time taken
for one full volume of the aeration tank to pass out of the aeration tank after completion of process.
HRT=Volume of the aeration tank/flow rate of the water
Commonly, HRT for **conventional activated sludge processes is about 2-24 hours**[7].

We wanted to achieve this by modifying amino acid residues through an in-silico version of site directed
mutagenesis.
We wanted to identify the key residues involved in the catalytic action and improve the binding affinity of the
substrate to the enzyme, which we had initially believed might increase the efficiency of the enzyme.

Build:

The TccA amidase enzyme has not been fully characterized in literature. Therefore, the first step before
building our genetic circuit was to understand the structure, binding site and working of the enzyme.

We predicted the structure of the TccA amidase as well as the binding sites. Since most of our data was not
obtained via experimentation, our Human practices team reached out to many experts, including Mr. Omkar Khade
and Dr. Gurunath Ramanathan, to learn more about the feasibility of our project idea.

#### Catalytic Triad

Through literature survey, we found the catalytic triad of the amidase family to be Ser-Ser-Lys. To
identify exactly which triad (Ser-Ser-Lys) of the amidase family was involved, we used multiple
alignments.

GRASP (collection of tools to perform and analyze ancestral sequence reconstruction) shows the amino
acid residues that most probably form the binding pocket of the enzyme. Here, Ser 155 and Ser 179 showed
85% confidence scores showing that it interacts with TCC and Lys 79 with 59% confidence score showing it
is not actively involved in bond cleavage but does help in stabilizing the interactions.

#### **Alphafold**

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/model-which-was-used-for-this-predicted-by-alpha-fold-and-present-in-uniprot.png)

*Model which was used for this (predicted by Alpha Fold and present in UniProt)*

We predicted structures by different softwares like SWISS-MODEL, Alphafold and iTASSER. For the test
stage, we proceeded with the Alphafold model as it showed the highest percentage of residues lying
within the accepted region of the Ramachandran plot.

Test:

We performed docking studies with the predicted binding site. The docking results from Autodock Vina and
SeamDock showed ΔG values of -7.51 kcal/mol and -4.7 kcal/mol respectively. This implies that TCC has a good
affinity towards the TccA amidase protein.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/2d-visualization-of-our-docked-structure-in-discovery-studio-visualizer-dsv.jpg)

*2D visualization of our docked structure in Discovery Studio Visualizer (DSV)*

These ΔG values imply that there is a stable binding of TCC to the TccA amidase. This suggests that the
predicted binding sites were correct.

Learn:

Through iHP meetings with Mr. Omkar Khade and Dr. Gurunath Ramanathan, we learnt that
manually altering the amino acids in close proximity to the catalytic triad requires a comprehensive
understanding of the individual functions of each amino acid involved. A random mutagenesis approach would be
difficult to perform in-silico as such experiments are usually only performed in-vitro. As a result, we felt the
best way forward was to focus on the initial problem statement-dealing with TCC in wastewater, with an
alternative approach.

---

MODULE 1

Design:

Ensuring our focus on the problem statement, and taking note of everything learnt in Module 0, we looked back
towards our previous human practices interactions. One of the major questions that had been posed to us was the
formation of toxic byproducts 4-chloroaniline and 3,4-dichloroaniline on biodegrading TCC. Considering these
changes, we decided to focus our project on breaking down the TCC into non-toxic byproducts.

Through literature surveys, we studied the presence of gene clusters as a part of other pathways in bacteria
that could degrade the above mentioned by-products.
However, insertion of such large cassettes within known model organisms could cause severe metabolic load on the
cell and may lead to stunted growth or loss of plasmid.

Hence, we looked for potential model organisms that had not been characterized for this purpose. An important
parameter was the existence of pathways that could degrade the chloroaniline byproducts of TCC breakdown.
Through extensive literature survey, we narrowed down our possible chassis options to three species capable of
degrading the byproducts -*Acinetobacter baylyi* [3],  *Pseudomonas putida* [4] [5]and  *Pseudomonas
fluorescens* [6].
All three of these organisms had the natural capability to degrade the toxic by-products. The other major
criteria we considered included:

* Survivability of the bacteria in the pH range of the wastewater and sludge(6.5-8)
* Survivability of the bacteria in the temperature range of the sludge tank of the WWTP(20-37° C)
* Survivability at different TCC concentration ranges present in the sludge(13,000-21,000 ng/ml of TCC or
  13-21 ppm of TCC)

Aim: To design experiments to check which of our three chosen bacterial species: *Pseudomonas putida,
Pseudomonas fluorescens*, and *Acinetobacter baylyi* had the best survivability in wastewater
treatment plant conditions

As TCC does not uniformly dissolve in small volumes of water, we dissolve TCC in acetonitrile at first and then
dilute this mixture in water. To ensure that acetonitrile does not have any adverse effect on the growth of our
bacteria, we take acetonitrile-containing media as a condition as well.

Build:

**Growth Curves** To perform these experiments, we inoculated the bacteria in media that simulated the
conditions of the sludge tank.The growth was measured by checking the arbitrary units of optical density at
600nm. To convert to absolute units, a calibration curve was plotted for wet weight of the cell vs OD.

1. **pH**
   The growth of the bacterial species at pH range of 6 to 8 was growth in LB media.
2. **Temperature**
   For temperature studies, we inoculated the bacteria in LB broth media and studied the growth over a
   temperature range of 24°C to 37°C.
3. **Acetonitrile**
   To dissolve and dilute TCC in water, we need to dissolve it in acetonitrile first as TCC has extremely low
   solubility in water. Since acetonitrile will be in the media that our bacteria grow in, we have to make sure
   the presence of this compound does not impede the steady growth of our organisms.
4. **TCC (Sludge concentration)**
   To discern whether our organisms can survive and be effective in the sludge tank, we need to know whether they
   can survive in the concentration of TCC within the sludge tank and effectively break down this TCC and its
   constituent degradation products. Hence, we inoculated our bacteria in media containing a range of TCC
   concentrations that is generally present in the sludge (13000 - 21000ng\ml).

Test:

The growth curves obtained from all the experiments we performed for all three of our possible chassis’ have
been listed below:

#### **Effect of Acetonitrile**

***Pseudomonas putida***

1. Growth curve of *Pseudomonas putida* in the presence of acetonitrile
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-putida-acetonitrile.png)

   Inference: The presence of acetonitrile in the growth media does not affect the growth of
   *Pseudomonas putida*.

2. Growth curve of *Pseudomonas fluorescens* in presence of acetonitrile.
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-fluorescens-acetonitrile.png)

   Inference: It was found that the presence of acetonitrile does not appreciably affect the growth of
   *Pseudomonas fluorescens*.

3. Growth curve of *Acinetobacter baylyi* in the presence of acetonitrile
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/a-baylyi-acetonitrile.png)

   Inference: It was observed that acetonitrile affects the growth of *Acinetobacter baylyi* and
   acts as an inhibitor.

#### **Effect of pH**

1. Growth curve of *Pseudomonas putida* at different pH values
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-putida-temp.png)

   Inference: The presence of acetonitrile in the growth media does not affect the growth of
   *Pseudomonas putida*.

2. Growth curves of *Pseudomonas fluorescens* at different pH values
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-fluorescens-ph.png)

   Inference: The optimal pH for growth of *Pseudomonas fluorescens* was observed to be pH 8.

3. Growth curve of *Acinetobacter baylyi* at different pH values
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/a-baylyi-ph.png)

   Inference: It was observed that the growth of *Acinetobacter baylyi* was not deterred by
   variation in the pH. Thus we infer that it would thrive within this pH range typically found in
   wastewater treatment plants.

#### **Effect of Temperature**

1. Growth curve of *Pseudomonas putida* at 24° C and 37° C
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-putida-temp.png)

   Inference: It is observed that *Pseudomonas putida*  exhibits superior growth at 24 degrees
   Celsius, whereas its growth is adversely affected at 37 degrees Celsius.

2. Growth curve of *Pseudomonas fluorescens* at 24° C and 30° C
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-fluorescens-temp.png)

   Inference: It was observed that *Pseudomonas fluorescens* exhibits better growth at 24° C.

3. Growth curve of *Acinetobacter baylyi* at 30° C and 37° C
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/a-baylyi-temp.png)

   Inference: It was observed that the growth of *Acinetobacter baylyi* was not deterred by
   variation in the temperature. Thus we infer that it would thrive within this temperature range typically found in
   wastewater treatment plants.

#### **Effect of TCC in Sludge Level Concentrations**

1. Growth curve of *Pseudomonas putida* at different concentrations of TCC (sludge concentration)
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-putida-tcc-sludge.png)

   Inference: Growth of *Pseudomonas putida* was stunted in the presence of TCC as compared to the
   control.

2. Growth curve of *Pseudomonas fluorescens* in the presence of TCC (sludge concentration)
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/p-fluorescens-tcc-sludge.png)

   Inference: It was observed that TCC did not deter the growth of *Pseudomonas fluorescens*.

3. Growth curve of *Acinetobacter baylyi* in the presence of different concentrations of TCC (sludge
   concentration)
   ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/a-baylyi-tcc-sludge.png)

   **Inference:** Presence of TCC at such high concentrations does not inhibit the growth of
   *Acinetobacter baylyi*. However, the TCC was dissolved in acetonitrile. Our final inference
   is that the inhibitory effect that existed due to the usage of acetonitrile appeared to be
   canceled out by the presence of TCC.

Learn:

#### ***Pseudomonas putida:***

* It has a lag phase of roughly 4 hours, after which its growth plateaus become steady.
* We found that it showed optimal growth at 30ºC and 7.5pH
* Its growth is stunted under the presence of TCC.
* It shows reduced growth at 37ºC.

**Conclusion:**

After taking these observations into account, our final conclusion was to eliminate this strain as it
does not proliferate well in the temperature conditions observed in the sludge tank of a wastewater
treatment plant.

#### ***Pseudomonas fluorescens:***

* Shows good growth at 30ºC, 7.5 pH.
* Growth is stunted under the presence of TCC.

**Conclusion:**

After taking these observations into account, our final conclusion was to rule this strain out as it
does not proliferate well in the pH range observed in the sludge tank of a wastewater treatment plant.

#### ***Acinetobacter baylyi:***

* It has a lag phase of 8-9 hours using primary inoculum
* The lag phase for the secondary inoculation was about 2-3 hours. We tested its growth by altering
  the temperature, pH, and TCC concentration.
* We found that 30ºC, 7.5 pH were the ideal conditions for growth and surprisingly, the presence of
  such high concentrations of TCC did not impede its exponential growth. Hence, *A. baylyi* can
  proliferate and grow uninhibited in the presence of TCC.

**Conclusion:**

Due to its ability to proliferate in environments containing near-toxic levels of TCC as well as with
all the other parameters that it would be subjected to within the sludge tank of a wastewater treatment
plant, we were able to come to the conclusion that *Acinetobacter baylyi* is the best choice for
our bacterial chassis. The other two bacteria were promptly rejected due to their growth being stunted
in the temperature and pH range of the sludge tank.

---

MODULE 2

Design:

To carry out the wet lab experiments, including cloning, we started out by designing our part and plasmid.
Aim: To create a genetic circuit containing TccA amidase and insert it in  *E.coli*  to study its
expression and characterise the enzyme.

Part Design

The purpose of the amidase protein [(Part number:
BBa\_K4641000)](http://parts.igem.org/Part%3ABBa_K4641000) in the chassis is to breakdown Triclocarban (3,4,4′-Trichlorocarbanilide) into
3,4-dichloroaniline and 4-chloroaniline. Our target protein is naturally constitutively expressed in
*Ochrobactrum sp. TCC-2*

![]()
![]()

Plasmid Design

The CarbanEl system is based on a Gibson assembly cloning system. The gene fragment is designed and synthesized
such that the overhangs to integrate the gene into pET22b+ are already added. For the characterization and study
of the enzyme activity, we expressed the amidase gene in  *E. coli BL21* . However, our final intended
chassis is *Acinetobacter baylyi*

The plasmid design is as below:

![](https://static.igem.wiki/teams/4641/wiki/design-part-page/plasmid.png)

Genetic circuit design:

Gene regulation refers to how a particular gene expression can be controlled. It includes a variety of
mechanisms that can be used to increase or decrease the expression or production of specific gene products.

![](https://static.igem.wiki/teams/4641/wiki/design-part-page/genetic-circuit.jpeg)

*TccA Gene Regulation in Escherichia coli*

Build:

The first step was to build our genetic circuit in  *E.coli DH5alpha* to study the expression and
degradation kinetics of our part.

We started with amplifying our tccA fragment and performing PCR clean-up. We isolated pET22b+ plasmid and
proceeded with the Gibson Assembly to insert the TccA amidase gene.
The competent *E. Coli E.coli DH5alpha* cells were prepared by CaCl2 method and the cells were
transformed.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/transformed-plate-in-dh5alpha.jpg)

*Transformed plate-contains tccA Gibson assembled pET22b+ transformed DH5alpha cells*

After a confirmatory colony PCR to check the presence of our insert, we transformed the Gibson-assembled plasmid
into  *E.coli BL21*  and then we moved towards expression studies.

Test:

Cloning:
Double digestion was then performed on the isolated plasmid of the transformed cells to confirm the presence of
the fragment which was successful. We then went on to expression studies. SDS-PAGE was carried out but distinct
bands were not observed. This could have been due to the extended time for destaining.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/sds-gel-image.png)

*SDS Page*

**Genetic circuit**

The genetic circuit represents the regulation of TccA gene in *E.coli*. Our gene, including RBS, is 1562
bps long; assuming that the degradation of mRNA is negligible the time taken to transcribe our gene is 6.25
secs.

We modeled the protein production in  *E.coli* . However, in the future, we can study and
model the protein production rate in our preferred chassis *Acinetobacter baylyi.*

#### **Modeling the genetic Circuit:**

![](https://static.igem.wiki/teams/4641/wiki/model-dl/eqn-1.png)

| Parameter | Explanation | Units |
| --- | --- | --- |
| k1 | Transcription rate of T7 RNA polymerase | bps/sec |
| k2 | k2 is the translation rate of a ribosome in E.coli | AAs/sec |
| d1 | Degradation rate of mRNA | bps/sec |
| d2 | Degradation rate of protein | AA/sec |

Assuming that degradation rate of mRNA is low and almost negligible, d1 ≈ 0

Assuming that degradation of protein is low and almost negligible, d2 ≈ 0

k1 is Transcription rate of T7 RNA polymerase = 250 bps/s

The length of TccA gene,
including the RBS and PelB = 1562 bps

k1 = 1562/250 = 6.25s

[mRNA] = k1 [Gene] - d1[mRNA] [mRNA] = k1 [Gene]
**mRNA = 6.25 [Gene]**

k2 is the translation rate of a ribosome in *E.coli*

Length of the mRNA = 1562 bps

[Protein] = k2 [mRNA]- d2[Protein]

[Protein] = k2

Assuming that the translation rate of a ribosome in *E.coli* is 12. 1 AA/sec,

Our protein = 515 AA (including RBS and PelB)

k2
= 42.56 secs

**Protein = 42.56 [mRNA]**

**Design equation based on degradation kinetics**

The rate expression is related to the rate at which the TccA amidase enzyme from the
*Ochrobactrum sp.* can degrade TCC. Since our chassis for implementation is *Acinetobacter baylyi*,
which is also gram-negative like *Ochrobactrum sp.*TCC-2, the rate of transport or the passive diffusion of
TCC into the cell is assumed to be similar in both bacteria. The rate of degradation of the TCC inside the cell
by the TccA amidase enzyme produced by our chassis will be very similar. The time calculated here for 21 PPM
(the highest concentration of TCC found in sludge) will be used to model the bioreactor.

#### **Study of the design equation**

In this study [1], we have used the values as shown in the tabular column below.
These values were used to calculate -rA (the rate of reaction with respect to reactant A or
in our case TCC) using differential calculation.

| Time (hours) | CA(concentration of TCC in 𝞵m ) | -rA |
| --- | --- | --- |
| 0 | 31.7 | 3.667 |
| 10 | 10.8 | 0.9375 |
| 24 | 1.3 | 0.15 |
| 48 | 0.2 | 0.014 |
| 72 | 0 | 0 |

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/concentration-of-substrate-vs-time.jpg)

*Concentration of substrate vs. time*

Assuming first order kinetics
-rA= K(CA)n
ln(-rA) = lnK+n (lnCA)

| ln (-rA) | ln (CA) |
| --- | --- |
| 3.456 | 1.299 |
| 2.379 | -0.0645 |
| 0.262 | -1.897 |
| -1.609 | -4.566 |
| - | - |

![]( https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/ln-ca-vs-ln-ra.png)

From the above graph, we know that the following data:
Slope = n = 1.1198 ≈ 1, which implies that it follows first-order kinetics.
Intercept = ln k = -2.5633
Therefore, k = e-2.5633
k= 0.082, which is the calculated rate expression.
From the literature, we learned the value of k [1].
k = 0.112

Learn:

On comparing our values to the literature, we get that the k values are approximately equal, thus validating our
differential calculus method.

Hence, TccA amidase follows first-order kinetics.

Future cycles
would involve the validation of these kinetics through experimentation.

---

MODULE 3

Summary

As mentioned in our [implementation](https://2023.igem.wiki/mit-mahe/project-implementation), we aim
to introduce a bioreactor in the wastewater treatment plant to treat the primary sludge and clear it of our
target contaminant, Triclocarban (TCC).

From the results of our previous module of the DBTL cycle, *Acinetobacter baylyi* GFJ2 was the most viable
choice for a chassis because it had the best growth in high concentrations of TCC and a good survivability for
the widely varying conditions of wastewater treatment plants. Additionally, the bacteria has the natural ability
to degrade the toxic byproducts produced by the degradation of TCC into non toxic byproducts (cis-muconic acid)
that can be taken by the Krebs cycle [3].

Design:

The team came up with multiple bioreactor designs for the implementation of our project that take into account
the degradation kinetics of the enzyme, the growth kinetics of the bacteria, and the adsorption kinetics of TCC
onto the packing material.

We have currently proposed a lab scale bioreactor due to the fact that we need to conduct further optimisation
experiments to upscale into a bioreactor that is feasible on an industrial scale.

We wanted to design a sludge based bioreactor where the primary sludge becomes the feed. The primary sludge is
almost 97-99% water [4]. We ideated this after a visit to a wastewater treatment plant in West Bengal, India.

We wanted the bacteria that degraded the TCC to be immobilized within a matrix. Literature suggests that cell
immobilised systems are far superior to free cell suspensions as it avoids cell washout and improves reaction
rates [5].

We found that biochar, a carbon-rich solid product produced from the pyrolysis of biomass residues, was an
attractive option for an immobilisation matrix due to its low cost, easy availability and ability to immobilize
cells efficiently[6].

#### ***Design 1- Continuous flow packed-bed reactor***

The design of this reactor would be such that the primary sludge would continuously flow through the
packed bed over the span of two days.

The primary sludge would be pumped from the bottom of the reactor and exited from the top. The reactor
top would be perforated to allow airflow, and the reactor would be continuously sparged with air to
increase mass transfer and provide oxygen to the microbes for proliferation.

The reactor would be fitted with a pressure gauge in order to measure and maintain the pressure drop
across the reactor.

The reactor will operate in continuous flow under unsterile conditions.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/packed-bed-reactor.png)

#### ***Design 2 - Continuous stirred tank reactor***

In this design, the primary sludge would enter from the top of the reactor and exit from the bottom of
the reactor.

The biochar with the immobilized bacteria would be fixed onto the walls of the reactor using a
honeycomb-like scaffold.

The reactor would have a stirrer that would agitate the sludge and aerate it to promote mass transfer
and adsorption onto the biochar. To prevent excess heat from being generated, the reactor would have
cooling jackets surrounding it. A temperature gauge would be added to measure the temperature and
regulate it to promote the exponential growth of microorganisms.

A level gauge would be added in order to prevent the overflow of the sludge in the reactor. A pipe at
the top would be introduced to recycle the overflow into the tank. The reactor will operate in
continuous flow under unsterile conditions.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/stirred-tank-reactor.png)

#### ***Design 3 - Batch stirred tank reactor***

This reactor would be similar to the continuous stirred tank bioreactor, except the sludge would stay in
the tank for longer to allow more time for mass transfer. The rpm of the stirrer would be slower, and a
sparger would be added to increase aeration. The rest of the elements would remain the same as in Design
2. The reactor will be operated in batch mode in unsterile conditions.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/batch-stirred-tank.png)

#### ***Design 4 - batch packed bed reactor***

The packed bed reactor would consist of a packed bed with biochar and immobilized bacteria as packing
material between two perforated plates. The primary sludge would enter from the top of the reactor and
flow to the bottom with the help of gravity. This eliminates the need for a pump to fill the sludge in
the reactor. A sparger at the bottom center would aerate the sludge and would promote a vertical
circular flow due to Bernoulli's principle. This will help increase mass transfer efficiency.

The sparger would have a valve to prevent the backflow of sludge into it, and the sparging would begin
before the inflow of sludge into the reactor began. The outlet pipe will also have a valve to prevent
sludge from leaving the tank before the specified time. The valve in the inlet pipe is to control the
flow rate of the sludge entering the tank. The reactor would be fitted with a pressure gauge to measure
and maintain the pressure drop across the reactor. The reactor will be operated in batch mode in
unsterile conditions.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/batch-packed-bed-reactor.png)

#### ***Design 5 - fluidized bed***

This design would consist of a fluidized bed with the packing material. The fluidized bed will have a
sparging system with appropriate tubing to control the flow rate of air. This would allow an upward,
circular flow of the sludge. This agitation will increase mass transfer efficiency.

The continuous flow would be due to the movement of the liquid phase (primary sludge) and gaseous phase
(air from the sparger). Both of these factors will induce movement of the solid, i.e., the packing
material. The reactor will operate in continuous flow under unsterile conditions.

![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/fluidised-bed.png)

Packing Material:

The packing material for the packed and fluidized bed is chemically modified biochar with bacteria
immobilized on it. The team decided to make the biochar into the shape of a hollow cylinder called a
Raschig ring. This type of structure would increase the area of adsorption and improve mass transfer
efficiency. The hollow cylinder pipe-like structure would prevent the problem of choking the reactor due
to larger particles that may be present in the sludge. While cylindrical pellets are more commonly used
and available, we propose the novel idea of a biochar Raschig ring.

Build:

*Reactor*

* The material of the reactors needs to be non-reactive to the compounds that will be present in the primary
  sludge that will enter the reactors. In response to this, we had a few options available to us, those being
  mild steel, stainless steel, glass, etc. Since our model is a lab-scale reactor, we chose to use glass to make
  the body of the lab-scale reactor.
* The inlet, outlet, and recycle pipes will be made of PVC.
* The stirrer would have a slow speed of 50 rpm.
* We would use generic industrial temperature and pressure gauges for the reactors.
* The sparger would be a perforated sparger for uniform aeration and would be placed at the bottom of all the
  reactor designs except for the continuous flow fluidized bed. The fluidized bed will have tube sparging with
  appropriate tubing to control the airflow for more aeration and, hence, more mass transfer efficiency.

*Packing Material*

* The packing material would consist of biochar. The biochar will be modified with a 1:1 ratio of biochar to
  KOH[8]. According to literature, the biochar modified with KOH showed higher adsorption of the target compound
  TCC compared to other ratios and just plain biochar.
* The biochar would be made into the shape of a Raschig ring using clay as a binding agent. The dimensions of
  the raschig ring are in the ratio 1:1 of the diameter to the height, and the inside diameter will be 1cm as
  shown in the figure below. These ideas were validated by meetings with Dr. Bhat and Dr. Kevin Sowers.
* The Raschig ring would then be fired at 110°C to 130°C to harden the ring so that it does not dissolve in
  the primary sludge.
* The genetically modified bacteria would then be immobilized on the biochar raschig rings.
* The biochar used will be sourced from a local rice mill by processing rice husk that is produced as waste
  at the mill.

Test:

#### ***Design 1- Continuous flow packed-bed reactor***

This design would allow us to treat a large amount of sludge in less time. The sparger would provide
enough aeration for our bacteria to grow optimally, and degradation would occur efficiently.

However, according to our adsorption and degradation kinetics the minimum contact time required for
efficient degradation of our target contaminant TCC would be 2 days. This large amount of time required
a longer packed bed would increase our cost of building. Additionally, since it would be a continuous
flow chances of choking at the outlet pipe increased.

#### ***Design 2 - Continuous stirred tank reactor (CSTR)***

To overcome the issue of a large amount of packing material required we came up with the design of the
CSTR. in this reactor we designed a scaffold that would be 3D printed and placed on the wall of the
reactor. The purpose of the scaffold was to hold the biochar in place for adsorption of our target
contaminant TCC to occur.

The stirring would increase the mass transfer efficiency and there would be sufficient aeration because
of it as well. However, this did not solve the issue we had with the contact time and additionally the
regeneration of the adsorbent i.e. the biochar proved to be difficult in this case.
According to literature, since the degradation of the compound by the enzyme produced by the bacteria is
very slow as shown by the kinetics in the [models](https://2023.igem.wiki/mit-mahe/model)
page the type of reactor most suitable for slow reactions was a batch process.

#### ***Design 3 - Batch stirred tank reactor***

This design would allow the sludge to remain in contact with the biochar for the required amount of
time. And regeneration of the biochar would not prove to be an issue, however the the cost of this
design was more than anticipated, causing us to reject this design.

#### ***Design 4 - batch packed bed reactor***

This design proved to be the most suited to our requirements. The reactor being a batch process allowed
us to maintain the required contact time of 2 days. The sparging allowed aeration and sufficient flow
for efficient mass transfer. The inflow of the sludge from the top of the reactor removed the cost of
the pump as gravity would help the fluid flow down the column.

The cost analysis concluded this design to be the cheapest option for us.

#### ***Design 5 - fluidized bed***

This design proved to be the most suited to our requirements. The reactor being a batch process allowed
us to maintain the required contact time of 2 days. The sparging allowed aeration and sufficient flow
for efficient mass transfer. The inflow of the sludge from the top of the reactor removed the cost of
the pump as gravity would help the fluid flow down the column.

The cost analysis concluded this design to be the cheapest option for us.

Learn:

For a lab scale reactor we will use the batch packed bed reactor. The kinetics of enzyme degradation, TCC
adsorption onto the biochar and the bacterial growth kinetics have been fitted into the equations for a batch
reactor as given in the [models](https://2023.igem.wiki/mit-mahe/model) page.

We learnt that design 1 would not be feasible due to low hydraulic retention time of the primary sludge in the
tank, hence we tried design 2. However, through a human practices visit with Dr. Subbulaxmi we learnt that the
efficiency of the biochar would be reduced significantly.

Design 3 significantly improved contact time; however, it would not be enough to compensate for the low
absorption efficiency of the biochar after our other meetings with Dr. Murty.

Design 4 incorporated all the suggestions from Dr. Bhat and Dr. Subbulaxmi and led us to our most ideal design
that gave us a high hydraulic retention time of the primary sludge in the tank as well as a high adsorption
affinity of the biochar.

However, we learnt from Dr. Kevin Sowers that this model would only be feasible for a lab scale model. Since at
industry level we would be required to treat a larger amount of primary sludge per day we proposed design 5
which would have comparable efficiency to design 4 and be able to treat large quantities of sludge due to its
larger volume and continuous operation.

---

MODULE 4

Design:

Our goal was to design a detection system that would go hand in hand with our bioreactor set-up and was
accurate, highly sensitive, and cost-effective. In our design stage, we have explored numerous ideas, highlights
of which include:

* *Electrochemical sensors* - were explored, as they are one of the most prevalent detection systems.
  However, we found that it would not be feasible to achieve the desired selectivity towards triclocarban.
* *Enzyme based sensors*- we identified an enzyme from Ochrobactrum sp.TCC-2; but due to the trace levels
  of TCC found in effluent this option would not be sensitive enough.
* *Aptasensors*- Aptamers specific for TCC are not available in the literature, due to which, we would be
  required to isolate and identify one de-novo using SELEX. This is expensive and would not be feasible within
  our time constraint.
* *Nanobody based sensors*- three nanobodies which were specific to TCC were identified. We realised that
  immobilizing the nanobody on the sensor and making required modifications would be expensive and within the
  given time frame, building a prototype and testing didn’t seem like a viable option.
* *Molecularly imprinted polymer based sensors(MIP)*- we found an MIP which would detect triclocarban and
  triclosan combined. Making an MIP specific to triclocarban in the lab would require extensive experimental
  data and testing.
* *Nanomaterial based sensors*- Nanomaterial based sensors can detect endocrine disrupting compounds and
  since TCC is one of them, this drew our attention. However, after thorough literature review we found that
  there was no nanomaterial isolated specifically for TCC detection.

[Ideation Page](https://2023.igem.wiki/mit-mahe/hardware#section3)

Toward the end, we decided to simplify our approach by using Raman Spectroscopy to identify light of a certain
wavelength range specific to TCC and design a simplified prototype of an optical detection system using a laser
and a photodiode to detect this range of wavelengths.

Build:

We used the Raman spectroscopy setup in our secondary PI, Kapil Sadani’s laboratory to conduct experiments and
isolate the specific peaks for our compound. Using the data from these experiments, we calculated the specific
wavelength for the IR filter and photodiode and built a theoretical set-up that can be employed for the
detection of TCC.

![](https://static.igem.wiki/teams/4641/wiki/engineering-success-dbtl/block-diagram.jpeg)

*Block Diagram of our set-up*

This set-up would include a laser of 785 nanometer monochromatic light, two focal lenses, a mirror with a hole
to cleanly pass the focused laser, a sample stage (cuvette), an IR filter, a photodiode, and a microcontroller.
The sample would have to be manually collected and brought to this set-up.

Test:

We ran experiments for various samples as follows :

1. Raman spectroscopic analysis for Triclocarban dissolved in 5% acetone.
2. Surface Enhanced Raman Spectroscopy (SERS) for Triclocarban dissolved in 5% acetone.
3. Lake water sample, that was suspected to have triclocarban dissolved in it, to baseline and isolate the
   specific peaks for TCC using SERS.

From this we found the specific wavelength for our Infrared filter and photodiode.

[Hardware Notebook](https://static.igem.wiki/teams/4641/wiki/hardware/h-s-wiki-notebook-docx.pdf)

* Experiment 1: 1, 10, 100 ppm (parts per million) of TCC in 5% acetone (standard)
  ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/raman-spec-1.png)
  Figure: Surface Enhanced Raman Spectroscopy of triclocarban dissolved in 5% acetone. (1, 10, 100 ppm)
* Experiment 2: 1, 10, 100 ppm (parts per million)of TCC in lake water sample
  ![](https://static.igem.wiki/teams/4641/wiki/dbtl-growth-curves/raman-spec-2.png)
  Figure: Surface Enhanced Raman Spectroscopy of triclocarban dissolved in lake water. (1, 10, 100 ppm)

Learn:

From the experimental data, we learned that the unique peaks (unique wavenumber) for Triclocarban is observed at
758.3 cm-1and 1445.2cm-1. Moreover, the second peak observed at 1445.2 cm-1 can be
concluded as an overtone of the peak observed at 758.3 cm-1 as the ratio of the intensities at
various concentrations remains constant.

In the lake water sample, we noticed peaks within the range of 750 and 760 nanometers. We can assume that the
lake water sample initially had dissolved triclocarban. After experiments with spiked triclocarban were done, we
noticed an amplification in the baseline peaks.

These preliminary results are indicative of a starting point for proving our proof of concept of a detection
system selective and sensitive towards TCC and easily adaptable in all kinds of bioreactor setups.

Success:

* *Acinetobacter baylyi* was identified as the most viable chassis for the incorporation of TccA amidase
* A successful clone of TccA in pET22b+ was obtained in  *E.coli DH5alpha*  and then transformed into *E.coli BL21* .
* We successfully found the wavelength of the IR filter which can be used in our detection system set up to
  detect TCC in sludge
* A packed bed batch reactor was found to be the most viable option for a lab scale bioreactor while a
  fluidised bed reactor was preferred for future implementation.

References:

[1]Yun H, Liang B, Qiu J, Zhang L, Zhao Y, Jiang J, Wang A. Functional Characterization of a Novel Amidase
Involved in Biotransformation of Triclocarban and its Dehalogenated Congeners in Ochrobactrum sp. TCC-2. Environ
Sci Technol. 2017 Jan 3;51(1):291-300. <https://doi.org/10.1021/acs.est.6b04885>. Epub 2016 Dec 14.
PMID: 27966913.

[2]Hülter, N., Sørum, V., Borch-Pedersen, K. et al. Costs and benefits of natural transformation in
Acinetobacter baylyi . BMC Microbiol 17, 34 (2017). <https://doi.org/10.1186/s12866-017-0953-2>

[3]Hongsawat, Parnuch, and Alisa S. Vangnai. “Biodegradation Pathways of Chloroanilines by Acinetobacter Baylyi
Strain GFJ2.” Journal of Hazardous Materials, vol. 186, no. 2–3, Elsevier BV, Feb. 2011, pp. 1300–07. <https://doi.org/10.1016/j.jhazmat.2010.12.002.>

[4] Nazari, L., Sarathy, S., Santoro, D., Ho, D., Ray, M. B., & Xu, C. (2018). Recent advances in energy
recovery from wastewater sludge. In Elsevier eBooks (pp. 67–100). <https://doi.org/10.1016/b978-0-08-101029-7.00011-4>

[5] Zhu, Y. (2007). Immobilized cell fermentation for production of chemicals and fuels. In Elsevier eBooks (pp.
373–396). <https://doi.org/10.1016/b978-044452114-9/50015-3>

[6]Wu, C., Zhi, D., Yao, B., Zhou, Y., Yang, Y., & Zhou, Y. (2022). Immobilization of microbes on biochar for
water and soil remediation: A review. Environmental Research, 212, 113226. <https://doi.org/10.1016/j.envres.2022.113226>

[7] Eggen, T., & Vogelsang, C. (2015). Occurrence and fate of pharmaceuticals and personal care products in
wastewater. In Comprehensive Analytical Chemistry (pp. 245–294). <https://doi.org/10.1016/b978-0-444-63299-9.00007-7>

{% endblock %}

{% extends "my-layout.html" %}
{% block sidebar\_content %}

* [Protocol of experiments](#section1)
* [Troubleshooting](#section2)

{% endblock %}
{% block heading %}Experiments{% endblock %}
{% block page\_content %}

**Protocol of experiments**

## **TCC Detection: Preparation of Standards (Using Water as Solvent)**

#### **Objective:**

To prepare a standard solution of Triclocarban (TCC) using water as the solvent.

#### **Materials:**

* 1.0 mg of Triclocarban(TCC)
* 10 ml of distilled water
* Test tube(27ml)
* Vortex mixer
* Water bath
* Thermometer

#### **Procedure:**

##### **Preparation of TCC Solution**

1. Measure 1.0 mg of TCC using a precision balance and record the exact mass.
2. Carefully transfer the measured TCC into a clean and dry test tube.

##### **Dissolution of TCC**

1. Add 10 ml of water to the test tube containing TCC.
2. Ensure that the water completely covers the TCC powder.
3. Attach a cap or stopper to the test tube securely.
4. Gently vortex the mixture for dissolution at 3000rpm. Ensure that the TCC powder is thoroughly dispersed in the water.

##### **Heating the Solution**

1. Set up a water bath and bring it to a temperature of 60 °C.
2. Carefully place the capped test tube containing the TCC and water mixture into the heated environment.
3. Allow the solution to heat at 60 °C for 30 minutes. (Note: Record the exact heating time for consistency.)

##### **Observation**

1. After heating, carefully remove the test tube from the water bath using appropriate tools (e.g., tongs or gloves) to prevent burns.
2. Observe the solution in the test tube.
3. Record your observation. If any turbidity is present, TCC has not dissolved. If you have a clear solution, then TCC has been dissolved.

##### **Comments:**

After adding the TCC in water, we observed that TCC did not dissolve at all. We tried heating it and vortexing after with no success. This is why we decided to change solvents.

## **TCC Detection: Preparation of Standards (Using Acetone as Solvent)**

#### **Objectives:**

To prepare a series of TCC standard solutions in acetone, perform parallel dilutions, detect absorbance peaks using a UV spectrophotometer, and establish a wavelength for absorbance measurement.

#### Materials:

* 1.0 mg of TCC
* 10 ml of acetone
* Graduated cylinders or pipettes for dilution
* Vortex mixer
* UV spectrophotometer
* Cuvettes (3.5 ml)
* Wavelength selection guide
* Spectrophotometer software

#### **Procedure:**

1. ##### **Preparation of Initial TCC Solution**

   1. Measure 1.0 mg of TCC using a precision balance and record the exact mass.
   2. Carefully transfer the measured TCC into a clean and dry container (e.g., beaker or vial).
   3. Add 10 ml of acetonitrile to the container with TCC, creating a 100 ppm (parts per million) solution.
2. ##### **Parallel Dilution**

   1. Prepare a series of dilutions of (1ppm, 5ppm, 10ppm, 15ppm, 20ppm, 30ppm) by taking an aliquot of the 100 ppm TCC solution and diluting it with acetone.
   2. To prepare the standard solutions, refer to the table below

      | **TCC Concentration (ppm)** | **Volume of 100 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
      | --- | --- | --- |
      | 1 | 0.1 | 9.9 |
      | 5 | 0.5 | 9.5 |
      | 10 | 1.0 | 9.0 |
      | 15 | 1.5 | 8.5 |
      | 20 | 2.0 | 8.0 |
      | 30 | 3.0 | 7.0 |
   3. Continue parallel dilutions until you obtain a 1 ppm TCC solution. Record the volumes and concentrations of each dilution.
3. ##### **Homogenization and Initial Measurements**

   1. Homogenize each TCC solution by gently mixing with a vortex mixer at 3000rpm.
   2. Using a UV spectrophotometer, run a scan program from 210 to 300 nm to find the peak of your TCC. Take the average peak value and set it as your fixed wavelength.(We have used 260)
   3. Document the results, including the absorbance and peak information.
4. ##### **Parallel Dilution and Absorbance Measurements (25 ppb-250 ppb Range)**

   1. Perform additional parallel dilutions of the TCC solution to create concentrations from 25 ppb to 250 ppb.
   2. Repeat the absorbance measurements at 260 nm for each dilution using the UV spectrophotometer. Record the absorbance and peak information.
   3. Document the results, including each concentration's absorbance and peak information within the specified range.
5. ##### **Multiple Readings and Peak Detection at 260 nm**

   1. Using the fixed wavelength of 260 nm, perform multiple readings at different concentrations of TCC.
   2. Record the absorbance values for each concentration and observe the peak characteristics.
   3. Document the results, including the absorbance and peak information at 260 nm.

#### **Comments:**

The results we got were mostly negative. We suspect the reasons to be the high volatility of acetone and its similar peak wavelength (280nm). The volatility keeps changing the properties of the solution.

## **TCC Detection: Preparation of Standards (Using Acetonitrile as Solvent)**

#### **Objectives:**

1. To prepare a series of TCC standard solutions in acetonitrile, including a 100 ppm stock solution.
2. To create standard solutions with different concentrations for calibration.

#### **Materials:**

* 1.0 mg of TCC
* 10 ml of acetonitrile
* Graduated cylinders or pipettes for dilution
* UV spectrophotometer
* Quartz cuvettes(3.5ml)
* Wavelength selection guide
* Spectrophotometer software

#### **Procedure:**

1. ##### **Preparation of Initial TCC Solution**

   1. Measure 1.0 mg of TCC using a precision balance and record the exact mass.
   2. Carefully transfer the measured TCC into a clean, dry container (e.g., beaker or vial).
   3. Add 10 ml of acetonitrile to the container with TCC, creating a 100 ppm (parts per million) solution.
   4. Label the solution with the concentration (100 ppm).
2. ##### **Acetonitrile as Blank:**

   Set aside a sample of acetonitrile to be used as a blank for this experiment.
3. ##### **Preparation of Standards**

   Prepare a series of dilutions using the 100 ppm TCC solution and acetonitrile to achieve the desired concentrations:

   | **TCC Concentration (ppb)** | **Volume of 100 ppm solution (ml)** | **Volume of Acetonitrile (ml)** |
   | --- | --- | --- |
   | 50 | 0.005 | 9.995 |
   | 100 | 0.01 | 9.99 |
   | 250 | 0.025 | 9.975 |
   | 1000 | 0.1 | 9.9 |
   | 2500 | 0.25 | 9.25 |
   | 5000 | 0.5 | 9.5 |
   | 7500 | 0.75 | 9.25 |
   | 10000 | 0.1 | 9.0 |
4. ##### **Calculation of Limit of Detection (LOD)**

   1. To determine the LOD, quality curve is required. Perform absorbance measurements for each of the prepared standards at a fixed wavelength.
   2. Record the absorbance values for each standard solution.
   3. Perform absorbance measurements for at least six different concentrations to create a calibration curve.
5. ##### **Spectrometer's Error Value**

   1. Since the first absorbance value is 0.011, it is necessary to determine the spectrometer's error value. This can be done by measuring the absorbance of a blank solution (acetonitrile) and calculating the error.
   2. Record the absorbance value obtained for the blank solution.

## **Reviving of Culture**

#### **Objective:**

To revive a culture stored in a glass ampoule by transferring it into suitable growth media, distributing it into multiple containers, and incubating it under optimal conditions for further use.

#### **Materials:**

* Glass ampoule containing the culture
* Sterile blade
* Heat sterilised forceps
* 0.5ml liquid media
* Pipette
* Test tubes(27ml)
* Petri dishes with nutrient broth agar
* Incubator
* Labels

#### **Procedure:**

1. ##### **Aseptic Handling**

   1. Begin by ensuring aseptic conditions in the laboratory. Disinfect the work area by wiping the surfaces with 70% ethanol, follow LAF sterilisation procedures and wash your hands thoroughly.
   2. Wear appropriate personal protective equipment (PPE), including lab coats, gloves, and safety goggles.
2. ##### **Ampoule Disintegration**

   1. Take the glass ampoule containing the culture.
   2. Using a sterile blade, carefully file the neck of the ampoule to create a score mark.
   3. Hold the ampoule firmly, and using the sterile blade, break the neck along the score mark to release the culture into the bottom of the ampoule.
3. ##### **Cotton Plug Removal:**

   Using heat-sterilized forceps, carefully remove the cotton plug from the ampoule.
4. ##### **Resuspension in Liquid Media**

   1. Using a pipette, add 0.5 ml of the appropriate liquid growth media into the ampoule.
   2. Gently resuspend the culture by pipetting up and down, ensuring even distribution of the culture in the liquid media.
5. ##### **Distribution into Containers**

   1. Distribute the revived culture into multiple containers for further growth and experimentation:
      1. Transfer a portion of the resuspended culture into two separate test tubes containing nutrient broth.
      2. Transfer another portion into two Petri dishes containing nutrient broth agar.
   2. Label each container appropriately with the culture name, date, and any other necessary information.
6. ##### **Incubation**

   Place the labelled containers into an incubator set to the required optimum temperature and time period as specified by the culture suppliers.
7. ##### **Monitoring**

   1. Periodically monitor the containers for growth and observe for any signs of contamination.
   2. Note any changes in appearance, such as the presence of colonies or turbidity in the liquid media.
8. ##### **Streaking of a new plate**

   1. Take a heat sterilized inoculation loop and dip it into the eppendorf tube containing the culture.
   2. Quadrant streak the culture present on the loop onto a petri plate.

#### **Comments:**

Make sure to keep extra plates, because the pour plates we incubated had lawns of the culture, keep the extra plate to streak it as well or to add less concentration of the pure culture.

## **Confirmatory test- Catalase test**

#### **Objective:**

To perform the catalase test and determine if the colony exhibits a positive catalase reaction.

#### **Materials:**

* Cell colony
* Clean, dry glass slide
* Loop or sterile wooden stick
* 3% hydrogen peroxide (H2O2)
* Biohazard glass disposal container

#### **Procedure:**

1. ##### **Preparation**

   1. Begin by ensuring proper laboratory safety measures. Wear appropriate personal protective equipment (PPE), including lab coats and safety goggles.
   2. Work in a designated area for microbiological tests.
2. ##### **Colony Transfer**

   Using a loop or a sterile wooden stick, transfer a small amount of the colony to the surface of a clean, dry glass slide.
3. ##### **Catalase Reaction Test**

   1. Place a drop of 3% hydrogen peroxide (H2O2) directly onto the colony on the glass slide.
   2. Mix the colony and hydrogen peroxide gently by gently stirring or tapping with the loop or sterile stick.
   3. Observe the reaction carefully
   4. Look for the rapid evolution of oxygen, which is evidenced by the formation of bubbles. This should occur within 5-10 seconds.
   5. If bubbling occurs within the specified time frame, the test is considered positive for catalase activity.
4. ##### **Disposal:**

   Dispose of the glass slide used for the test in a designated biohazard glass disposal container.

## **Confirmatory test- Gram Staining**

#### **Objective:**

To perform a Gram stain on a bacterial smear and observe the results, specifically identifying the bacteria as Gram-negative.

#### **Materials:**

* Clean glass slide
* Bacterial culture or smear
* Bunsen burner or slide warmer
* Crystal violet stain
* Iodine solution
* 95% ethyl alcohol (decolorization solution)
* Safranin stain
* Microscope with oil immersion objective lens
* Microscope immersion oil
* Distilled water or tap water

#### **Procedure:**

1. ##### **Preperation of Smear**

   1. Take a clean glass slide and place a small amount of the bacterial culture or smear on it.
   2. Using a sterile loop or wire, spread the bacterial material into a thin, even smear on the slide.
   3. Allow the smear to air dry completely.
   4. Heat fix the smear by passing it through the flame of a Bunsen burner or using a slide warmer. Ensure that the slide is not overheated, which could lead to distortion of bacterial morphology.
2. ##### **Staining Procedure**

   1. Flood the heat-fixed smear with crystal violet stain, ensuring that the entire smear is covered. Allow the crystal violet to stand for 1 minute.
   2. Rinse the slide with tap water to remove excess crystal violet.
   3. Flood the smear with iodine solution, covering the entire smear. Allow the iodine to stand for 1 minute. The iodine serves as a mordant and enhances staining.
   4. Rinse the slide with tap water to remove excess iodine.
   5. Decolorize the smear by gently flooding it with 95% ethyl alcohol. Allow the alcohol to stand for approximately 10 seconds. Monitor the decolorization closely, as over-decolorization can lead to false results.
   6. Rinse the slide with tap water immediately to stop the decolorization process.
   7. Counterstain the smear by flooding it with safranin stain. Allow the safranin to stand for 45 seconds.
   8. Rinse the slide with tap water to remove excess safranin.
3. ##### **Observation**

   1. Allow the slide to air dry completely.
   2. Examine the dried slide under a microscope using the oil immersion objective lens.
   3. Observe the bacterial morphology and the color of the cells after staining.
4. ##### **Gram Staining Result**

   Based on the observation, record the Gram staining result. In this case, if the cells appear pink or red, record the result as Gram-negative.

## **Confirmatory test- Indole spot test**

#### **Objective:**

To perform the indole spot test on a culture and determine the result as negative for the presence of indole.

#### **Materials:**

* Cell culture (18-24 hour growth)
* Petri dish cover
* Whatman filter paper
* Kovács reagent

#### **Procedure:**

1. ##### **Preparation of cell culture:**

   Inoculate a culture and incubate it for 18 to 24 hours at 31 °C to allow for growth.
2. ##### **Indole Spot Test Preparation**

   1. To conduct the indole spot test, begin by placing a small piece of Whatman filter paper in a clean and dry petri dish cover.
   2. Saturate the filter paper with Kovács reagent, adding approximately 1 to 1.5 ml of the reagent to ensure the paper is thoroughly soaked.
3. ##### **Smeared Sample Preparation**

   1. Obtain a small amount of cell paste from the 18 to 24-hour culture. This can be achieved by gently scraping a loop or sterile wooden stick along the surface of the culture.
   2. Smear the cell paste onto the saturated Whatman filter paper in the petri dish cover.
4. ##### **Incubation**

   1. Carefully cover the petri dish to create a closed environment.
   2. Incubate the prepared filter paper at the appropriate temperature and time period (31 °C for 18-24 hours).
5. ##### **Observation:**

   After the incubation period, carefully examine the filter paper for any color change or visible indication of indole production.
6. ##### **Indole Spot Test Result:**

   Based on the observation, record the result of the indole spot test. If there is no visible color change or indication of indole production, record the result as negative.

## **Confirmatory test- Methyl Red and Voges-Proskauer test**

#### **Objective:**

To prepare Methyl Red and Voges-Proskauer (MR-VP) broth medium, inoculate it and incubate the test tubes at 37 °C for 24 hours in preparation for the Methyl Red and Voges-Proskauer tests.

#### **Materials:**

* MR-VP broth medium (prepared in advance)
* Test tubes (three)(27 ml)
* Autoclave
* Culture
* Inoculation loop or sterile wooden stick
* Static incubator set at 37 °C
* Labels

#### **Procedure:**

1. #### **Preparation of MR-VP Broth Medium**

   1. Prepare MR-VP broth medium in three separate test tubes.
   2. Ensure that the medium is properly mixed and sterilized by autoclaving.
   3. Label the test tubes as "MR," "VP," and "Control" for identification.
2. #### **Inoculation of Culture**

   1. Using sterile technique, ensure that all equipment and materials are free from contamination.
   2. Inoculate each labeled test tube with bacterial culture as follows:
      1. Using an inoculation loop or sterile wooden stick, transfer a small amount of bacterial culture into the "MR" labeled test tube containing MR-VP broth medium.
      2. Similarly, inoculate the "VP" labeled test tube with bacterial culture.
      3. The "Control" tube will not receive any inoculation and will serve as a control.
3. #### **Incubation**

   1. Place all the labeled test tubes in an incubator set at 37 °C.
   2. Allow the test tubes to incubate for a period of 24 hours.
4. #### **Monitoring**

   1. Periodically check the test tubes during the incubation period to ensure that they remain at the correct temperature.
   2. Observe for any signs of growth or changes in the medium.

## **Preparation of Standards for TCC Detection (99% Water and 1% TCC + Acetonitrile)**

#### **Objective:**

To prepare a series of TCC standard solutions using a mixture of 99% water and 1% TCC in acetonitrile, perform dilutions, and detect the absorbance of these samples at 260nm using a UV spectrophotometer.

#### **Materials:**

* 1.0mg of TCC
* 10ml acetonitrile
* 10ml water
* Graduated cylinders or pipettes for dilution
* UV spectrophotometer
* Cuvettes (3.5 ml)
* Wavelength selection guide
* Spectrophotometer software
* Labels

#### **Procedure:**

1. ##### **Preparation of Initial TCC Solution**

   1. Measure 1.0 mg of TCC using a precision balance and record the exact mass.
   2. Carefully transfer the measured TCC into a clean and dry container (e.g., beaker or vial).
   3. Add 10 ml of acetonitrile to the container with TCC, creating a 100 ppm (parts per million) solution.
2. ##### **Blank Solution:**

   Prepare a blank solution using acetonitrile, which will serve as a reference during absorbance measurements.
3. ##### **Dilution for 10 ppm Solution**

   1. Pipette 0.1 ml of the 100 ppm TCC solution into a clean test tube.
   2. Add 9 ml of the blank solution to the same test tube, creating a 10ppm TCC solution.
4. ##### **Preparation of 100 ppb-3000 ppb Samples**

   1. Pipette 0.1 ml of the 10 ppm TCC solution into a clean test tube containing 9.9 ml of water. This creates a 100 ppb TCC solution.
   2. Repeat the process, each time using the 10 ppm TCC solution but varying the volume of water added as follows:
      1. 0.25 ml of solution 2 with 9.75 ml of water (250 ppb).
      2. 0.5 ml of solution 2 with 9.5 ml of water (500 ppb).
      3. 1 ml of solution 2 with 9 ml of water (1000 ppb or 1 ppm).
      4. 2 ml of solution 2 with 8 ml of water (2000 ppb or 2 ppm).
      5. 3 ml of solution 2 with 7 ml of water (3000 ppb or 3 ppm).
5. ##### **Absorbance Measurements**

   1. Using a UV spectrophotometer, set the wavelength to 260 nm.
   2. Prepare cuvettes with the various TCC standard solutions and the blank solution.
   3. Measure the absorbance of each sample at 260 nm using the UV spectrophotometer.

## **Growth curve for growth of bacteria in the presence of Acetonitrile**

#### **Objective:**

To create a growth curve for bacteria in the presence of acetonitrile by measuring optical density (OD) at different time intervals.

#### Materials:

* Three conical flasks
* 25 ml LB broth per flask
* Autoclave
* 1 ml overnight culture of bacterial
* 1% acetonitrile solution
* Spectrophotometer
* Cuvettes (3.5 ml)
* Timer or stopwatch
* Labels

#### **Procedure:**

1. ##### **Preparation of LB Broth**

   1. Take three conical flasks and add 25 ml of LB broth to each flask.
   2. Autoclave the flasks to sterilize the LB broth medium.
2. ##### **Flask Setup**

   1. Label the three flasks as follows: "Blank," "Control," and "Culture + Acetonitrile."
   2. Prepare the contents of each flask as follows:
      1. **Blank:** Contains only LB broth medium.
      2. **Control:** Add 1 ml of the overnight culture of bacteria to the LB broth. Mix well.
      3. **Culture + Acetonitrile:** Add 1 ml of the culture to the LB broth and then add 1% acetonitrile solution to the same flask. Mix well.
3. ##### **Incubation**

   1. Place each flask in an incubator set to the appropriate temperature for the growth of bacteria.
   2. incubate the flasks under controlled conditions.
4. ##### **Observation and OD Measurements**

   1. At regular intervals (e.g., every hour), remove each flask from the incubator.
   2. Using a spectrophotometer, measure the optical density (OD) of the cultures at 600nm.
   3. Record the OD readings for each flask at each time interval.
   4. Keep the cultures in the incubator between measurements.
5. ##### **Growth Curve Plotting:**

   Plot the OD readings against time to create a growth curve for bacteria in the presence of acetonitrile.
6. ##### **Data Analysis**

   Analyze the growth curve to observe and interpret the growth pattern and the effect of acetonitrile on bacterial growth.

## **Preparation of standards for TCC detection (for LCMS)**

#### Objective:

To prepare a series of TCC standard solutions for LCMS (Liquid Chromatography-Mass Spectrometry) analysis by diluting a stock TCC solution in acetonitrile, and then mixing it with water to achieve various concentrations ranging from 100 ppm to 3000 ppb

#### **Materials:**

* 1.0 mg of TCC
* 10 ml acetonitrile
* 9 ml water
* Pipettes (0.1 ml, 0.05 ml)
* Test tubes(27ml)
* Labels

#### **Procedure:**

1. ##### **Preparation of Stock TCC Solution**

   1. Measure precisely 1.0 mg of TCC using a precision balance and record the exact mass.
   2. Transfer the measured TCC into a test tube.
   3. Add 10 ml of acetonitrile to the container with TCC, creating a 100 ppm (parts per million) TCC stock solution. Label the solution with its concentration (100 ppm).
2. ##### **Blank Solution:**

   Prepare a blank solution using 10 ml of acetonitrile. This solution will serve as a reference during LCMS analysis.
3. ##### **Preparation of 500 ppb-5000 ppb Samples**

   1. Pipette 0.05 ml of Solution into a clean test tube labeled "Solution 1."
   2. Pipette 0.1 ml of Solution into a clean test tube labeled "Solution 2."
   3. Pipette 0.2 ml of Solution into a clean test tube labeled "Solution 3."
   4. Pipette 0.3 ml of Solution into a clean test tube labeled "Solution 4."
   5. Pipette 0.4 ml of Solution into a clean test tube labeled "Solution 5."
   6. Pipette 0.5 ml of Solution into a clean test tube labeled "Solution 6."
4. ##### **Final Dilution**

   To each test tube containing the various solutions (1 to 6), add enough water to reach a total volume of 10 ml, ensuring proper mixing.

## **Growth curve for culture at a chosen pH**

#### Objective:

To create a growth curve for bacterial culture at a chosen pH by measuring optical density (OD) at different time intervals.

#### **Materials:**

* Four conical flasks
* 50 ml LB broth per flask
* Autoclave
* 1N NaOH solution
* 1N HCl
* Bacterial culture culture
* Spectrophotometer
* Cuvettes (3.5 ml)
* Timer or stopwatch
* pH meter
* Labels

#### **Procedure:**

1. ##### **Preparation of LB Broth at chosen pH**

   1. Take four conical flasks and add 50 ml of LB broth to each flask
   2. Adjust the pH of the LB broth in the flasks to chosen pH using 1N NaOH or 1N HCl respectively.
   3. Autoclave the flasks to sterilize the LB broth medium.
2. ##### **Flask Setup**

   1. Label the four flasks as follows: "Blank," "chosen pH - 1," "chosen pH - 2," and "chosen pH - 3."
   2. Prepare the contents of each flask as follows:
      1. **Blank:** Contains only LB broth medium at chosen pH.
      2. **Chosen pH- 1**: Inoculate the flask with one colony of bacterial culture. Mix well.
      3. **Chosen pH- 2**: Inoculate the flask with another colony of bacterial culture. Mix well.
      4. **Chosen pH - 3**: Inoculate the flask with a third colony of bacterial culture. Mix well.
3. ##### **Incubation**

   1. Place each flask in an incubator set to the appropriate temperature for the growth of bacteria.
   2. Incubate the flasks under controlled conditions.
4. ##### **Observation and OD Measurements**

   1. At regular intervals (e.g., every hour), remove each flask from the incubator.
   2. Measure the optical density (OD) of the cultures at 600nm using a spectrophotometer.
   3. Record the OD readings for each flask at each time interval.
   4. Keep the cultures in the incubator between measurements.
5. ##### **Growth Curve Plotting:**

   Plot the OD readings against time to create a growth curve for bacteria at chosen pH.
6. ##### **Data Analysis**

   Analyze the growth curve to observe and interpret the growth pattern of bacteria at chosen pH.

## **Dissolving Lyophilized IDT TccA Gene Fragment**

#### Objective:

To dissolve a lyophilized IDT tccA gene fragment, measure its concentration, and prepare a stock solution for storage at -20°C.

#### Materials:

* Lyophilized IDT tccA gene fragment
* 100 μl autoclaved Milli-Q water
* Microcentrifuge tube
* Vortex mixer
* Centrifuge
* Ice
* UV-Vis spectrophotometer
* Cuvette(3.5 ml)
* Pipettes (25 μl, 75 μl)
* Milli-Q water
* -20°C freezer
* Labels

#### **Procedure:**

1. ##### **Dissolving the Lyophilized Gene Fragment**

   1. Add 100 μl of autoclaved Milli-Q water to the lyophilized IDT tccA gene fragment in a microcentrifuge tube.
   2. Very gently shake the tube to ensure the gene fragment comes into contact with the water.
   3. Vortex the sample for 1 second to aid in dissolving the gene fragment.
   4. Centrifuge the sample at 14,000 rpm for 30 seconds to pellet any remaining undissolved material.
   5. Immediately place the sample in an ice bath to maintain its integrity.
2. ##### **Measurement of OD (Optical Density):**

   Measure the optical density (OD) of the sample using a UV-Vis spectrophotometer at 260nm for nucleic acids.
3. ##### **Dilution of Gene Fragment:**

   1. Pipette 25 μl of the dissolved gene fragment solution and transfer it into a new microcentrifuge tube.
   2. Add 75 μl of Milli-Q water to the tube, resulting in a total volume of 100 μl.
   3. Ensure that the diluted solution is thoroughly mixed.
4. ##### **Concentration Calculation:**

   Calculate the concentration of the dissolved gene fragment. For example, if the OD measurement indicates a concentration of 10 ng/μl, record this value.
5. ##### **Storage**

   1. Label the microcentrifuge tube containing the dissolved gene fragment with the concentration value (e.g., 10 ng/μl) and any other relevant information.
   2. Store the tube in a -20°C freezer for long-term storage.

## **Dissolving lyophilized tccA primers**

#### **Objective:**

To dissolve lyophilized tccA primers, measure their concentration, and prepare a stock solution for storage at -20°C.

#### **Materials:**

* Lyophilized Forward Primer
* Lyophilized tccA Reverse Primer
* Autoclaved Milli-Q water
* Microcentrifuge tubes (two)
* Vortex mixer
* Centrifuge
* Ice
* UV-Vis spectrophotometer
* Cuvette (3.5 ml)
* Pipettes (10 μl, 90 μl)
* Milli-Q water
* -20°C freezer
* Labels

#### Procedure:

1. ##### **Dissolving the Lyophilized Primers**

   1. Add 368.9 μl of autoclaved Milli-Q water to the lyophilized Forward Primer (100 pmol/ μL) in a microcentrifuge tube.
   2. Add 249.3 μl of autoclaved Milli-Q water to the lyophilized tccA Reverse Primer (100 pmol/ μL) in a separate microcentrifuge tube.
   3. Gently mix each tube to ensure the primer comes into contact with the water.
   4. Vortex each sample for 1 second to aid in dissolving the primers.
   5. Centrifuge each sample at 14,000 rpm for 30 seconds to pellet any remaining undissolved material.
   6. Immediately place each sample in an ice bath to maintain primer integrity.
2. ##### **Dilution of Primers**

   1. For each primer, pipette 10 μl of the dissolved primer solution and transfer it into a new microcentrifuge tube.
   2. Add 90 μl of Milli-Q water to each tube, resulting in a total volume of 100 μl.
   3. Gently pipette mix each tube to ensure thorough mixing.
3. ##### **Measurement of Concentration**

   1. Measure the concentration of each primer using a UV-Vis spectrophotometer at an appropriate wavelength for nucleic acids (e.g., 260 nm).
   2. Verify that the concentration of each primer is approximately 10 μM.
4. ##### **Storage**

   1. Label each microcentrifuge tube containing the dissolved primer with the primer name and a concentration value (e.g., Forward Primer 10 μM, tccA Reverse Primer 10 μM).
   2. Store the tubes in a -20°C freezer for long-term storage.

## **PCR Reaction Mix Preparation for TccA Gene Amplification**

#### **Objective:**

To prepare PCR reaction mixtures for tccA gene amplification, including master mix preparation, dividing it into separate tubes, and creating samples for both negative and positive controls.

#### **Materials:**

* PCR reagents (e.g., Taq polymerase, dNTPs, buffer, primers)
* Template DNA
* Microcentrifuge tubes (0.2 ml)
* Pipettes (various sizes)
* PCR tube rack
* Labels

#### **Procedure:**

1. ##### **Master Mix Preparation**

   1. Prepare a master mix for a 50 μl PCR reaction mixture in a 0.2 ml PCR tube. The master mix should include all necessary PCR reagents, excluding the template DNA and primers as mentioned below.
   2. Mix the components of the master mix thoroughly to ensure homogeneity.
   3. Calculate the total volume of the master mix, which should be 50 μl.
2. ##### **Dividing the Master Mix:**

   Divide the total 50 μl PCR reaction mixture into two separate 0.2 ml PCR tubes, with each tube containing 25 μl of the master mix.
3. ##### **Sample Creation:**

   Divide each of the two 25 μl PCR reaction mixtures into three separate samples. You will have a total of six samples.
4. ##### **Control Samples**

   1. For the negative control samples:
      1. In one of the 25 μl reaction mixtures, add no template DNA. This will serve as the "No template DNA" negative control.
      2. In another 25 μl reaction mixture, add all components of the master mix except the DNA polymerase (e.g., Q5 High-Fidelity DNA Polymerase). This will serve as the "No Q5" negative control.
   2. In another 25 μl reaction mixture, add all components of the master mix except the DNA polymerase (e.g., Q5 High-Fidelity DNA Polymerase). This will serve as the "No Q5" negative control.
5. ##### **Labeling:**

   Label each sample tube appropriately, indicating the type of sample (e.g., Negative Control - No template DNA, Negative Control - No Q5, Positive Control - Mam7 gene) and any other relevant information.

| Component | Volume | Concentration Available | Volume | For 10 Reactions |
| --- | --- | --- | --- | --- |
| Q5 High-Fidelity 2X Master Mix | 12.5 ul | 2X | 1.25 ul | 125 ul |
| 10uM Forward Primer | 1.25 ul | 10 mM | 1.25 ul | 12.5 ul |
| 10uM Reverse Primer | 1.25 ul | 10 mM | 1.25 ul | 12.5 ul |
| Template DNA | 1 pg/10ng |  | 10ng (1 full) |  |
| Nuclease-Free Water | To make 25 ul |  | To make 25 ul | 90 ul |
| Total Volume | 25 ul |  | 25 ul |  |

| Component | Negative-1 | Negative-2 | Positive |
| --- | --- | --- | --- |
| Q5 High-Fidelity 2X Master Mix | 12.5 ul |  | 1.25 ul |
| 10uM Forwad Primer | 1.25 ul | 1.25 ul | 1.25 ul |
| 10uM Forward Primer | 1.25 ul | 1.25 ul | 1.25 ul |
| Template DNA |  | 5 ng (0.5 ul of 10ng/ul) | 10ng (1 full) |
| Nuclease-Free Water | To make 25 ul | To make 25 ul | To make 25 ul |
| Total Volume | 25 ul | 25 ul | 25 ul |

## **Gradient PCR for TccA gene**

#### **Objective:**

To prepare a master mix for gradient PCR, aliquot it into PCR tubes for multiple reactions, and set up samples for gradient PCR, including negative and positive controls.

#### **Materials:**

* 2X Q5 High-Fidelity Master Mix
* PCR primers
* Template DNA (if required)
* Autoclaved Milli-Q water
* Eppendorf tube
* PCR tubes (9 or as required)
* Pipettes (various sizes)
* PCR tube rack
* Labels

#### **Procedure**

1. ##### **Master Mix Calculation:**

   Calculate the amount of each component required for the master mix, including water, 2X Q5 High-Fidelity Master Mix, and primers. Ensure that the master mix is sufficient for n+1 reactions, where n is the number of reactions required.
2. ##### **Master Mix Preparation**

   1. Prepare the master mix by combining the calculated amounts of water, 2X Q5 High-Fidelity Master Mix, and primers in an Eppendorf tube.
   2. Mix the components thoroughly to ensure homogeneity.
3. ##### **Aliquoting the Master Mix:**

   Aliquot the prepared master mix into 9 separate PCR tubes. These tubes will be used for 8 samples for gradient PCR and one for a negative control (sample 11) with no DNA.
4. ##### **Sample Preparation:**

   Set up the following samples in the PCR tubes containing the master mix:

   * Samples 1 to 8: These are your experimental samples for gradient PCR. Ensure that each tube contains a different DNA template concentration, as per your gradient PCR design.
   * Sample 9: Negative control with 10 ng of tccA (if applicable) and water. Mix thoroughly.
   * Sample 10: Positive control with the MAM7 gene (if applicable). Mix thoroughly.
   * Sample 11: Negative control with no DNA. This serves as a blank control.
5. ##### **Labeling:**

   Label each PCR tube appropriately, indicating the sample number, type of sample, and any other relevant information.

## **Plasmid Isolation**

#### **Objective:**

To isolate plasmid DNA from bacterial cells using the QIAprep 2.0 spin column-based plasmid isolation method and measure the concentration of the isolated plasmid.

#### **Materials:**

* Bacterial culture samples (1-10)
* QIAprep 2.0 spin column kit
* Microcentrifuge tubes
* Pipettes (various sizes)
* Table-top microcentrifuge
* Collection tubes
* Buffer P1
* Buffer P2
* Buffer N3
* Buffer PE
* Buffer EB (10 mM Tris-HCl, pH 8.5)
* Spectrophotometer
* Labels

#### **Procedure:**

1. ##### **Pelleting Bacterial Cells:**

   Centrifuge the bacterial culture samples (1-10) at 13,000 rpm for 10 minutes at 4°C to pellet the bacterial cells.
2. ##### **Resuspension and Lysis**

   1. Resuspend the pelleted bacterial cells in 250 μl of Buffer P1. Transfer the resuspended cells to a microcentrifuge tube.
   2. Add 250 μl of Buffer P2 to the cell suspension. Mix thoroughly by inverting the tube 4–6 times until the solution becomes clear.
   3. Add 350 μl of Buffer N3 immediately and mix immediately and thoroughly by inverting the tube 4–6 times.
   4. Centrifuge the mixture for 10 minutes at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
3. ##### **Binding and Washing**

   1. Add 800 μl of the supernatant to a QIAprep 2.0 spin column by pipetting.
   2. Centrifuge the column for 30–60 seconds at 13,000 rpm and discard the flow-through.
   3. Wash the QIAprep 2.0 spin column by adding 0.75 ml of Buffer PE. Centrifuge for 30–60 seconds at 13,000 rpm.
4. ##### **Elution of Plasmid DNA**

   1. Transfer the QIAprep 2.0 spin column to a collection tube.
   2. Centrifuge the column for 1 minute to remove the residual wash buffer.
   3. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube.
   4. To elute DNA, add 30 μl of Buffer EB (10 mM Tris-HCl, pH 8.5) to the center of the QIAprep 2.0 spin column. Let it stand for 5 minutes, then centrifuge for 1 minute at 13,000 rpm.
   5. Repeat the elution step by adding another 15 μl of Buffer EB to the column, letting it stand for 4 minutes, and then centrifuging for 1 minute at 13,000 rpm.
5. ##### **Measurement of Plasmid Concentration:**

   Measure the concentration of the isolated plasmid using a spectrophotometer at 260nm.

## **PCR Cleanup**

#### **Objective:**

To purify amplified tccA DNA fragments using a QIAquick column-based PCR cleanup method and determine the gene's concentration by UV spectrophotometry.

#### **Materials:**

* Amplified tccA DNA
* Buffer PB
* QIAquick column
* 2 ml collection tube
* Buffer PE
* Autoclaved 1.5 ml Eppendorf tube
* Buffer EB (10 mM Tris·Cl, pH 8.5)
* UV spectrophotometer

#### **Procedure:**

1. ##### **Purification of Amplified tccA DNA**

   1. Take each separate 25 μL of the amplified tccA DNA
   2. Add 125 μL of Buffer PB to each of the amplified tccA mixes and mix thoroughly.
   3. Transfer each mixture to a separate QIAquick column, placing each column in a provided 2 ml collection tube.
   4. Centrifuge each column for 30–60 seconds at 13,000 rpm, 32°C.
   5. Discard the flow-through and place the QIAquick columns back in their respective collection tubes.
   6. Add 750 μL of Buffer PE to each QIAquick column and centrifuge for 30–60 seconds at 13,000 rpm, 32°C for washing.
   7. Centrifuge each QIAquick column for an additional 1 minute at 13,000 rpm, 32°C in a new 2 ml collection tube to remove residual wash buffer.
   8. Place each QIAquick column in an autoclaved 1.5 ml Eppendorf tube.
   9. Add 25 μL of Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each QIAquick membrane and let it stand for 1 minute.
   10. Centrifuge each QIAquick column for 1 minute at 13,000 rpm, 32°C to elute the DNA.
   11. To the same QIAquick membrane, add 15 μL of Buffer EB to the center and place it in a new 2 ml collection tube that was provided. Centrifuge for 1 minute at 13,000 rpm, 32°C to elute the DNA again.
2. ##### **Concentration Determination by UV Spectrophotometry:**

   Measure the concentration of the purified gene using a UV spectrophotometer at 260 nm.

## **Testing enzyme effectivity with single and and double digestion**

#### **Objective:**

To test the effectiveness of enzyme digestion using both single and double digestion reactions.

#### **Materials:**

* Template DNA (for 250 ng of vector)
* 10X CutSmart Buffer
* BamHI enzyme
* XhoI enzyme
* Milli-Q water
* Microcentrifuge tubes
* PCR tube rack
* Thermal cycler
* DNA ladder (1 kb ladder)
* Gel electrophoresis apparatus
* Gel-loading dye

#### **Procedure**

* ##### **Single Digestion Reaction Mix Preparation**

  1. Components for the single digestion of the gene:
     + Template DNA (for 250 ng insert): 10 μl
     + 10X CutSmart Buffer: 2.5 μl
     + Restriction enzyme of choice: 0.25 μl
     + Milli-Q water: Make up to 25 μl
  2. Prepare the single digestion reaction mixtures as specified above and mix them thoroughly. Incubate the reactions at the appropriate temperature for restriction enzyme digestion (typically 37°C) for 3 hours
* ##### **Double Digestion Reaction Mix Preparation**

  1. Components for the double digestion of the gene and plasmid vector:
     + Template DNA (for 250 ng insert): 10 μl
     + 10X CutSmart Buffer: 2.5 μl
     + Restriction enzyme 1 of choice: 0.25 μl
     + Restriction enzyme 2 of choice: 0.25 μl
     + Milli-Q water: Make up to 25 μl
  2. Prepare the double digestion reaction mixture as specified above and mix it thoroughly.
* ##### **Thermocycling Conditions for Double Digestion**

  1. Perform the following thermocycling steps for the double digestion reaction:
     + Incubation at 37°C for 3 hours for enzyme digestion.
     + Enzyme inactivation at 65°C for 20 minutes.
     + Hold the reaction at 4°C until further use.
* ##### **Gel Electrophoresis**

  1. Load the digested DNA samples along with a 1 kb DNA ladder onto an agarose gel.
  2. Perform gel electrophoresis using appropriate electrophoresis conditions

## **Gibson Assembly**

#### **Objective:**

To perform Gibson assembly by combining the required components to create a reaction mixture and follow the protocol provided in the NEB (New England Biolabs) Gibson Assembly protocol (Reference: [NEB Gibson Assembly Protocol](https://www.neb.com/en/protocols/2012/12/11/gibson-assembly-protocol-e5510)).

#### **Materials**

* Gibson Assembly Master Mix (2X)
* Template DNA (50 ng/μl)
* Insert DNA (38 ng/μl)
* Nuclease-free water
* Microcentrifuge tubes
* Pipettes (10 μl, 2 μl, 0.25 μl, 7.25 μl)
* thermocycler
* Incubator (50°C)

#### **Procedure**

1. ##### **Component Preparation:**

   | Component | Volume(μl) | Concentration Available |
   | --- | --- | --- |
   | Gibson Assembly Master Mix | 10 | 2X |
   | Template DNA | 2 | 50 ng/μl |
   | Insert DNA | 0.25 | 38 ng/μl |
   | Nuclease-free Water | 7.25 |  |
   | Total Volume | 20 |  |
2. ##### **Reaction Mix Preparation**

   1. In a microcentrifuge tube, add the following components in the order listed:
      * 10 μl of Gibson Assembly Master Mix (2X)
      * 2 μl of Template DNA
      * 0.25 μl of Insert DNA
      * 7.25 μl of Nuclease-free Water
   2. Mix the components gently by pipetting up and down, ensuring thorough mixing. Avoid introducing air bubbles.
3. ##### **Gibson Assembly Reaction**

   1. Follow the Gibson Assembly protocol provided in the NEB reference link ([here](https://www.neb.com/en/protocols/2012/12/11/gibson-assembly-protocol-e5510)) for the specific temperature and incubation times required for the reaction.
   2. Typically, this will involve incubating the reaction mixture at 50°C for a certain duration to allow for assembly and then cooling to 4°C or placing on ice to terminate the reaction.
4. ##### **Transformation (If Required):**

   If the next step involves transforming the assembled DNA into a host organism (e.g., E. coli), follow the appropriate transformation protocol.

## **Competent cell preparation and Transformation of Gibson assembled tccA + pET22b+ to E.coli dh5alpha/BL21 using CaCl2 method**

#### **Objective:**

To transform Gibson assembled tccA + pET22b+ into E. coli DH5alpha using the CaCl2 method, followed by plating and incubation.

#### **Materials:**

* 0.1M CaCl2
* Anhydrous CaCl2 (11.1g)
* Autoclaved distilled water
* PVDF filter (0.22μm)
* LB Agar
* LB agar powder (6g)
* Ampicillin (0.25μL)
* Distilled water
* LB Broth
* LB broth powder (2.5g)
* E. coli DH5-alpha culture
* Glycerol stock
* Falcon tubes (15ml)
* Ice
* 1.5ml Falcon tubes
* Plasmid (10ng)
* LB media (pre-warmed)
* LB ampicillin plates

#### **Procedure:**

1. ##### **Chemical and Media Preparation**

   1. **0.1M CaCl2** a. Weigh out 11.1g of anhydrous CaCl2 and add it to 80mL of autoclaved distilled water. b. Mix the solution until the CaCl2 is fully dissolved. c. Top up the solution to a final volume of 100mL. d. Take 10mL of the above solution and dilute it by adding 90mL of autoclaved distilled water for a 1:10 dilution. e. Filter sterilized the solution through a PVDF filter with a pore size of 0.22μm.
   2. **LB Ampicillin Plates** a. Weigh out 6g of LB agar powder and add it to 250ml of distilled water. b. Autoclave the LB agar solution. c. Dissolve 0.25μL of ampicillin into the LB agar solution. d. Pour 25ml of LB-ampicillin solution into each Petri plate.
   3. **LB Ampicillin Plates** a. Weigh out 6g of LB agar powder and add it to 250ml of distilled water. b. Autoclave the LB agar solution. c. Dissolve 0.25μL of ampicillin into the LB agar solution. d. Pour 25ml of LB-ampicillin solution into each Petri plate.
2. ##### **E. coli DH5-alpha Culture Preparation**

   Inoculate 200μL of E. coli DH5-alpha from the glycerol stock onto 5ml of LB. b. Incubate the culture at 37°C and 200rpm for 16 hours. c. Measure the growth of the DH5-alpha culture using a spectrophotometer at absorbance A600. d. Transfer the culture to 15ml falcon tubes placed on ice. e. Centrifuge the falcon tubes at 4°C at 5600g for 10 minutes to pellet the DH5-alpha culture. f. Discard the supernatant. g. Resuspend each pellet in 1ml of ice-cold 0.1M CaCl2. h. Incubate the 2ml cell suspension on ice for 30 minutes. i. Centrifuge the suspension at 4°C at 5600g for 10 minutes. j. Discard the supernatant. k. Resuspend each pellet in 500μL of ice-cold 0.1M CaCl2. l. Prepare five 100μL aliquots of competent cells in ice-cold 1.5ml falcon tubes. m. Store the prepared competent cells at -80°C.
3. ##### **Transformation**

   * Add 10ng of plasmid to each aliquot for the required transformant and positive control.
   * Incubate the cells on ice for 30 minutes.
   * Place the cells in a 42°C water bath for exactly 30 seconds.
   * Place the cells on ice for 2 minutes.
   * Add 1ml of pre-warmed LB media to each aliquot.
   * Incubate the aliquots of cells at 37°C, 200rpm for 1 hour.
4. ##### **Plating and Incubation**

   * Spread plate 100μL of the transformed cells from the aliquots onto LB ampicillin plates.
   * Incubate the plates at 37°C for 12-16 hours.

## **Colony PCR**

#### **Objective:**

To perform colony PCR of transformed cells, including colony cell suspension preparation, PCR reaction mix preparation, and setting thermocycling conditions.

#### **Materials:**

* Transformed cell colonies
* Autoclaved toothpick
* Eppendorf tubes
* Nuclease-free (autoclaved Milli-Q) water
* PCR reagents (e.g., Q5 Master Mix, T7 Forward Primer, tccA Reverse Primer)
* PCR tubes
* Thermocycler
* Labels

#### **Procedure:**

1. ##### **Colony Cell Suspension Preparation**

   1. Using a freshly autoclaved toothpick, touch it onto a colony of transformed cells.
   2. Dip the toothpick into an Eppendorf tube containing 100 μl of nuclease-free (autoclaved Milli-Q) water to create a colony cell suspension. Ensure the toothpick is fully submerged in the water.
   3. Mix the suspension by gentle vortexing or pipetting.
2. ##### **PCR Reaction Mix Preparation:**

   Prepare a PCR reaction mix for colony PCR with the following components in a PCR tube:

   * Q5 Master Mix (2X): 12.5 μl
   * T7 Forward Primer: 1.5 μl
   * tccA Reverse Primer: 1.5 μl
   * Autoclaved Milli-Q Water: 8.5 μl
   * Colony Cell Suspension: 1 μl

   Mix the components by gentle pipetting or vortexing.
3. ##### **Thermocycling Conditions for Colony PCR:**

   Set up the thermocycler with the following cycling conditions:

   * **Initial Denaturation:**
     + Temperature: 98°C
     + Time: 3 minutes
   * **Cycling (30 cycles):**
     + **Denaturation**
       - Temperature: 98°C
       - Time: 15 seconds
     + **Annealing:**
       - Temperature: 51°C
       - Time: 30 seconds
     + **Extension:**
       - Temperature: 72°C
       - Time: 90 seconds
   * **Final Extension:**
     + Temperature: 72°C
     + Time: 10 minutes
   * **Hold:**
     + Temperature: 4°C
4. ##### **Sample Loading:**

   Load 25 μl of each PCR reaction mix, including the colony PCR reactions (for 10 colonies) and the positive control, into separate PCR tubes.
5. ##### **Thermocycler Run:**

   Start the thermocycler program to run the colony PCR reactions.
6. ##### **Analysis:**

   After the PCR run is complete, analyze the PCR products as needed (e.g., gel electrophoresis, DNA sequencing).

## **Running of Gel Electrophoresis**

#### **Objective:**

To prepare a 1% agarose gel and run gel electrophoresis for DNA samples, including DNA templates and a ladder.

#### **Materials:**

* Agarose (0.3 g)
* 1X TAE buffer (30 ml)
* SYBR Safe dye (2 μl)
* Electrophoresis tank with combs
* DNA templates (Colony 1-10)
* 1 kb ladder
* Micropipettes and tips
* Electrophoretic power supply
* Power cord
* Labels

#### **Procedure:**

1. ##### **Preparation of 1% Agarose Gel**

   1. Weigh 0.3 g of agarose and add it to 30 ml of 1X TAE buffer in a flask to make a 1% agarose gel.
   2. Heat the mixture until the agarose completely dissolves and the solution becomes clear.
   3. After dissolving, add 2 μl of SYBR Safe dye to the agarose solution and mix gently.
2. ##### **Electrophoresis Tank Setup**

   1. Set the electrophoresis combs to be approximately 2 cm away from the cathode side of the gel.
   2. Ensure that the gel tank is clean and dry.
3. ##### **Pouring the Agarose Gel**

   1. When the agarose solution reaches a temperature of approximately 60 degrees Celsius, pour it into the central part of the gel tank.
   2. Allow the gel to solidify by keeping it undisturbed at room temperature.
4. ##### **Adding 1X TAE Buffer:**

   Pour 1X TAE buffer into the gel tank until the buffer level stands at 0.5-0.8 cm above the gel surface.
5. ##### **Well Formation:**

   Gently lift the combs from the gel, ensuring that the wells remain intact for loading samples.
6. ##### **Sample Loading:**

   Load the DNA samples into the wells as follows for both gels:

   * Well 1 (of both gels): 1 kb ladder (6 μl)
   * Wells 2-7 (gel 1): Colony 1-6 DNA templates (25 μl each)
   * Wells 2-5 (gel 2): Colony 7-10 DNA templates (25 μl each)
7. ##### **Electrophoresis**

   1. Connect the power cord to the electrophoretic power supply.
   2. Set the electrophoresis parameters as needed (e.g., voltage, run time) and start the electrophoresis.
   3. Allow the electrophoresis to run until the DNA samples have migrated sufficiently to achieve the desired separation.
8. ##### **Visualization and Documentation**

   1. After electrophoresis, remove the gel from the tank and place it on a UV transilluminator or gel documentation system.
   2. Visualize the separated DNA bands using UV light.
9. ##### **Data Recording:**

   Document the gel image and record the positions and sizes of DNA bands.
10. ##### **Safety Precautions:**

    Follow appropriate safety measures when working with UV light sources and SYBR Safe dye.

## **SDS-PAGE for protein analysis**

#### **Objective:**

To perform SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) for protein analysis.

#### **Materials:**

* Gel casting unit with glass plates and spacers
* Deionized water
* Ethanol
* 30% Acrylamide
* 1M Tris (pH 8.8)
* 10% SDS
* 10% APS(freshly prepared)
* TEMED
* 0.5M Tris (pH6.8)
* Ethanol (for gel surface overlay)
* Comb for creating wells in the gel
* Sample loading buffer
* Protein samples (Induced and Uninduced)
* Electrophoresis apparatus
* Coomassie Blue dye
* Destaining solution
* Plastic container
* Shaker

#### **Procedure:**

1. ##### **Preparation of Gel Casting Unit**

   1. Clean the glass plates and spacers of the gel casting unit with deionized water and ethanol.
   2. Assemble the plates with the spacers on a stable, even surface.
2. ##### **Preparation of Resolving Gel**

   1. Prepare the resolving gel solution using the following volumes for a 12.5% gel:
   2. | Component | Resolving Gel |
      | --- | --- |
      | MilliQ water | 1.151mL |
      | 30% Acrylamide | 2.079mL |
      | 1M Tris: pH 8.8 | 1.663mL |
      | 10% SDS | 50μl |  |
      | 10% APS(freshly prepared) | 50μl |  |
      | TEMED | 5μl |  |
   3. Pour the gel solution into the plates assembled with spacers. To maintain an even and horizontal resolving gel surface, overlay the surface with ethanol.
   4. Allow the gel to set for about 20-30 minutes at room temperature.
3. ##### **Preparation of Stacking Gel**

   1. Prepare the stacking gel solution using the following volumes:
   2. Allow the gel to solidify by keeping it undisturbed at room temperature.
   3. | Component | Stacking Gel |
      | --- | --- |
      | MilliQ water | 1.752mL |
      | 30% Acrylamide | 0.369mL |
      | 0.5M Tris: pH 6.8 | 0.324mL |
      | 10% SDS | 25μl |  |
      | 10% APS(freshly prepared) | 25μl |  |
      | TEMED | 3μl |  |
   4. Discard the overlaid ethanol on the resolving gel.
   5. Add the 5% stacking gel solution until it overflows. Insert the comb immediately ensuring no air bubbles are trapped in the gel or near the wells.
   6. Allow the gel to set for about 20-30 minutes at room temperature.
4. ##### **Sample Preparation and Loading**

   1. Add 1 mL of loading buffer to each of the samples (Induced and Uninduced) of 1 mL each and incubate at 99 degrees for 20 minutes.
   2. Load these samples onto the gel along with two ladder markers.
   3. Perform electrophoresis at 50V for 3 hours.

- ##### **Staining and Destaining**

  1. Carefully remove the gel and place it in a plastic container.
  2. Add Coomassie Blue dye to the gel and leave it overnight on a shaker.
  3. The next day, add a destaining solution and keep it for 4 hours.
  4. Observe the gel under white light.

**Troubleshooting**

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}