iGEM 2024 — Team hongkong-jss

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block extraStyles %}
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{% block page\_content %}

# Notebook

![](https://static.igem.wiki/teams/5152/banner/notebook.webp)

## Side Menu

[Experiment Log](#section1)
[Hardware Log](#section2)
[Human Practice Log](#section3)

## Experiment Log

3/7/2024(Wed)

Preparation of linearized pUC19 for HiFi Assembly.

Preparation of ampicillin plates for positive controls.

4/7/2024(Thu)

PstI & EcoRI double digestion of pUC19 (Labeled pUC19 digested)

|  |  |
| --- | --- |
| COMPONENT | 50 μl REACTION |
| DNA | 20 uL |
| 10X NEBuffer r2.1 | 5 uL |
| EcoRI-HF | 2 |
| PstI | 2 |
| Nuclease-free Water | 21 |

* Incubate 37oC for 1 hr, put into 4oC for Friday Material Preparation

**Material preparation**

* Prepared LB/Amp plate and LB/plate ~20 each
* Prepared 20mLLBAmpx3in4oC
* Opened new AMP x 2 from -80oC

**gBlock resuspension**

* Resuspended gBLOCKs of 5 +ve controls by TE from purelink miniprep (old), 100 uL each to dilute 1000 ng into 10 ng/L (according to IDT suggestion)
* Overnighted at 50oC (mistake)

5/7/2024(Fri)

**Material preparation**

* Minipreped pUC19 x 6
* PreparedLB/Amp (Last batch Control test failed on OP50)

**HiFi Assembly**

* Ran gel on digested pUC19 + Gel purification x 4
* Used Hebe's linearized pUC19

![](https://static.igem.wiki/teams/5152/notebook2/note01.webp)

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Components | amilCP | cjBlue | eforRed | tsPurple | dTomato | Positve  (+Ve) | Remarks |
| Vector | 2 | 2 | 2 | 2 | 2 | 10 |  |
| Insert | 5.7 | 5.8 | 5.6 | 5.7 | 5.8 |  | All added ~6 ul |
| HiFiMM | 10 | 10 | 10 | 10 | 10 | 10 |  |
| H2O | 2.3 | 2.2 | 2.4 | 2.3 | 2.2 |  |  |
| Total | 20 | 20 | 20 | 20 | 20 | 20 |  |

* Incubate at 50oC for 60 mins
* Heat shock transformation into TOP10 x 6 (10 uL DNA, 10 min on ice, 42C 45s, 500 uL SOC), spread on LB/AMP (4:30 PM)

6/7/2024(Sat)

Time: ~1:30 pm

**Positive tests result**

* cjBlue, tsPurple - no colonies / dried plates
* dTomato, amilCP, eforRed - small colonies, with color
* +ve - no colonies

**Postive test**

* dTomato, amilCP, eforRed respread again on LB/AMP/ARA from 0524
* cjBlue, tsPurple, +ve Hifi, respread again from transformed comp cells on LB/AMP/ARA from 0524

**Plate checking**

* Relabelled all plate stacks A-E
* Each chose 2 plates to spread pUC19 / *E. coli*
* *E. coli* added SOC at 37oC to grow up for stock

![](https://static.igem.wiki/teams/5152/notebook2/note02.webp)

9/7/2024(Tue)

**5 +ve control results:**

All +ve controls showed positive results, with respective colors (cjBlue - Green, tsPurple - Purple, amilCP - Blue, dTomato - Red, eforRed - Red)

**Testing plate:**

All amp working, except stack A with an unknown colony, discard stack A

**Work today:**

* Prep LB/broth to culture single colony of the +ve controls for Miniprep of the plasmids for glycerol stock
* Order experimental constructs

10/7/2024(Wed)

* Incorrectly inoculated 2 dTomato but no cjBlue
* Spread from the inoculated broths on LB/AMP plates
* Picked a colony from original plate of cjBlue to spread

11/7/2024(Thu)

**Results**

* All 4 spreaded plate showed color
* eforRed most vivid in broth but less in plate

(After 18 hours)

![](https://static.igem.wiki/teams/5152/notebook2/note03.webp)![](https://static.igem.wiki/teams/5152/notebook2/note04.webp)

![](https://static.igem.wiki/teams/5152/notebook2/note05.webp)

**Work**

* Prep 20 mL LB/AMP x 7, 4 for growing dTomato, amilCP, eforRed, tsPurple stock 3 for selecting cjBlue since it doesn't express very well

![](https://static.igem.wiki/teams/5152/notebook2/note06.webp)

* Miniprep dTomato, amilCP, eforRed, tsPurple
* Ordered 6 constructs for experiments, refer to IDT fact sheet

12/7/2024(Fri)

**Results**

7 LB broth:

* 3 x cjBlue (no observable colouration)
* Grew dTomato, amilCP, eforRed, tsPurple in 20 mL at 37oC for 16 hrs for glycerol stock

**Work**

* Preparation of glycerol stock (tsPurple, amilCP each x 3)
* Respread cjBlue from colonies of original plates
* eforRed and dTomato re-culture in 20 mL broth to ensure the colouration is correct

(after 48 hours)

![](https://static.igem.wiki/teams/5152/notebook2/note07.webp)

6/8/2024(Tue)

* Resuspended gBLOCKs of 6 experimental constructs by TE from purelink miniprep (new), 100 uL each to dilute 1000 ng into 10 ng/L (according to IDT suggestion)
* Incubate at 50oC for 10 mins

**HiFi Assembly**

* Used LP puC19 (digested + gel purified) \*\*\* (Hebe's not found)

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Component | pZnt | PbrR | YodA | MerR (Hg) | Cd 2 | Cd 1 |
| Vector | 2 | 2 | 2 | 2 | 2 | 2 |
| Insert | 6 | 6 | 6 | 6 | 6 | 6 |
| HiFiMM | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 2 | 2 | 2 | 2 | 2 | 2 |
| Total | 20 | 20 | 20 | 20 | 20 | 20 |

* Incubated at 50oC for 30 mins
* Heat shock transformation into TOP10 x 7 (with 1 -ve control) (10 uL DNA, 10 min on ice, 42C 45s, 500 uL SOC), spread on LB/AMP (12:30 PM)

8/8/2024(Thu)

* All plates have no colonies
* Redo starting from the ligation part

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Component | pZnt | PbrR | YodA | MerR (Hg) | Cd 2 | Cd 1 |
| Vector | 2 | 2 | 2 | 2 | 2 | 2 |
| Insert | 6 | 6 | 6 | 6 | 6 | 6 |
| HiFiMM | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 2 | 2 | 2 | 2 | 2 | 2 |
| Total | 20 | 20 | 20 | 20 | 20 | 20 |

* Incubated at 50oC for 30 mins
* Heat shock transformation into TOP10 x 8 (with 1 -ve control) (10 uL DNA, 10 min on ice, 42C 45s, 500 uL SOC), spread on LB/AMP (3:30 PM)

9/8/2024(Fri)

Yesterday incorrectly spread on LB plate without amp, all formed lawn

Today poured 20 new LB/AMP plates, may consider re-spread yesterday's transformed cells Nevertheless, today perform a new set from miniprep of pUC19 cells

* Miniprep 2 x 100 uL pUC19 from 6 mL *E. coli*
* Ran gel to confirm the purity of miniprep product (but the bands were very curved and it seems there is some contamination in the wells? Clean the gel cask more thoroughly next
* Proceed to PstI and EcoRI digestion.

|  |  |
| --- | --- |
| Components | 1X (uL) |
| Buffer 2.1 | 5 |
| PstI | 3 |
| EcoRI | 3 |
| DNA (pUC19) | 15 uL (~3 ug) |
| H2O | 24 |
| Total | 50 |

* Run gel to check and directly proceed to gel purification Gel: Ladder, Digested pUC19

![](https://static.igem.wiki/teams/5152/notebook2/note08.webp)

14/8/2024(Wed)

* Due to gel always showing faint bands and smears, we suspect the pUC19 stock is degraded. Start from the beginning.
* Today we transformed pUC19 to TOP10 again. pUC19 obtained from invitrogen kits.
* 10 uL used for heat shock transformation.

15/8/2024(Thu)

Picked transformed colonies to inoculate 2 x 50 mL LB/AMP, 37oC overnight 180 rpm

16/8/2024(Fri)

* Miniprep x 4 pUC19 from 3mL cells from yesterday
* on re-spreaded plates with experimental construct - Cd1, pYodA, PbrR, +ve (others no colonies)

|  |  |  |
| --- | --- | --- |
| Components | 25 uL | x16 |
| Forward M13 | 0.5uL | 8 |
| Reverse M13 | 0.5uL | 8 |
| Template | Colonies |  |
| 2X OneTaq | 12.5 | 200 |
| H2O | 11.5 | 184 |
| Total | 25 |  |

PCR condition:

|  |  |  |
| --- | --- | --- |
| Stage | Temp | Time |
| Initial denaturation | 94 | 30 |
| Denaturation | 94 | 20 |
| Annealing | 55 | 40 |
| Extension | 68 | 60 |
| Final extension | 68 | 300 |
| Cycle | 30 |  |

* Then, proceed to run gel
* Gel results show no bands, but very sharp primer bands. Forgot to dilute the primer stock (100 uM were used!!)
* New pUC19 Proceed to PstI and EcoRI digestion.

![](https://static.igem.wiki/teams/5152/notebook2/note09.webp)![](https://static.igem.wiki/teams/5152/notebook2/note10.webp)

|  |  |
| --- | --- |
| Components | 1X (uL) |
| Buffer 2.1 | 5 |
| PstI | 3 |
| EcoRI | 3 |
| DNA (pUC19) | 15 uL (~3 ug) |
| H2O | 24 |
| Total | 50 |

* Run gel to check and directly proceed to gel purification

21/8/2024(Wed)

HiFi Assembly

Used LP puC19( digested + gel purified)

|  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Components | pZnt | PbrR | YodA | MerR  (Hg) | Cd 2 | Cd 1 | +ve |
| Vector | 1 | 1 | 1 | 1 | 1 | 1 | 10 |
| Inserts | 6 | 6 | 6 | 6 | 6 | 6 |  |
| Hifi MM | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 3 | 3 | 3 | 3 | 3 | 3 |  |
| Total | 20 | 20 | 20 | 20 | 20 | 20 | 20 |

Incubated at 50oC for 60 mins

Heat shock transformation into TOP10 x 8 (with 1 -ve control) (10 uL DNA, 10 min on ice, 42C 45s, 200 uL SOC), spread on LB/AMP (12:30 PM)

again but no bands in all lanes. All faint bands ~100 bp are primers? (Seems no primer band in - ve)

![](https://static.igem.wiki/teams/5152/notebook2/note11.webp)

PCR validation to check if our PCR set is available

![](https://static.igem.wiki/teams/5152/notebook2/note12.webp)

|  |  |  |  |
| --- | --- | --- | --- |
| Components | 25ul | x1.5 volume | x3 |
| Forward M13 | 0.5 | 0.75 | 2.25 |
| Reverse M13 | 0.5 | 0.75 | 2.25 |
| Template | 2 | 3 |  |
| 2X OneTaq | 12.5 | 18.75 | 56.25 |
| H2O | 9.5 | 14.25 | 42.75 |
| Total | 25 | 37.5 | 103.5 |

PCR condition and primer sets are working as expected, band size within expectation. PCR no problem.

30/8/2024(Fri)

New order from IDT arrive, sequence (5' overlap 20 nt) corrected

Spin down with max speed for 1 min, 100 ul ddH2O resuspend, 50oC 10 mins for dissolving, stored at -20C

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Components | PbrR | MerR-  K174200 | MerR-pMerT | +ve  (dTomato) | -ve |
| Vector | 2 | 2 | 2 | 2 | 10 |
| Insert | 6 | 6 | 6 | 6 |  |
| Hifi MM | 10 | 10 | 10 | 10 | 0 |
| H2O | 2 | 2 | 2 | 2 | 10 |
| Total | 20 | 20 | 20 | 20 | 20 |

Incubated at 50oC for 90 mins

Heat shock transformation into TOP10 x 5 (10 uL DNA, 30 mins on ice, 42C 45s, 500 uL SOC), spread on LB/AMP (5:30 PM), incubate for 24 hrs

All HiFi assemble kit used up. Order new one (50rx) from BioArrow

2/9/2024(Mon)

Carry out experiment to characterize the expression of the chromoproteins (dTomato, tsPurple, eforRed, and amilCP)

Set different time points (4, 8, 12, 18, 24 hrs)

Carry out on new transformants, Correct colonies identified from all 3 plates (refer to gel photos)

18hrs:

![](https://static.igem.wiki/teams/5152/notebook2/note13.webp)

12hrs:

![](https://static.igem.wiki/teams/5152/notebook2/note14.webp)

4, 8 shows no pellets.`

![](https://static.igem.wiki/teams/5152/notebook2/note15.webp)

 Proceed to carry out functional study - PbrR (PbNo3), MerR-K174200 (CdCl2), MerR-pMerT (HgCl2) (5:00 pm)

19 mL of culture + 1 mL of metal solution to reach final conc. (Pb: 100 uM, Cd: 200 uM, Mer: 100 uM, Zn: 200 uM)

11/9/2024(Wed)

Result observation (after 16 hours, 9am)

 1 mL of treated culture, spin down 8000g, 2 mins

 Positive result found on PbRr-pPbRr construct (colony 3, 5), repeat

![](https://static.igem.wiki/teams/5152/notebook2/note16.webp)

 Positive result found on MerR-K174200 construct (colony 2, 3), repeat

![](https://static.igem.wiki/teams/5152/notebook2/note17.webp)

 No growth observed in pMerT + Hg culture, showing that this final conc. inhibits the growth of *E. coli* completely.

![](https://static.igem.wiki/teams/5152/notebook2/note18.webp)

New DNA from Twist arrived

New HiFi assemble kit arrived (50 rx)

Prepare to re-do the assembles

12/9/2024(Thu)

Positive result found on PbRr-pPbRr construct repeat.

![](https://static.igem.wiki/teams/5152/notebook2/note19.webp)

However, both MerR-K174200 +ve and -ve turned color in the repeat. Repeat again.

![](https://static.igem.wiki/teams/5152/notebook2/note20.webp)left to right: -ve, pUC19, Cd+

New Twist DNA fragments assemble:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Component | pZnt | pYodA | K174015 | +ve  (dTomato) | -ve |
| Vector | 2 | 2 | 2 | 2 | 10 |
| Insert | 6 | 6 | 6 | 6 |  |
| HiFi MM | 10 | 10 | 10 | 10 |  |
| H2O | 2 | 2 | 2 | 2 | 10 |
| Total | 20 | 20 | 20 | 20 | 20 |

Incubated at 50oC for 60 mins

Heat shock transformation into TOP10 x 5 (10 uL DNA, 30 mins on ice, 42C 45s, 500 uL SOC), spread on LB/AMP (5:30 PM), incubate for 24 hrs (9:15 am)

Lawn formed on plates, suspect that ampicillin went wrong

Resuspended new ampicillin in from stock (15 ml water into 200 mg ampicillin salt)

Doing control tests on the new ampicillin - pUC19 vs TOP10 in LB/AMP broth

MerR-K172400 repeat with 200 uM cadmium works this time.

![](https://static.igem.wiki/teams/5152/notebook2/note21.webp)

13/9/2024(Fri)

No colonies found on re-spread transformants

Ampicillin concentration seems incorrect, no growth shown in both setups

Test on ampicillin concentration - 2X, 5X, 10X, 20X dilution

Found that 5X dilution of the ampicillin stock is suitable

![](https://static.igem.wiki/teams/5152/notebook2/note22.webp)

Re-spreaded the transformed *E. coli*

Pour LB/Amp plates again

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Component | pZnt | pYodA | K174015 | +ve  (dTomato) | -ve |
| Vector | 2 | 2 | 2 | 2 | 10 |
| Insert | 6 | 6 | 6 | 6 |  |
| HiFi MM | 10 | 10 | 10 | 10 |  |
| H2O | 2 | 2 | 2 | 2 | 10 |
| Total | 20 | 20 | 20 | 20 | 20 |

Incubated at 50oC for 60 mins

Heat shock transformation into TOP10 x 5 (10 uL DNA, 30 mins on ice, 42C 45s, 500 uL SOC), spread on LB/AMP (5:30 PM), incubate for 24 hrs

However, out of the old batch of TOP10 comp cells (Jun 2023). Use a new batch of TOP10 cells (from Feb 2024) (ABE)

14/9/2024(Sat)

Colonies found on pZnt, pYodA plates, except K174015.

![](https://static.igem.wiki/teams/5152/notebook2/note23.webp)

16/9/2024(Mon)

Repeat functional study of PbrR in 15 mL falcon tubes to see if it can also be done to save incubator spaces, however, we doubt it since it is hard to shake in 15 ml falcon tubes. (7:30 am)

Just found out that pMerT was incorrectly ordered, it ordered the same sequence as pPbr…

![](https://static.igem.wiki/teams/5152/notebook2/note24.webp)

After 12 hours, an observable red colour difference can be observed from +ve compared with -ve setups (without Pb and Pb+pUC19 only). However, the cell pellets are obviously smaller and some black colour was observed, this may indicate serious cell death ?

![](https://static.igem.wiki/teams/5152/notebook2/note25.webp)

17/9/2024(Tue)

Carry out with M13 primer sets.

All with correct band sizes observed, may carry on the functional study

![](https://static.igem.wiki/teams/5152/notebook2/note26.webp)

19/9/2024(Thu)

Set-up functional study on pZnT, pYodA. (9:15 am)

 05:30 pm pZnT and pYodA show no growth of cells in both metal added and no metal setup, unknown reason why. May wait until tomorrow and see.

20/9/2024(Fri)

pZnT and pYodA setups still show no sign of growth. (???) Repeat.

Repeat pPbr successful colony.  However, after 24 hours + incubation, although the Cd added culture shows a much brighter red colour, but -ve also show a mild leaky expression of red colour.

![](https://static.igem.wiki/teams/5152/notebook2/note27.webp)

left to right: Cd+, pUC19, -ve

MerR-K172400 construct repeat successful colony. With controls (pUC19 + Cd) and (K172400 without Cd)

![](https://static.igem.wiki/teams/5152/notebook2/note28.webp)

Testing pPbr construct with different concentration of PbNO3 (0.01 uM, 0.1 uM, 1 uM, 10 uM, 500 uM, and 1000 uM), with -ve control and pUC19 cell only control (06:30 pm)

Functional assay on pZnt and pYodA with 200 uM of Zn and 100 uM of Cd. (07:00 pm)

21/9/2024(Sat)

After 14 hours (8:30 am),

harvest result of pPbr. There is an observable colour difference in the culture without spinning down into pellets. From 0.01 - 10 uM, there is an obvious colour change. However, in 500 uM, the colour is not much different from 10 uM. Meanwhile, 1000 uM shows a paler colour. Suspect it's due to the high concentration of Pb's inhibition effect.

After spinning down the pellets, the colour of pellets from 1 - 500 uM show not much difference.

![](https://static.igem.wiki/teams/5152/notebook2/note29.webp)

![](https://static.igem.wiki/teams/5152/notebook2/note30.webp)

left to right: -ve, 0.01 uM, 0.1 uM, 1 uM, 10 uM, 500 uM, 1000 uM

From 0.01 - 10 uM there is an increase, but 500 uM and 1000 uM decreased, maybe due to cell death??

In pZnt setups, after spinning down, both setups shown pale red colour, while +ve (with Zn) added, showed a deeper red colour. Need repeat to confirm.

![](https://static.igem.wiki/teams/5152/notebook2/note31.webp)

In pYodA setups, no growth seen in the +ve setup (Cd added), unknown why. However, the -ve setup shows an obvious blue colour from the amilCP. Doubt it will work. Need repeat to confirm.

![](https://static.igem.wiki/teams/5152/notebook2/note32.webp)

23/9/2024(Mon)

Repeat on the concentration effect of pPbr cells. (7:30 am)

After 12 hours, there is no significant colour difference.

24/9/2024(Tue)

After 24 hours, the colour becomes very obvious.

![](https://static.igem.wiki/teams/5152/notebook2/note33.webp)left to right: pUC19, -ve , 100 uM

The -ve still showed leaky expression. However, it is easily distinguishable the difference between -ve and with Pb added.

![](https://static.igem.wiki/teams/5152/notebook2/note34.webp)

left to right: -ve, 0.1 uM, 1 uM, 10 uM, 100 uM

End of the experiments, work on reports now.

**The end of experiment log.**

## Hardware Engineering log

Mid-April to Early May

Brainstorm and develop the first draft of the device.

• Use a colorimeter to detect the color change of GM *E. coli*.

• Use an LED bulb to indicate whether the sample solution contains excessive heavy metals.

Early May to Mid-June

Redesign the prototype.

Incorporate suggestions from human practices into the design.

• Use Al vision instead of a colorimeter to enhance the effectiveness and accuracy of the device.

• Apply loT technology to notify users via SMS.

• Replace the LED bulb with an LCD screen for direct reading of detection results and additional information.

• Install a temperature control unit to maintain optimal *E. coli* culture conditions.

Mid-June to Late July

Improve the functionality of the device.

Incorporate suggestions from human practices into the design.

• Add a magnetic stirrer to promote oxygen penetration in the chamber.

• Equip a UV light for sterilization before detection.

• Install a bleach dispensing system for eliminating the GM *E. coli* after use.

Late July to Late September

Turn the machine from draft into reality.

• Prepare and purchase all required materials.

• Build and assemble the prototype using laser cutting.

• Write a program to instruct the device.

• ⁠Develop a website and a software for backend management.

• Test the functionality of the device and debug it.

• Introduce the device in detail to all teammates.

**The end of hardware log.**

## Human Practice Log

| Date | Sector | Event | Description |
| --- | --- | --- | --- |
| March 2024 | Public | Online survey (n=357) | We did an online survey to study public perception of heavy metals pollution. |
| April 2024 | Science | Prof. Wong Ming Hung | Prof. Wong's extensive experience in heavy metal pollution has been invaluable in guiding our selection of key metals for the study. |
| April 2024 | Science | Prof. Wang Weng-Xiong | Prof. Wang noted that genetic variations in *E. coli* may affect gene expression and influence testing results. |
| May 2024 | Science | Mr. Wong Cheuk Hon | Mr. Wong recommended testing various heavy metal-inducible promoters and selecting robust reporters to enhance success and facilitate observation. |
| May 2024 | Public | Public survey I (n=188) | We conducted a public survey to evaluate the acceptance of using GM *E. coli* for detecting heavy metals. |
| June 2024 | Science | Mr. Chan Ping Ho | Mr. Chan guided us in applying AI vision and IoT technology to automate our device, enhancing its user-friendliness. |
| June 2024 | Regulation | Team meeting: government policy | We examined regulations on GMO use in high school research and investigated government perceptions of heavy metal contamination issues in Hong Kong. |
| June 2024 | Industry | Dr. Ng Siu Man | Dr. Ng offered several suggestions about the business model and practical applications of our heavy metals detection method. |
| June 2024 | Science | Prof. Tsui Tsz Ki | Professor Tsui provided valuable suggestions concerning the design of our experiments and the development of the hardware. |
| June 2024 | Regulation | Water Supplies Department | Mr. Yu and Mr. Chan detailed the 2015 lead in drinking water incident, enhancing our understanding of the situation and discussing testing standards. |
| June 2024 | Industry | Dr. Leung Wai Mui | Dr. Leung provided insights on using GM *E. coli* to detect heavy metals, shaping our education program and business model. |
| July 2024 | Science | Prof. Ngo Chi Ki | Professor Ngo advised on optimising chromoprotein expression in *E. coli*. |
| September 2024 | Public | Public survey II | We conducted interviews to assess public willingness to use our method for detecting heavy metals, aiming to understand community acceptance and concerns. |
| September 2024 | Regulation | Environmental Protection Department | Dr. Mak noted that our device could enhance low-cost screening for heavy metals but emphasised the need for accuracy to prevent public concern. |

**The end of human practice log.**

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
{% block page\_content %}

# Engineering

![Engineering Banner](https://static.igem.wiki/teams/5152/banner/engineering.webp)

## Side Menu

[Cycle 1: Design of Project Plan](#section1)
[Cycle 1: Build of Chromoprotein References](#section2)
[Cycle 1: Test of Reporter Signals](#section3)
[Cycle 1: Learn from the Reporters](#section4)
[Cycle 2: Design of Metal Biosensors](#section5)
[Cycle 2: Build of Metal Biosensors](#section6)
[Cycle 2: Test of Metal Biosensors](#section7)
[Cycle 2: Learn from the Results](#section8)

![Engineering Cycles](https://static.igem.wiki/teams/5152/engineering/working-photos.webp)

## **Engineering cycles**

During the experimental phase of our project, we followed the principles of the engineering cycle to organize our project workflow, experimental design, and problem-solving methods. We broke our project down into smaller, manageable phases, allowing us to apply the engineering cycle multiple times, which helped us practice and refine our approach.

This structured framework was especially helpful when we faced challenges during the cloning process. In the next section, we'll explain how we successfully carried out our project by integrating the core elements of engineering design thinking.

## **1. Design**

**Identifying the local problem and solution - our inspiration**

After interviewing various experts and Hong Kong government bodies, we discovered that a major source of heavy metal contamination is the improper disposal of building and construction materials by workers. This leads to heavy metals leaking into Hong Kong's water sources, directly affecting the local population. Furthermore, we noticed that traditional Chinese medicine is widely used in Hong Kong, and there have been reports of heavy metal contamination, especially zinc and mercury, in some herbal medicines. This discovery highlighted the importance of developing a convenient and affordable method to detect these pollutants.

Taking these factors into account, we decided to focus our project on developing biosensors for detecting heavy metals using synthetic biology. Biosensors offer several advantages, including being more cost-effective, environmentally friendly since they do not require toxic chemicals for sample preparation, and user-friendly due to the lack of special equipment or advanced techniques needed for operation. Additionally, their portability allows for convenient on-site detection. After conducting research, we identified lead, cadmium, mercury, and zinc as the most prevalent and hazardous heavy metal pollutants in Hong Kong.

As a result, our project aims to create biosensors specifically designed to detect these four heavy metals.

**Designing a workflow that can facilitate problem-solving**

Our primary goal is to develop a biosensor that can be visually observed with the naked eye, allowing individuals without specialized equipment or technical expertise to easily interpret the results—even in settings like our school laboratory, which lacks advanced instruments like a spectrometer.

Although our initial research on biosensors revealed that fluorescent proteins like GFP and RFP, as well as luminescent signals from luciferase, are often more sensitive and responsive, we chose to use chromoproteins. These proteins produce visible colour changes in *E. coli*, making them ideal for our biosensor design.

To begin, we conducted a preliminary study of various chromoproteins available in the iGEM registry. Thanks to the hard work of previous iGEM teams, the registry offers a long and detailed list of chromoprotein candidates, along with valuable information about each. However, we weren"t sure if the expression and culturing conditions in our basic laboratory setup would produce the same results as those reported in the registry. Therefore, we decided to test and briefly characterize the chromoproteins ourselves as the first part of our project.

From the range of colours available, we selected chromoproteins that offered the greatest contrast with the natural colour of bacterial cultures. Green, yellow, and orange proteins were too similar to the pale or yellowish colour of the broth we use, so we eliminated those from consideration. Instead, we chose amilCP ([BBa\_K592009](https://parts.igem.org/Part%3ABBa_K592009)) (dark blue), cjBlue ([BBa\_K592011](https://parts.igem.org/Part%3ABBa_K592009)) (dark green), tsPurple ([BBa\_K1033906](https://parts.igem.org/Part%3ABBa_K1033906)) (bright purple), dTomato ([BBa\_K4813000](https://parts.igem.org/Part%3ABBa_K4813000)) (sharp pink), and eforRed ([BBa\_K592012](https://parts.igem.org/Part%3ABBa_K592012)) (dark red) due to their high contrast and bright colours, as reported in the literature.

Next, we designed five expression constructs for these chromoproteins, utilizing constitutive promoters ([BBa\_J23100](https://parts.igem.org/Part%3ABBa_J23100)) and a strong ribosome binding site ([BBa\_B0032](https://parts.igem.org/Part%3ABBa_B0032)). We recognize that using strong promoters might not fully represent the expression conditions of our final metal biosensor constructs, but the results will provide a rough reference for further investigation.

More importantly, we recognize that problem-solving is the most challenging and crucial aspect of the engineering cycle. Therefore, we created these designs to serve several key purposes as well:

1. Ensuring our bacterial strain, lab conditions, and experimental setup can successfully express these proteins.
2. Verifying that the colour of the expressed proteins matches the expected results from previous reports.
3. Using these five constructs to verify that our experiment design, protocols and materials are correct and functional.
4. Employing these constructs as positive controls in future experiments, providing a reference point for troubleshooting any issues that arise when we create the biosensor constructs.

In our later experiments, these constructs proved to be invaluable for troubleshooting and ensuring that our cloning process was smooth and effective. They provided us with a reliable reference point, helping us confirm that our cloning techniques were working as intended.

## **2. Build**

**Cloning and characterization of chromoprotein expressions**

First, we designed constitutive expression constructs for the chromoproteins *amilCP*([BBa\_K5152002](https://parts.igem.org/Part%3ABBa_K5152002)), *cjBlue* ([BBa\_K5152001](https://parts.igem.org/Part%3ABBa_K5152001)), *tsPurple* ([BBa\_K5152000](https://parts.igem.org/Part%3ABBa_K5152000)), *dTomato* ([BBa\_K4813005](https://parts.igem.org/Part%3ABBa_K4813005)), and *eforRed*([BBa\_K5152003](https://parts.igem.org/Part%3ABBa_K5152003)). For the dTomato part, it was created by our previous iGEM team, we further validated its functions and expressions in this project and documented it in the part registry ([BBa\_K4813000](https://parts.igem.org/Part%3ABBa_K4813000)).

The graphic representation of these constructs is shown below.

 ![](https://static.igem.wiki/teams/5152/engineering/first-pic-1.webp)

The chromoprotein CDS (coding sequences) are driven by the strong Anderson family constitutive promoter ([BBa\_J23100](https://parts.igem.org/Part%3ABBa_J23100)), paired with a strong RBS ([BBa\_B0034](https://parts.igem.org/Part%3ABBa_B0034)), and terminated by a strong double terminator ([BBa\_B0015](https://parts.igem.org/Part%3ABBa_B0015)). Additionally, the constructs include a 5' 20 nt overlapping region and a 3' 20 nt overlapping region to facilitate the HiFi assembly process with the pUC19 vector, using the restriction sites PstI and EcoRI. We opted for double enzyme digestion to linearize the plasmids, as this reduces the likelihood of self-ligation during the assembly process.

The pUC19 vector was sourced from the glycerol stocks of our previous project. It was purified using a miniprep procedure and then subjected to restriction digestion for 2 hours. After digestion, the product was run on a gel electrophoresis, and the band corresponding to the vector (as illustrated in the diagram below) was extracted and purified using a gel purification kit.

![Gel Purification of pUC19](https://static.igem.wiki/teams/5152/engineering/1-gel-purification-of-puc19.webp)

**Figure 1:** Gel photo showing the linearized pUC19 vector (~2.6 kbp) after digestion with PstI and EcoRI. The highlighted band corresponds to the digested vector, which will be extracted and purified using a gel purification kit.

Since we do not have access to spectrophotometry machines due to their high cost, we estimated the concentration of DNA in our samples by comparing the brightness intensity of the bands with the DNA ladder on the gel. Based on this estimation, we calculated the ratio of insert to vector to be approximately 3:1 and proceeded with the assembly process. The chromoprotein inserts were synthesized as gBlocks from IDT.

The five chromoprotein expression constructs were then assembled and transformed into TOP10 *E. coli* strains using the heat shock transformation method. The transformants were selected on LB agar plates containing ampicillin.

After 16 hours of incubation, most of the colonies showed significant colouration corresponding to their constructs, except for *cjBlue*. However, after incubating for more than 24 hours, *cjBlue* also began to express its colour.

![Constitutive Chromoprotein](https://static.igem.wiki/teams/5152/engineering/2-constitutive-chromoprotein.webp)

**Figure 2:** E. coli colonies transformed with the constitutive chromoprotein expression constructs (cjBlue, tsPurple, amilCP, eforRed, and dTomato) on LB/AMP selection plates.

![cjBlue Expression](https://static.igem.wiki/teams/5152/engineering/3-cjblue-weak.webp)

**Figure 3:** E. coli cells transformed with the cjBlue expressing construct show a strange pattern of chromoprotein expression after 16 hours of incubation at 37°C.

![Pellet of Chromoproteins](https://static.igem.wiki/teams/5152/engineering/4-pellet-of-chromoproteins.webp)

**Figure 4:** Cell pellets of E. coli expressing different chromoproteins obtained by centrifugation (8000 g, 2 minutes) of 1 mL of cell culture. Each pellet displays the characteristic colour of the expressed chromoprotein.

We inoculated the colonies from all the plates and repeated the spreading process. The same results were observed, confirming that cjBlue expression was not working as expected in our laboratory conditions. As a result, we decided to exclude cjBlue and selected the remaining chromoproteins as candidates for our biosensor construction. Additionally, since dTomato has been successfully utilized in previous projects by our lab, we are familiar with its expression properties, and it will be used as a positive control in future cloning processes.

## **3. Test**

**Using the workflow and conditions as a positive control**

With the successful expression of the chromoproteins, we proceeded to further characterize their expression properties, specifically focusing on the time required for visible result reading.

We cultured the cells and harvested them at different time points: 4 hours, 8 hours, 12 hours, 18 hours, and 24 hours. At each time point, we collected 1 mL of the culture and centrifuged it at 8,000 g for 2 minutes to obtain the cell pellets for observation.

The results at 12 hours and 18 hours time point are shown in the diagrams for reference below.

![Chromo After 12 and 18 Hours](https://static.igem.wiki/teams/5152/engineering/fig-5.webp)

**Figure 5:** The cell pellets after 12 hours and 18 hours of incubation at 37°C. The pellets after 18 hours of incubation exhibit significantly higher colour intensity compared to those at 12 hours. pUC19 represents the cells transformed with the pUC19 empty vector as a control.

We recognize that using a constitutive promoter might not perfectly reflect chromoprotein expression when transitioning to metal-inducing promoters. However, this data provides a useful preliminary range for our study.

Additionally, we cultured the cells at room temperature without shaking, which resulted in a slower colour change. Noticeable colour appeared only after at least 24 hours of incubation.

## **4. Learn**

From our results, we drew several key conclusions:

1. **Influence of Culture Conditions:** The expression of chromoproteins is highly affected by culture conditions and may not always match the results reported in the literature. In our experiments,*cjBlue* consistently only showed expression after 24 hours of incubation, making it impractical to use as a reporter gene in our project. As a result, we decided to exclude *cjBlue*from our biosensor design.
2. **Optimizing Expression with Temperature and Shaking:** We observed that incubating the cultures at 37°C with shaking significantly enhanced chromoprotein expression. This discovery led us to collaborate with our hardware team, requesting them to integrate temperature control and shaking functions into our hardware device to optimize the performance of our biosensor.
3. **Time Required for Result Reading:** We found that a minimum of 12 hours is required for the colour intensity of the cells to provide a clear contrast for result reading. While longer incubation times (around 18 hours) produce more obvious results, we also discovered that in cells with metal-sensing constructs, extended incubation can lead to leaky expression of the chromoproteins, resulting in false positives. Therefore, it is essential to establish a specific time frame and a threshold for measurement in our hardware device to ensure accurate detection without prolonged incubation.

After confirming that the chromoproteins have stable expression and that our measurement method is reliable, we moved on to the next phase of our project: designing our biosensor devices. This step involves incorporating the validated chromoprotein reporters into constructs that respond to the presence of the target heavy metals, ensuring accurate and practical detection.

## **5. Design**

**Designing Heavy Metal-Inducing Expression Constructs**

With the knowledge gained, we proceeded to create our heavy metal biosensors.

To achieve this, we conducted a thorough literature review on metal-inducible promoters and evaluated their potential for use in our project. By studying various sources and exploring the BioBrick registry, we identified several promising candidates for our biosensor design, including:

1. *pPbr* operon ([BBa\_I721001](https://parts.igem.org/Part%3ABBa_I721001)) (Lead-inducible)
2. *pCadA* operon ([BBa\_K1724000](https://parts.igem.org/Part%3ABBa_K1724000)) ([BBa\_K174015](https://parts.igem.org/Part%3ABBa_K174015)) (Cadmium-inducible)
3. *pYodA* operon ([BBa\_K896008](https://parts.igem.org/Part%3ABBa_K896008)) (Cadmium-inducible)
4. *pMerT* operon ([BBa\_K346002](https://parts.igem.org/Part%3ABBa_K346002)) (Mercury-inducible)
5. *pZnt* operon ([BBa\_K346002](https://parts.igem.org/Part%3ABBa_K346002)) (Zinc-inducible)

**Characterizing and Designing Biosensor Constructs**

We then analyzed the characteristics of each operon to tailor our biosensor constructs to meet the goals of our project.

For *pYodA*, *pZnt*, and *pCadA* operons, the literature reported that they are native operons in *E. coli*, meaning there is no need to ectopically express their regulatory proteins.

However, for the *pPbr*, a synthetically modified *pCadA*, and *pMerT* operons, which are not naturally found in *E. coli*, we needed to co-express their regulatory proteins:

* PbrR repressor ([BBa\_I721002](https://parts.igem.org/Part%3ABBa_I721002)) for *pPbr* (lead-inducible)
* A modified cadmium-sensitive MerR repressor ([BBa\_K174015](https://parts.igem.org/Part%3ABBa_K174015)) for the *pCadA* operon
* Mercury-binding MerR ([BBa\_K346002](https://parts.igem.org/Part%3ABBa_K346002)) activator for *pMerT*

The illustrations of the regulatory mechanisms of the above operons are shown below:

1. *pPbr* operon (Positive regulation)
 ![](https://static.igem.wiki/teams/5152/engineering/2-webp-new.webp) 2. *MerR-pCadA* operon (Negative regulation)
 ![](https://static.igem.wiki/teams/5152/engineering/3-webp-new.webp) 3. *MerR-pMerT* operon (Positive regulation)
 ![](https://static.igem.wiki/teams/5152/engineering/4-webp-new.webp)

**Balancing Expression of Regulatory Proteins**

As mentioned, when expressing regulatory proteins, we carefully considered expression strength. Overexpressing a repressor can reduce the biosensor's sensitivity, while under-expressing it may cause leaky expression and false positives. Conversely, overexpressing an activator can lead to false positives, while under-expressing it results in low sensitivity.

After designing the constructs, we initially had 7 designs ready. We ordered these constructs in the form of gBlocks from IDT. However, we encountered an issue with one of the lead sensor constructs—due to the presence of many repeating sequences, IDT was unable to synthesize it.

The 6 final designs of our constructs are illustrated below:

1. *PbrR-pPbr* lead sensing with dTomato reporter construct
 ![](https://static.igem.wiki/teams/5152/engineering/5-webp-new.webp) 2. *MerR-pCadA* cadmium sensing with amilCP reporter construct
 ![](https://static.igem.wiki/teams/5152/engineering/6-webp-new.webp) 3. *pCadA* cadmium sensing with amilCP reporter construct
 ![](https://static.igem.wiki/teams/5152/engineering/7-webp-new.webp) 4. *pYodA* cadmium sensing with amilCP reporter construct
 ![](https://static.igem.wiki/teams/5152/engineering/8-webp-new.webp) 5. *MerR-pMerT* mercury sensing with tsPurple reporter construct
 ![](https://static.igem.wiki/teams/5152/engineering/9-webp-new.webp) 6. *pZnt* zinc sensing with eforRed reporter construct
 ![](https://static.igem.wiki/teams/5152/engineering/10-webp-new.webp)

At this stage, everything seemed to be running smoothly, but soon we faced several major challenges that required significant time and effort to resolve. In the following section, we will discuss the hurdles we encountered and the problem-solving process that helped us overcome them.

**Experiment plans**

Initially, we aimed to not only demonstrate that our biosensors can detect the presence of heavy metals, but also to conduct the following investigations:

1. **Correlation Between Chromoprotein colour Intensity and Heavy Metal Concentration:** We planned to quantify the relationship between the intensity of the chromoprotein colour and the concentration of the corresponding heavy metal, allowing us to establish a calibration curve for more precise measurements.
2. **Specificity of the Biosensors:** We intended to test the specificity of our biosensors to ensure they only respond to their target heavy metals and not to other metals, preventing false positive results.
3. **Interaction of Mixed Biosensors:** We wanted to investigate what happens when different biosensors are mixed together. Specifically, we aimed to see if they can function simultaneously and detect different metals in the same environment without interfering with each other.
4. **Demonstration in our hardware device:** Finally, we planned to demonstrate the functionality of our biosensors in the auto-reporting machine developed by our hardware team, to show how the sensors could be incorporated into a fully automated monitoring system.

However, due to time constraints, we were only able to complete part of these experiments. Most of the planned investigations will be carried out in our future studies.

## **6. Build**

**Cloning attempts of metal-inducing expression constructs**

Upon receiving the gBlocks from IDT, we immediately began the cloning process. Building on our experience from cloning the constitutive chromoprotein expression strains, we followed the same protocol we had successfully used before.

During our first cloning attempt, we encountered an issue with lawn growth on all plates. (Fig. 6) After investigation, we discovered the problem was due to the ampicillin in our plates. We found that our ampicillin might have expired, as it was stored at -20°C instead of -80°C and had been stocked for over a year.

![No Amp Plates](https://static.igem.wiki/teams/5152/engineering/7-no-amp-plates.webp)

**Figure 6:** The plates showing lawns formed after the first cloning attempt. Upon investigation, we discovered that the issue was caused by the absence of ampicillin in the plates, which allowed non-selective bacterial growth.

To address this, we re-spread the remaining transformants onto a new set of plates containing the appropriate ampicillin concentration. This ensured that only the transformed colonies with the correct plasmids would survive and grow.

![Re-spread Plates](https://static.igem.wiki/teams/5152/engineering/copy-of-img-4136-1.webp)

**Figure 7:** The plates re-spread with the remaining transformants. No colonies were formed, prompting us to repeat the assembly process and eventually verify the DNA sequence to identify potential issues with the cloning.

After incubation, all plates were empty with no colonies, except for the positive controls. We initially suspected that the transformants had been stored in the fridge, which may have reduced their viability, preventing colony formation.

In response, we repeated the assembly process and transformed the products once more. This time, a few colonies appeared on the plates. After consulting with our teacher, we learned these colonies were most likely false positives. However, at that time, we believed that these colonies were successful, so we proceeded with colony PCR to verify the clones.

Unexpectedly by us, all the colonies from the colony PCR showed no bands, indicating that there was no insert in the plasmids. Additionally, we made a significant mistake during the PCR process by not adding positive controls to the reaction, which could have helped us interpret the results more accurately.

![Failed Colony PCR](https://static.igem.wiki/teams/5152/engineering/8-failed-colony-pcr.webp)

**Figure 8:** One of the gel results from the colony PCR of the first cloning attempt, showing no bands formed. This indicates that none of the colonies contained the desired insert we attempted to assemble. The PCR was run using the M13 standard primer for pUC19 vectors. The faint bands at the bottom of the gel represent the primers.

Learned from this experience, we had consistently used a positive control setup during the assembly process. These positive control assemblies were always successful to show colonies on the plates, which led us to suspect that the issue might be with the PCR process itself. As a result, we conducted an additional round of PCR verification to troubleshoot the problem further.

**Validation of PCR**

From the PCR results, we confirmed that our PCR process was functioning correctly. After consulting with our teacher, we also understood the critical importance of including positive controls in our PCR setups. This would help us better interpret the results and avoid unnecessary confusion in future experiments.

![PCR Validation](https://static.igem.wiki/teams/5152/engineering/9-pcr-validation.webp)

**Figure 9:** Gel photo showing the result of validating that our PCR conditions and machine are functioning properly. The DNA template used was from our team's previous iGEM project, and the primers were the same M13 primers used in our current project, under the same PCR conditions. All the band sizes are within the expected range, confirming that the PCR setup is working correctly.

Next, we needed to investigate other potential causes for the cloning failure. Since the PCR process was confirmed to be working properly, we shifted our focus to other aspects of the workflow to identify any issues.

**Identified the mistakes**

Since both the cloning process and PCR were verified to be error-free, we returned to the initial step and double-checked the DNA sequence we ordered. After careful review, we discovered that the 5' overlapping sequence was incorrectly designed to overlap with the EcoRI digestion site instead of PstI, causing the assembly process to fail.

As a result, we had to immediately re-design the constructs with the correct overlapping sequences and reorder the DNA. This step was crucial to ensuring the success of the cloning and moving forward with our project.

**The successful cloning attempt of metal-inducing expression constructs**

After receiving the newly synthesized DNAs from IDT and TwistBioscience (many thanks to them for their kind sponsorship!), we immediately proceeded with the assembly process again. Using the same workflow that we had successfully employed for the constitutive constructs, this time we managed to obtain colonies from five out of the six assembled constructs. These included:

1. Lead-inducing *pPbr* with the co-expression of PbrR activator construct ([BBa\_K5152004](https://parts.igem.org/Part%3ABBa_K5152004))
2. Cadmium-inducing *pCadA* with the co-expression of modified MerR repressor construct ([BBa\_K5152006](https://parts.igem.org/Part%3ABBa_K5152006))
3. Cadmium-inducing *pYodA* construct ([BBa\_K5152007](https://parts.igem.org/Part%3ABBa_K5152007))
4. Zinc-inducing *pZnt* construct ([BBa\_K5152009](https://parts.igem.org/Part%3ABBa_K5152009))
5. Mercury-inducing *pMerT* with co-expression of MerR activator construct ([BBa\_K5152008](https://parts.igem.org/Part%3ABBa_K5152008))

Following this, we proceeded with colony PCR to verify the presence of the correct inserts in the clones.

![Colony Tracking](https://static.igem.wiki/teams/5152/engineering/10-colony-tracking.webp)

**Figure 10:** We tracked the clones by picking and inoculating them on new agar plates. As shown in the picture, some of the clones exhibited leaky expression (visible colour without induction). To avoid potential issues with false positives, we decided not to select clones showing colour for further study.

**Clones verified by colony PCR**

From the gel photos below, some of the colony PCR results showed bands corresponding to the expected sizes of the inserts. Therefore, we selected these colonies for our subsequent functional studies, ensuring that only the correctly assembled constructs would be used for further experiments.

![PbrR Colony PCR](https://static.igem.wiki/teams/5152/engineering/11a-ppbr-colony-pcr.webp)
![MerR-pCadA Colony PCR](https://static.igem.wiki/teams/5152/engineering/11b-merr-pcada-colony-pcr.webp)
![pZnt-pYodA Colony PCR](https://static.igem.wiki/teams/5152/engineering/11c-pznt-pyoda-colony-pcr.webp)

**Figure 11:**

**(a):** PbrR construct colony PCR result. The expected band size of ~1.5 kbp was observed in lanes 3, 4, and 5.

**(b):** MerR-pCadA construct colony PCR result. The expected band size of ~1.6 kbp was observed in lanes 4, 5, and 6.

**(c):** pZnt construct and pYodA colony PCR result. The expected band size of ~600 bp was observed in the corresponding lanes.

Despite some successful colony PCR results, we encountered several unexpected results that raised concerns. Specifically, some colonies showed no insert, which was unexpected given the steps we took to minimize self-ligation:

Gel purification was done after double enzyme digestion of the pUC19 vector, significantly reducing the chance of self-ligation. Thus, there should not have been any clones carrying antibiotic resistance unless they contained the assembled plasmids.

However, several colonies lacked inserts, which led us to consider potential explanations:

1. Plasmid contamination during the transformation process, despite our efforts to prevent contamination.
2. Inappropriate antibiotic concentration in the selection plates. To investigate this possibility, we tested our ampicillin plates by spreading them with *E. coli* carrying the pUC19 vector and *E. coli* without resistance.

Nevertheless, we proceeded by selecting the colonies that showed correct band sizes in the colony PCR for our functional assays.

**Functional studies of the *E. coli* heavy metal biosensors**

One key observation from our transformed cells was that some exhibited leaky expression of the chromoprotein even without metal induction, especially when incubated for too long (typically ≥ 18 hours). To address this, we carefully selected colonies that did not show leaky expression for the functional assays and kept the measurement time within 18 hours in all subsequent experiments.

After overcoming several hurdles, we finally obtained the correct clones. However, due to previous errors in DNA design, equipment limitations, and various challenges during the experiment, the process was delayed. For example:

* We lacked a refrigerated centrifuge, making it impossible to prepare our own competent cells. Thankfully, the Amgen Biotechnology Experience Program Hong Kong generously sponsored us with competent cells when we ran out.
* It was already September by the time we had the correct clones, leaving us with limited time before the wiki freeze.
* Our small shaking incubator could only hold 12 small conical flasks, which required careful planning to optimize time and space usage as the deadline approached.

Given the time constraints, we decided to focus on proving that all our constructs could respond to the corresponding heavy metals. To maximize our chances of success, we conducted research on the concentration of heavy metals used in literature reports and chose the highest reported concentration for each metal that had been shown to induce a response. This approach ensured that the metal concentration would be sufficient to activate the chromoprotein expression and demonstrate proof of concept.

The concentrations of heavy metals used in our experiments are listed in the table below:

| **Heavy metal salt** | **Final concentration added to the biosensor culture (uM)** |
| --- | --- |
| Lead (II) Nitrate | 100 |
| Cadmium (II) Chloride | 200 |
| Zn (II) Chloride | 200 |
| Mercuric Chloride | 100 |

We also planned to explore whether the cells could be cultured in 15 mL Falcon tubes, as this would significantly reduce the amount of space required, allowing us to conduct more experiments simultaneously. However, during our trials, we found that this culturing method was not feasible. The specific reasons for this will be discussed in the section below.

After these preliminary tests, we proceeded to use the selected heavy metal concentrations for our experimental setups.

## **7. Test**

**Characterization of the biosensors**

For each biosensor, our initial experimental setup involved adding the corresponding heavy metal (at the final concentrations listed in the table above) and comparing the results with a control setup where no heavy metal was added.

In the later stages of our investigation, following a suggestion from our teacher, we added an additional control setup: *E. coli* containing only the pUC19 plasmid, with the corresponding heavy metal added. This control was crucial to demonstrate that the heavy metal alone would not cause any colour change in *E. coli* without the biosensor constructs.

This additional control was particularly important because we encountered an unexpected issue during our experiments. When we grew the cells in 15 mL Falcon tubes with the heavy metal, the cells exhibited a strange blackened and brown colouration. Concerned that this could cloud our judgment on the experimental results, we conducted a control experiment using *E. coli* with only the pUC19 plasmid in conical flasks. In these experiments, the unusual colouration was not observed, suggesting that the problem was specific to the Falcon tube culturing method (as shown in the figure below).

Given this observation, we decided to discontinue the use of Falcon tubes for culturing, as the method could lead to misleading results. However, this decision significantly slowed our experimental progress due to the limited space available in the shaking incubator when using conical flasks.

![Black Pellet](https://static.igem.wiki/teams/5152/engineering/12-ab-black-pellet.webp)

**Figure 12:**

**(a):** The biosensor culture grown in a 15 mL Falcon tube with lead (Pb) added. Initially, based on the appearance of the culture, we thought there was a colour change due to the biosensor ([BBa\_K5152004](https://parts.igem.org/Part%3ABBa_K5152004)) successfully detecting the metal. However, this was not the case when we spun down the culture to observe the pellet.

**(b):** A strange colouration appeared in the cell pellet when incubated in 15 mL Falcon tubes with heavy metal. This unusual colour change could potentially cloud our judgment of the experimental results. Therefore, we decided to discontinue using Falcon tubes for the experimental setup. Notably, this colour change was not observed in *E. coli* carrying pUC19 when incubated in conical flasks.

We suspect that the unusual colouration observed in the cells grown in 15 mL Falcon tubes with heavy metals is due to poor aeration during incubation. This lack of sufficient oxygen may have created an anaerobic environment, leading to the strange appearance of the cells. However, this is just a hypothesis, and further investigation would be needed to confirm this phenomenon and understand the underlying cause.

After conducting the functional studies, we obtained the following results:

## I. Lead Biosensor ([BBa\_K5152004](https://parts.igem.org/Part%3ABBa_K5152004))

In the lead biosensor, the results were promising. To ensure the reliability of the findings, we repeated the experiment three times.

Upon adding 100 µM of lead and incubating for 12 hours, the experimental setup showed a clear red colouration in the pellet, indicative of dTomato expression, compared to the white pellet in the control. Additionally, a mild red colour was visible in the culture itself, further supporting the successful detection of lead by the biosensor (as shown in the figure below).

![PbrR-pPbr Functional Test](https://static.igem.wiki/teams/5152/engineering/13-ab-ppbr-functional-100-um.webp)

**Figure 13:**

**(a):** By naked-eye observation, the cultures of the *PbrR-pPbr* lead biosensor show a distinguishable red colouration compared to the negative control setup, where no lead was added.

**(b):** The red colouration is even more pronounced in the pellet, highlighting the expression of the reporter gene (*dTomato*) ([BBa\_K4813005](https://parts.igem.org/Part%3ABBa_K4813005)). This confirms that our biosensor is successfully responding to the presence of lead (Pb).

Although the colour intensity of culture medium after 12 hours of incubation was not significantly different from the control, we predicted that extending the incubation time would result in a more pronounced colour change in the culture medium. This prediction was confirmed (as shown in the figure below).

After 18 hours of incubation, the red colouration in the experimental setup became much more obvious. However, a mild red colour was also observed in the no heavy metal control setup, indicating leaky expression. This leaky expression is a potential issue that we aim to investigate in further experiments.

![PbrR-pPbr after 18 hours](https://static.igem.wiki/teams/5152/engineering/14-ppbr-100-um-after-18-hours.webp)

**Figure 14:** The PbrR-pPbr lead biosensor shows a significant red colouration after 18 hours of incubation with 100 µM of lead. However, when compared to the pUC19-only control setup, the PbrR-pPbr biosensor without added lead also displayed a mild red colouration, indicating that extended incubation leads to a mild leaky expression of the red chromoprotein.

Next, we investigated whether the expression of the biosensor is concentration-dependent. We added final concentrations of 0.01, 0.1, 1, 10, 500, and 1000 µM of lead nitrate solution to the biosensor cultures.

Without needing to harvest the cells, we observed that the biosensor's response was indeed concentration-dependent within the range of 0.1 to 10 µM. A higher concentration of lead resulted in a more intense colour signal from the reporter. This demonstrates that our biosensor's chromoprotein expression correlates with the lead concentration (as shown in the figure below).

![Pb Concentration](https://static.igem.wiki/teams/5152/engineering/15-pb-concentration.webp)

**Figure 15:** The red colour intensity of the lead biosensor increases with an observable difference as the concentration of lead rises from 0.1 µM to 10 µM. However, when the concentration exceeds 500 µM, there appears to be a negative effect on the colour intensity, potentially due to toxicity or inhibition of cellular functions at high lead concentrations.

![Pb Concentration Pellet](https://static.igem.wiki/teams/5152/engineering/16-pb-concentration-pellet.webp)

**Figure 16:** When the cultures with different concentrations of lead were spun down into pellets, the colour intensities were all too high, making the differences indistinguishable to the naked eye for concentrations greater than 0.1 µM of lead. This suggests that the reporter signal becomes saturated at higher lead concentrations, making it difficult to visually differentiate between higher levels of induction.

Meanwhile, in the 500 µM and 1000 µM setups (as shown in Fig. 15), the colour intensity appeared to decrease. We hypothesize that this is due to the toxicity of these high lead concentrations, which likely exceeded the cells' resistance to lead poisoning. As a result, the cells may have experienced poor growth, leading to reduced expression of the reporter protein.

## II. Cadmium Biosensor

We designed three cadmium biosensors, with the first utilizing a MerR repressor protein, which was originally a mercury-binding activator but was modified by the 2015 iGEM Team SCUT to specifically bind cadmium and repress the expression of *pCadA*. This modified MerR repressor is coupled with a *pCadA* promoter ([BBa\_K5152006](https://parts.igem.org/Part%3ABBa_K5152006)), which contains the modified MerR binding site, to drive the expression of the amilCP reporter protein.

When cadmium binds to the modified MerR, it releases its repression on the *pCadA* promoter, allowing the expression of the downstream reporter protein, resulting in a visible blue colouration.

The functional study successfully demonstrated that this cadmium biosensor works as expected. We repeated the experiment three times, cells treated with 200 µM cadmium chloride after 12 hours of incubation already showed a blue colouration, while the control cells remained white (as shown in the figure below).

However, it's important to note that in one of the repeats, even though the cells were harvested in under 18 hours, both the control and experimental setups showed blue colouration, although the experimental setup exhibited a higher intensity of blue. This indicates that the biosensor exhibits leaky expression, a property that we will need to address in future studies.

![MerR-pCadA Functional Test](https://static.igem.wiki/teams/5152/engineering/17ab-merr-pcada-functional.webp)

**Figure 17:**

**(a):** A mild blue colouration from the amilCP protein is observed in the cadmium biosensor culture when 200 µM cadmium is added.

**(b):** In the pellet form, an obvious blue colouration from the amilCP protein is observed in the pellet of the biosensor culture with 200 µM cadmium added, confirming the activation of the cadmium biosensor.

In contrast, the other design utilizing the *pYodA* promoter ([BBa\_K5152007](https://parts.igem.org/Part%3ABBa_K5152007)), which is reported to be cadmium-specific in *E. coli*, demonstrated a severe leaky expression issue. In both of our experimental repeats, while the cadmium-added setups consistently showed a deeper blue colouration, the negative control setups (without cadmium) also exhibited blue colouration (as shown in the figure below).

This significant leaky expression makes *pYodA* unsuitable as a promoter target for an effective biosensor, as it compromises the sensor's specificity and reliability.

![pYodA Functional Leaky Expression](https://static.igem.wiki/teams/5152/engineering/18-pyoda-functional-leaky-expression.webp)

**Figure 18:** Leaky expression of the amilCP blue protein was observed in the *pYodA*-containing cells, even when cadmium was not added. This indicates that the *pYodA* promoter exhibits significant leaky expression, making it less suitable for use in a cadmium biosensor.

The final design involved a modified pCadA promoter with ArsR and CrzA binding sites added upstream ([BBa\_K5152005](https://parts.igem.org/Part%3ABBa_K5152005)), a design originally developed by the 2009 iGEM Team Newcastle. Unfortunately, we were unable to successfully assemble this construct. Despite attempting the cloning process three times, no colonies formed after transformation.

At this point, we still cannot determine the exact reason for the failure, and further investigation will be needed to identify and resolve the issue.

## III. Zinc Biosensor ([BBa\_K5152009](https://parts.igem.org/Part%3ABBa_K5152009))

The design of the zinc biosensor is relatively simple, as it utilizes the *pZnt* promoter, which is part of the native Zinc regulatory operon in *E. coli*. This system works with the native ZntR repressor protein found in *E. coli*; when zinc is present, it releases the inhibition of ZntR on the *pZnt* promoter, allowing the expression of the downstream reporter gene, *eforRed*.

However, similar to the issues observed with the *pYodA* cadmium biosensor, we consistently observed leaky expression in all experimental repeats. The biosensor without zinc added showed a mild red colouration from the eforRed protein, indicating that there is leaky expression even in the absence of zinc. Although the cells with zinc consistently showed a deeper red colour than the controls, the leaky expression makes this design less than ideal as a reliable biosensor, as it could lead to false positives or false alarms (as shown in the figure below).

Further investigation is needed to address this issue and reduce the leaky expression, possibly by optimizing the promoter or regulatory elements in the system.

![pZnt Functional Leaky Expression](https://static.igem.wiki/teams/5152/engineering/19-pznt-functional-leaky-expression.webp)

**Figure 19:** Leaky expression of the eforRed red protein was observed in the pZnt-containing cells, even when zinc was not added. This indicates that the pZnt promoter is prone to leaky expression, making it less reliable as a biosensor for zinc detection due to the potential for false positives.

## IV. Mercury Biosensor ([BBa\_K5152008](https://parts.igem.org/Part%3ABBa_K5152008))

For this design, we made a major mistake, and we realized it when we observed that one of the colonies formed after transformation was bright pink in colour. Initially, we thought the pink colour was due to contamination from other plates, but after consistently seeing more red colonies forming, we decided to go back and check the DNA.

Upon review, we discovered that we had accidentally entered the sequence of the lead-sensing construct when ordering the DNA for the mercury biosensor construct from IDT (as shown in the pictures below). Unfortunately, by the time we identified this mistake, there was no time left to re-order the correct DNA sequence.

As a result, there are no further results for this biosensor.

![Mistake in DNA Order](https://static.igem.wiki/teams/5152/engineering/fig-20.webp)

**Figure 20:** Our careless mistake where we accidentally copied the same sequence of the PbrR construct (lead biosensor) when placing the order for the pMerT construct (mercury biosensor). This error resulted in the wrong DNA being synthesized, and as a result, we were unable to proceed with the experiments for the mercury biosensor.

However, before finding out the mistake, we performed the functional study on the incorrect colonies. Interestingly, we found that in multiple repeats, the cultures to which we added 100 µM of mercuric chloride showed no cell growth (as shown in the figure below). This is highly likely due to the extreme toxicity of mercury, which killed all the cells at this concentration.

This finding suggests that 100 µM of mercury is too high a concentration for such studies, and we must be very careful in future investigations involving mercury. Lower concentrations should be used to prevent complete cell death and to allow us to accurately assess the biosensor's response.

![](https://static.igem.wiki/teams/5152/engineering/21-hg-no-growth.webp)

**Figure 21:**  No growth of E. coli was observed in the culture after 16 hours of incubation. The supposed "MerR-pMerT" construct we were testing was actually the PbrR-pPbr lead-sensing construct, as we mistakenly ordered the wrong DNA sequence. The lack of growth is likely due to the high toxicity of 100 µM mercuric chloride, which killed all the cells.

## **8. Learn**

Through the experimental engineering cycles, we gained valuable insights into our project. We learned that employing an effective and systematic problem-solving approach was key to successfully cloning our constructs and conducting investigations on our designs. Each iteration of the cycle allowed us to refine our methods, troubleshoot issues, and adapt our experiments based on the challenges we faced, ultimately leading to a deeper understanding of both the engineering process and the functionality of our biosensors.

**Conclusions of our results**

1. **Validation of Chromoprotein Expression:** We successfully validated the expression of the chromoproteins and integrated them into new composite designs, showcasing their characteristics. However, we observed that the expression of cjBlue in *E. coli* seemed to cause difficulties in expression. Further investigation is needed to determine the underlying reasons for this issue.
2. **Lead Biosensor Success:** Our lead biosensor effectively detected the presence of lead and initiated the expression of chromoprotein as a reporter gene. The expression of the chromoprotein produced a visible signal that could be observed with the naked eye, without requiring any specialized equipment or techniques.
3. **Concentration-Dependent Lead Detection:** The lead biosensor demonstrated concentration-dependent behavior, where the intensity of the colour correlated with the concentration of lead within a certain range. This feature enhances the versatility and practicality of the biosensor, making it more useful for various applications.
4. **Cadmium Biosensor Success:** Our cadmium biosensor design (*MerR-pCadA*) was also able to detect the presence of cadmium, showing functional expression and detection capabilities.
5. **Leaky Expression in Other Biosensors:** Our other biosensor constructs, including *pYodA* and *pZnt*, showed some potential in detecting their respective heavy metals. However, due to leaky expression, we cannot yet draw definitive conclusions about their reliability. Further repeats and investigations are required to confirm their validity and reliability.
6. **Lessons from DNA Ordering:** A critical lesson we learned is the importance of carefully checking DNA sequences before placing an order. Our experience with ordering the wrong sequence for the mercury biosensor highlighted the need for more rigorous checks to avoid such errors in the future.

With these results and conclusions, we have successfully demonstrated that our concept is viable and proven that our biosensor designs can work as intended. However, we are also fully aware that there is still significant room for improvement and that further investigations are necessary to refine the system.

**Limitations and room for improvements of the studies**

There are several major considerations that we need to address in our project:

1. **Increasing the sensitivity to low concentrations:** Sensitivity is crucial for a biosensor. The acceptable lead level in drinking water is around 0.03 µM (5 ppb by FDA and/or 10 µg/L by Hong Kong Water Service Department). In our results, the 0.01 µM Pb concentration showed no significant difference from the control. Therefore, we need to investigate ways to enhance sensitivity to detect lead at these lower concentrations.
2. **Find out the sensitivity to different concentrations:** We aim to decrease the interval between tested concentrations to determine how precisely the biosensor responds to small changes in metal concentration. This will help us assess the sensitivity and accuracy of the biosensor across a broader range of concentrations.
3. **Integrate the biosensor into hardware and real-world applications:** To use our biosensor in the automated warning and growing device developed by the hardware team, we need time to test its compatibility and use bacterial results to train the AI model for colour recognition. This integration will require a significant amount of time for testing and optimization.
4. **Minimizing leaky expression in biosensors:** A common issue is that reporter genes may show leaky expression due to culturing conditions, like prolonged incubation. In some designs, such as *pZnt* and *pYodA*, this occurs consistently in negative controls. Enhancing the expression of regulatory proteins can help tighten control, but it might reduce biosensor sensitivity. Careful further investigation is needed to address this problem.
5. **Testing on solid medium / cell-free expression systems:** We aim to test if the biosensor functions effectively on solid media or in a cell-free system. Success in these systems would significantly increase the applicability of the biosensor, offering more flexible and safer usage options.
6. **Validate the other biosensor designs and assess combined use:** We plan to continue investigating the biosensor designs we couldn’t complete, as our goal is to develop sensors for the four most common heavy metal pollutants. Additionally, we are interested in testing what happens when different biosensors are mixed in the same culture to evaluate their combined response to heavy metals.
7. **Ensuring safety measures:** Safety is critical to our project. We need to implement measures like a kill switch or containment mechanisms when working with engineered organisms. Additionally, exploring cell-free expression systems could help mitigate concerns regarding the use of genetically modified organisms.

Therefore, we're beginning a new round of the engineering cycle to address these important points. By combining our biological system with the device, we aim to improve our product's functionality, applicability, and safety. It's crucial for us to thoroughly investigate every aspect to ensure that our system meets the necessary standards and regulations for real-world use.

**Major references**

1. Borremans B, Hobman JL, Provoost A, Brown NL, van Der Lelie D. (2001) Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. *J Bacteriol*. 2001 Oct;183(19):5651-8. doi: 10.1128/JB.183.19.5651-5658.2001. PMID: 11544228; PMCID: PMC95457.

2. Wei W, Liu X, Sun P, Wang X, Zhu H, Hong M, Mao ZW, Zhao J. (2014) *Simple whole-cell biodetection and bioremediation of heavy metals based on an engineered lead-specific operon. Environ Sci Technol.* 2014 Mar 18;48(6):3363-71. doi: 10.1021/es4046567. Epub 2014 Mar 3. PMID: 24564581.

3. Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C. (2005) *An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl.* 2005 Apr 29;44(18):2715-2719. doi: 10.1002/anie.200462443. PMID: 15800869.

4. Puškarova A, Ferianc P, Kormanec J, Homerova D, Farewell A, Nyström T. (2002) *Regulation of yodA encoding a novel cadmium-induced protein in Escherichia coli. Microbiology (Reading).* 2002 Dec;148(Pt 12):3801-3811. doi: 10.1099/00221287-148-12-3801. PMID: 12480884.

5. Brown, N. L., Stoyanov, J. V., Kidd, S. P., & Hobman, J. L. (2003). The MerR family of transcriptional regulators. *FEMS microbiology reviews*, 27(2-3), 145–163. https://doi.org/10.1016/S0168-6445(03)00051-2

{% endblock %}

# Experiments

![](https://static.igem.wiki/teams/5152/banner/experiments.webp)

## Side Menu

[Preparation of pUC19 Vector](#section1)
[Constructs Cloning](#section2)
[*E. coli* Culturing](#section3)
[Functional Assay of Heavy Metal](#section4)

## Experiment Protocols

### pUC19 Vector Preparation

#### Transformation of pUC19

##### Materials:

* pUC19 positive control DNA (Invitrogen)
* One Shot™ TOP10 Chemically Competent *E. coli* (ThermoFisher, Cat# C404003) / DH5α Competent Cells obtained from HKABE (CUHK)
* S.O.C Medium (ThermoFisher, Cat# 15544-034)
* LB agar (ThermoFisher, Cat# 22700-025) plates with ampicillin (Gibco™, Cat# 11593027) added

##### Procedures:

1. Mix 10 μL of DNA with 1 mL of competent cells in a polypropylene tube stored at -80°C.
2. Incubate the tube on ice for at least 10 minutes.
3. Heat shock the tube by exposing it to 42°C for 45 seconds.
4. Return on ice for 2 minutes.
5. Add 1 mL of pre-warmed SOC medium to the tube.
6. Incubate the tube at 37°C for 10 minutes for recovery.
7. Take 100-200 μL of the solution and spread it evenly on an ampicillin LB plate.
8. Incubate the plate at 37°C overnight.

#### Purification of pUC19 Plasmids

##### Materials:

* PureLink™ Quick Plasmid Miniprep Kit (ThermoFisher, Cat# K210010)

##### Procedures:

* Refer to manufacturer’s manual.
* 50 μL of elution buffer were used in all preparations.

#### Restriction Digestion of pUC19

##### Materials:

* EcoRI-HF (NEB, Cat# R3101S)
* PstI (NEB, Cat# R140S)
* NEBuffer™ r2.1 (NEB, Cat# R140S)

##### Procedures:

| Component | 50 µl Reaction (µL) |
| --- | --- |
| DNA | 20 |
| 10X NEBuffer r2.1 | 5 |
| EcoRI-HF | 2 |
| PstI | 2 |
| Nuclease-free Water | 21 |

1. Incubate at 37 °C for 1 hour.
2. Heat inactivation at 65 °C for 20 minutes.

#### Gel Purification of Digested pUC19

##### Materials:

* PureLink™ Quick Gel Extraction Kit (ThermoFisher, Cat# K210012)

##### Procedures:

* Refer to the manufacturer’s manual.
* 50 μL of elution buffer were used in all preparations.

### Constructs Cloning

#### HiFi Assembly

##### Materials:

* NEBuilder® HiFi DNA Assembly Master Mix (NEB, Cat# E2621S)

##### Procedures:

###### For constitutive expression constructs:

| Components (µL) | amilCP | cjBlue | eforRed | tsPurple | dTomato | Positive |
| --- | --- | --- | --- | --- | --- | --- |
| Vector | 2 | 2 | 2 | 2 | 2 | 10 |
| Insert | 5.7 | 5.8 | 5.6 | 5.7 | 5.8 |  |
| HiFi MM | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 2.3 | 2.2 | 2.4 | 2.3 | 2.2 |  |
| Total | 20 | 20 | 20 | 20 | 20 | 20 |

###### For biosensor constructs:

| Components (µL) | pPbr | MerR-pCadA | pCadA | pYodA | pMerT | pZnt | dTomato (+ve) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Vector | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Insert | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
| HiFi MM | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Total | 20 | 20 | 20 | 20 | 20 | 20 | 20 |

1. Incubate at 50°C for 1 hour.

#### Colony PCR

##### Materials:

* M13 forward and reverse primer (100uM, Tech Dragon)
* OneTaq® 2X Master Mix with Standard Buffer (NEB, Cat# M0482S)

##### Primers:

| Primers | Sequence |
| --- | --- |
| M13 forward | TGTAAAACGACGGCCAGT |
| M13 reverse | CAGGAAACAGCTATGACCATG |

##### Procedures:

1. Prepare mastermix of PCR reaction according to the table below:

| Components | X1 (µL) |
| --- | --- |
| Forward M13 | 0.5 |
| Reverse M13 | 0.5 |
| Template | Colonies |
| 2X OneTaq | 12.5 |
| H2O | 11.5 |
| Total | 25 |

2. Run the PCR with the condition below for 30 cycles.

| Stage | Time (s) |
| --- | --- |
| Initial Denaturation (94 °C) | 30 |
| Denaturation (94 °C) | 20 |
| Annealing (55 °C) | 40 |
| Extension (68 °C) | 60 |
| Final Extension (68 °C) | 300 |

#### Gel Electrophoresis

##### Materials:

* The MiniOne Electrophoresis System (MiniOne Systems, Cat #M1000)
* Gel Loading Dye Purple (6X) (NEB, Cat #B7024S)
* UltraPure™ Agarose (Invitrogen, Cat #16500500)
* AE Buffer (Tris-acetate-EDTA) (50X) (ThermoFisher, Cat# B49)
* Quick-Load® Purple 1kb Plus DNA ladder (NEB, #N0550S)
* GelGreen™ DNA Stain 50 µl (MiniOne Systems, Cat# M3113)

##### Procedures:

1. Add 5 μl of loading dye into each tube of DNA.
2. Add 25 μl of distilled water into a new tube and 5 μl of LD into the tube (negative control).
3. Set up the gel tank:
4. Prepare 1X TAE buffer for gel forming and gel tank buffer.
5. Prepare 1% agarose gel with 1X TAE buffer in a conical flask.
6. Microwave the gel for 1 min to make the agarose dissolve completely.
7. Cool down the conical flask with running water.
8. Add Gel Stain into agarose gel for DNA visualization (i.e. 2 μL in 20mL of gel).
9. After the gel forms, transfer it into the gel tank and add buffer to just cover the whole gel.
10. Add the DNA and ladder into the wells.

### *E. coli* Culturing

#### *E. coli* Growth Media

##### Materials:

* LB Broth Base (Lennox) (Invitrogen, Cat# 12780052)
* LB Agar (Lennox L Agar), powder (Invitrogen, Cat# 22700025)
* Ampicillin, sodium salt, irradiated (Gibco™, Cat# 11593027)

##### Procedures:

1. Prepare the media following the manufacturer's instructions using distilled water.
2. Sterilize the media using a pressure cooker (since we don't have an autoclave).
3. After cooling down, add ampicillin.
4. Unless otherwise noted, incubate the cultures at 37°C, 180 rpm.

### Functional Assay of Heavy Metals

#### Heavy Metal Functional Assays

##### Materials:

* Lead (II) Nitrate solution (2 mM)
* Cadmium (II) Chloride solution (4 mM)
* Mercury (II) chloride solution (2 mM)
* Zinc (II) chloride solution (4 mM)

##### Procedures:

1. Add 1 mL of the metal solution to 19 mL of LB/Ampicillin broth with the cultures.
2. Incubate for at least 12 hours at 37°C, 180 rpm.
3. Centrifuge 1 mL of the culture for 2 minutes at 8,000 g.
4. Observe the colour change in the pellet.

#### Lead Concentration-Dependent Response Assay

##### Materials:

* Lead (II) Nitrate solution (2 mM)

##### Procedures:

1. Dilute the stock metal solution to the desired concentration.
2. Add 1 mL of the metal solution to 19 mL of LB/Ampicillin broth with the cultures.
3. Incubate for at least 12 hours at 37°C, 180 rpm.
4. Centrifuge 1 mL of the culture for 2 minutes at 8,000 g.
5. Observe the colour change in the pellet.

{% 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 %}

# Results

![](https://static.igem.wiki/teams/5152/banner/results.webp)

## Side Menu

[Preparation of pUC19 Vector](#section1)
[Cloning of Chromoproteins](#section2)
[Study on Chromoprotein Expressions](#section3)
[Cloning of Biosensors](#section4)
[Functional Assays on Heavy Metal](#section5)
[Summary of Findings](#section6)

## **Preparation of pUC19 Vectors**

We began the transformation process by introducing pUC19 plasmids into TOP10 *E. coli* cells using the heat shock method, followed by selection on ampicillin LB plates. Afterward, plasmid DNA was extracted using a mini-prep procedure, and restriction digestion with EcoRI and PstI was performed to linearize the vector for subsequent HiFi assembly. The linearized plasmids were then purified through gel extraction.

The digestion of the pUC19 vector was performed according to the table below:

| **COMPONENT** | **50 µl REACTION** (µL) |
| --- | --- |
| DNA | 20 |
| 10X NEBuffer r2.1 | 5 |
| EcoRI-HF | 2 |
| PstI | 2 |
| Nuclease-free Water | 21 |

Figure 1 shows the gel electrophoresis results matched our expectations. The lanes containing purified, linearized pUC19 plasmid (~2.7 kb) displayed three distinct bands. We selected the plasmids from lane 4 as our candidate for assembly, as we did not have access to a spectrophotometer to measure DNA concentration. We assumed the concentration in lane 3 was at an average level, likely neither too low nor too high, which would avoid negatively impacting the assembly efficiency.

![Gel Electrophoresis of pUC19](https://static.igem.wiki/teams/5152/results/1-gel-purification-of-puc19.webp)

***Figure 1:** Photograph of result of gel electrophoresis showing the digested and gel purified pUC19 vectors. The first lane is showing the 1kbp+ ladder from NEB.*

## **Cloning of Constructs for Chromoprotein Proteins**

### **Insert preparation**

We planned to use the HiFi assembly method to insert our gene constructs into pUC19 vectors, with generous support from NEB through their sponsorship of HiFi assembly kits.

Our gene constructs were synthesized as gBlocks by IDT, and the ordered gBlocks are detailed in the table below:

| **Name** | **Description** | **Part Registry Entry** |
| --- | --- | --- |
| Constitutive tsPurple Chromoprotein Expression | Strong constitutive expression of tsPurple chromoprotein in *E.coli* | [BBa\_K5152000](https://parts.igem.org/Part%3ABBa_K5152000) |
| Constitutive cjBlue Chromoprotein Expression | Strong constitutive expression of cjBlue chromoprotein in *E. coli* | [BBa\_K5152001](https://parts.igem.org/Part%3ABBa_K5152001) |
| Constitutive amilCP Chromoprotein Expression | Strong constitutive expression of amilCP chromoprotein in *E. coli* | [BBa\_K5152002](https://parts.igem.org/Part%3ABBa_K5152002) |
| Constitutive eforRed Chromoprotein Expression | Strong constitutive expression of eforRed chromoprotein in *E. coli* | [BBa\_K5152003](https://parts.igem.org/Part%3ABBa_K5152003) |
| Constitutive dTomato Chromoprotein Expression | This part was designed by our previous team and has been shown to strongly express dTomato, a chromoprotein that produces a highly visible red color as a reporter signal. | BBa\_K4813005 |

Next, we diluted the purified fragments to a concentration of 100 ng/µl, following the manufacturer's instructions. We then proceeded with HiFi assembly, utilizing the linearized pUC19 vectors for the process.

### **HiFi assembly of chromoprotein expression constructs into pUC19**

To ensure the success of the HiFi assembly, it is crucial to calculate the molar ratio of the inserts to the vector. According to the manufacturer's protocol, a 2:1 molar ratio (inserts to vector) is recommended for assemblies involving 2-3 fragments, while a 1:1 molar ratio is suggested for 4-6 fragments. However, as previously mentioned, we lack the equipment to accurately measure DNA concentration. Instead, following the advice of our external advisor, we will estimate the concentration by comparing the relative brightness of the DNA bands with that of the ladder bands. This estimation may introduce some uncertainty in the molar ratio calculations.

We set up the assembly following the table below:

| **Components** (µL) | **amilCP** | **cjBlue** | **eforRed** | **tsPurple** | **dTomato** | **Positive** |
| --- | --- | --- | --- | --- | --- | --- |
| Vector | 2 | 2 | 2 | 2 | 2 | 10 |
| Insert | 5.7 | 5.8 | 5.6 | 5.7 | 5.8 |  |
| Hifi MM | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 2.3 | 2.2 | 2.4 | 2.3 | 2.2 |  |
| **Total** | 20 | 20 | 20 | 20 | 20 | 20 |

### **Transformation of Recombinant Plasmids**

We introduced the assembled plasmids into *E. coli* using heat shock transformation. The transformed cells were plated on LB/ampicillin agar to assess the functionality of the plasmids in expressing their respective chromoproteins.

![E. coli colonies with chromoprotein constructs](https://static.igem.wiki/teams/5152/results/2-constitutive-chromoprotein.webp)

***Figure 2:** *E. coli* colonies transformed with constitutive chromoprotein expression constructs (cjBlue, tsPurple, amilCP, eforRed, and dTomato) on LB/ampicillin plates.*

![Cell pellets of E. coli expressing chromoproteins](https://static.igem.wiki/teams/5152/results/3-pellet-of-chromoproteins.webp)

***Figure 3:** Cell pellets of *E. coli* expressing various chromoproteins obtained by centrifugation. Each pellet shows distinct coloration corresponding to the expressed chromoprotein.*

In **Figure 3**, the chromoproteins are ordered from left to right as follows: eforRed, dTomato, amilCP, and tsPurple. cjBlue was not included, as its color development required at least 24 hours, unlike the others, which showed color after 16 hours. **Figure 4** shows that cjBlue may not be expressed efficiently, suggesting it may not suit our application.

We suspect the delayed expression of cjBlue may be due to suboptimal conditions or cell health. Since optimizing expression is not our focus, we decided to proceed with the other four chromoproteins.

![E. coli cells with cjBlue expression](https://static.igem.wiki/teams/5152/results/4-cjblue-weak.webp)

***Figure 4:** *E. coli* cells transformed with the cjBlue expression construct displaying weak chromoprotein production after 16 hours at 37°C.*

## **Optimizing the observation time for Chromoproteins**

We investigated the optimal incubation time for chromoproteins, as they require varying durations for maturation and color development. This helps determine if their expression profiles suit our experiments.

We cultured cells expressing dTomato, amilCP, tsPurple, and eforRed in LB/Amp medium at 37°C, shaking at 180 rpm. Samples were taken at 4, 8, 12, 18, and 24 hours, with 1 mL of culture harvested at each point. The cells were pelleted by centrifugation, and the pellets were examined for color development.

At 4 and 8 hours, no significant color was observed. By 12 hours, visible pellets formed with faint coloration. After 18 hours, color intensity increased, suggesting maturation. (See Fig.)

![Cell pellets after incubation](https://static.igem.wiki/teams/5152/engineering/fig-5.webp)

***Figure 5:** Cell pellets after 12 and 18 hours of incubation at 37°C. The 18-hour pellets display higher color intensity. pUC19 shows cells with the empty vector as a control.*

We understand that using a constitutive promoter might not replicate conditions with metal-inducible promoters in later experiments. Nonetheless, these results provide valuable insights into chromoprotein maturation times and expression profiles.

## **Cloning of Biosensors**

For cloning our heavy metal biosensors, we followed the same protocols used for chromoprotein constructs. A constitutive chromoprotein expression setup was included as a positive control, consistently yielding reliable results.

Our biosensor constructs were synthesized by IDT and Twist Bioscience, listed in the table below:

| Name | Description | Part Registry Entry |
| --- | --- | --- |
| PbrR-pPbr lead sensing chromoprotein reporter device | Includes PbrR repressor regulating the pPbr lead-inducible promoter, activating dTomato expression in presence of lead. | [BBa\_K5152004](https://parts.igem.org/Part%3ABBa_K5152004) |
| pCadA cadmium sensing chromoprotein reporter device | Utilizes pCadA promoter with ArsR and CzrA binding sites, inducing amilCP expression in presence of cadmium. | [BBa\_K5152005](https://parts.igem.org/Part%3ABBa_K5152005) |
| MerR-pCadA cadmium sensing chromoprotein reporter device | Co-expresses MerR with pCadA promoter, activating amilCP expression in presence of cadmium. | [BBa\_K5152006](https://parts.igem.org/Part%3ABBa_K5152006) |
| pYodA cadmium sensing chromoprotein reporter device | Uses pYodA to drive amilCP expression without co-expressing regulatory proteins. | [BBa\_K5152007](https://parts.igem.org/Part%3ABBa_K5152007) |
| MerR-pMerT mercury sensing chromoprotein reporter device | Contains pMerT promoter, with MerR repressor, activating tsPurple in presence of mercury. | [BBa\_K5152008](https://parts.igem.org/Part%3ABBa_K5152008) |
| pZnt zinc sensing chromoprotein reporter device | Utilizes pZnt promoter to drive eforRed expression. | [BBa\_K5152009](https://parts.igem.org/Part%3ABBa_K5152009) |

Initial cloning attempts showed few colonies, though the positive control was successful. Colony PCR confirmed the correct insertion of constructs.

### **Failed Cloning Attempts**

Gel electrophoresis validated colony PCR results. Conditions are shown below:

| Components | X1 (µL) |
| --- | --- |
| Forward M13 | 0.5 |
| Reverse M13 | 0.5 |
| Template | Colonies |
| 2X OneTaq | 12.5 |
| H2O | 11.5 |
| **Total** | 25 |

| Stage | Time (s) |
| --- | --- |
| Initial Denaturation (94 °C) | 30 |
| Denaturation (94 °C) | 20 |
| Annealing (55 °C) | 40 |
| Extension (68 °C) | 60 |
| Final Extension (68 °C) | 300 |

| Primers | Sequence |
| --- | --- |
| M13 forward | TGTAAAACGACGGCCAGT |
| M13 reverse | CAGGAAACAGCTATGACCATG |

30 cycles of PCR reactions were done in all attempts.

Despite multiple rounds, no distinct bands were observed (**Fig. 6**), indicating failed cloning. We initiated troubleshooting to identify issues.

![Failed Colony PCR](https://static.igem.wiki/teams/5152/results/6-failed-colony-pcr.webp)

***Figure 6:** Gel result from colony PCR showing absence of bands, indicating no desired insert. Faint bands are primers.*

### **Validation of PCR**

We checked the PCR apparatus using validated DNA and primers from a previous project. Tests produced expected results (**Fig. 7**), confirming PCR apparatus functionality.

![PCR Validation](https://static.igem.wiki/teams/5152/results/7-pcr-validation.webp)

***Figure 7:** Gel image validating PCR conditions. Expected band sizes confirm apparatus functionality, ruling it out as the error source.*

Investigation revealed incorrect 5' overlap design in ordered DNA. New orders for corrected sequences were placed with IDT and Twist Bioscience.

### **Successful Cloning Attempts**

With new DNA fragments, we initiated cloning following the previous workflow. The assembly protocol is outlined below. The dTomato construct was used as a positive control.

| Components (µL) | pPbr | MerR-pCadA | pCadA | pYodA | pMerT | pZnt | dTomato (+ve) |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Vector | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| Insert | 6 | 6 | 6 | 6 | 6 | 6 | 6 |
| HiFi MM | 10 | 10 | 10 | 10 | 10 | 10 | 10 |
| H2O | 2 | 2 | 2 | 2 | 2 | 2 | 2 |
| **Total** | 20 | 20 | 20 | 20 | 20 | 20 | 20 |

We successfully obtained colonies for all designs except pCadA and MerR-pMerT. Colony PCR verified correct clones (**Fig. 8**).

![PbrR Colony PCR](https://static.igem.wiki/teams/5152/results/8a-ppbr-colony-pcr.webp)
![MerR-pCadA Colony PCR](https://static.igem.wiki/teams/5152/results/8b-merr-pcada-colony-pcr.webp)
![pZnt and pYodA Colony PCR](https://static.igem.wiki/teams/5152/results/8c-pznt-pyoda-colony-pcr.webp)

***Figure 8:***
***(a)** PbrR construct colony PCR result with expected ~1.5 kbp bands in lanes 3, 4, and 5.*
***(b)** MerR-pCadA construct result with expected ~1.6 kbp bands in lanes 4, 5, and 6.*
***(c)** pZnt and pYodA result with expected ~600 bp bands.*

Leaky expression was consistently observed, notably in pZnt and pYodA designs (**Fig. 9**).

![Colony Tracking](https://static.igem.wiki/teams/5152/results/9-colony-tracking.webp)

***Figure 9:** Some clones exhibited leaky expression (visible color without induction). To avoid false positives, we excluded these clones from further study.*

## Functional Assays on Heavy Metal Detection

We conducted functional assays to test whether our biosensors could respond to the presence of heavy metals. This step was crucial in determining the effectiveness of the constructs and their ability to detect and report the presence of target metals.

The concentrations of heavy metals used in our experiments are listed in the table below:

| Heavy Metal Salt | Final Concentration (µM) |
| --- | --- |
| Lead (II) Nitrate | 100 |
| Cadmium (II) Chloride | 200 |
| Zinc (II) Chloride | 200 |
| Mercuric Chloride | 100 |

For all experimental setups, we added 1 mL of the designated metal solution into 19 mL of LB/Ampicillin broth to reach the stated final concentration, with either the corresponding biosensor cells or control cells. This ensured consistent conditions across all experiments, allowing for accurate comparison of the biosensor responses.

### I. Lead Biosensor ([BBa\_K5152004](https://parts.igem.org/Part%3ABBa_K5152004))

**The Lead Biosensor Successfully Detects the Presence of Lead Through a Visible Colour Change**

In the lead biosensor *PbrR-pPbr*, upon adding 100 µM of lead and incubating for 12 hours, the experimental setup displayed a clear red coloration in the pellet, indicating dTomato expression. This was in stark contrast to the white pellet observed in the control. Additionally, a faint red hue was visible in the culture medium itself, further confirming successful lead detection by the biosensor **(Fig. 10)**.

![Lead Biosensor](https://static.igem.wiki/teams/5152/results/10-ab-ppbr-functional-100-um.webp)

***Figure 10:**
**(a):** Through naked-eye observation, the cultures expressing the *PbrR-pPbr* lead biosensor exhibited a distinguishable red coloration, in contrast to the negative control setup where no lead was added.
**(b):** The red coloration becomes even more prominent in the cell pellet, highlighting the expression of the reporter gene (*dTomato*). This observation confirms that our biosensor is successfully responding to the presence of lead (Pb).*

After 18 hours of incubation, the coloration of the culture medium became distinctly red, clearly indicating a response to lead. However, a faint red hue was also observed in the biosensor cell culture without lead added, suggesting a mild leaky expression effect. This unintended expression in the absence of lead highlights some basal activity of the biosensor system. **(Fig. 11)**

![Lead Biosensor after 18 hours](https://static.igem.wiki/teams/5152/results/11-ppbr-100-um-after-18-hours.webp)

***Figure 11:** The *PbrR-pPbr* lead biosensor shows a significant red coloration after 18 hours of incubation with 100 µM of lead. However, when compared to the pUC19-only control setup, the *PbrR-pPbr* biosensor without added lead also displayed a mild red coloration, indicating that extended incubation leads to a mild leaky expression of the red chromoprotein.*

**The Reporter Signal is Dependent on the Concentration of Lead**

To investigate whether the reporter signal of the biosensors reflects the concentration of lead, 0.01, 0.1, 1, 10, 500, and 1000 µM lead solutions were added to the biosensor cells. 1 mL of each metal solution was added to 19 mL of LB/Ampicillin broth, containing either the corresponding biosensor cells or control cells, to reach the desired final concentrations.

After 16 hours, the cultures showed no significant differences in colour. However, after 24 hours of incubation, noticeable differences in colour intensity were observed across cultures with varying lead concentrations. In the range of 0.01 µM to 10 µM, the intensity of the red colour increased with higher lead concentrations, indicating a concentration-dependent response.

In contrast, in the 500 µM and 1000 µM setups, the colour became paler, which may be attributed to the toxicity of the high metal concentrations, potentially causing cell death or impairing protein expression. **(Fig. 12)**

![Lead Concentration Response](https://static.igem.wiki/teams/5152/results/12-pb-concentration.webp)

***Figure 12:** The red colour intensity of the lead biosensor increases with an observable difference as the concentration of lead rises from 0.1 µM to 10 µM. However, when the concentration exceeds 500 µM, there appears to be a negative effect on the colour intensity, potentially due to toxicity or inhibition of cellular functions at high lead concentrations.*

From these results, it is evident that our lead biosensor design is highly promising, demonstrating significant potential for further development into a practical and reliable tool for lead detection.

### II. Cadmium Biosensor ([BBa\_K5152006](https://parts.igem.org/Part%3ABBa_K5152006))

We also successfully demonstrated that our *MerR-pCadA* ([BBa\_K5152006](https://parts.igem.org/Part%3ABBa_K5152006)) cadmium biosensor is capable of detecting the presence of cadmium. Upon adding 200 µM cadmium chloride to the biosensor culture, the treated cells developed a distinct blue coloration, whereas the control cells remained white **(Fig. 13)**. This confirms the functional response of the cadmium biosensor.

![Cadmium Biosensor](https://static.igem.wiki/teams/5152/results/13ab-merr-pcada-functional.webp)

***Figure 13:**
**(a):** In the pellet form, an obvious blue coloration from the *amilCP* protein is observed in the pellet of the biosensor culture with 200 µM cadmium added, confirming the activation of the cadmium biosensor.
**(b):** In the pellet form, an obvious blue coloration from the *amilCP* protein is observed in the pellet of the biosensor culture with 200 µM cadmium added, confirming the activation of the biosensor.*

### III. Other Biosensor Designs

For the other biosensor designs, the results did not meet our expectations. The specific issues and functional testing outcomes for these designs are detailed on our Engineering page, which outlines all the processes and challenges we encountered with the remaining biosensor constructs.

## **Summary of Key Findings:**

1. We successfully constructed expression constructs for chromoproteins and validated their expressions. Additionally, we studied the expression time required for the cells in our design.
2. Our *PbrR-pPbr* ([BBa\_K5152004](https://parts.igem.org/Part%3ABBa_K5152004)) lead biosensor demonstrated promising results, showing significant potential for practical lead detection. Importantly, the reporter signal was found to be concentration-dependent, indicating its sensitivity to varying lead levels.
3. The *MerR-pCadA* ([BBa\_K5152006](https://parts.igem.org/Part%3ABBa_K5152006)) cadmium biosensor successfully responded to the presence of cadmium, producing a clear colour change, confirming its functionality.
4. All constructs exhibited some degree of leaky expression, emphasizing the need to carefully optimize the timing and conditions for measuring the biosensors' readouts.

Based on these findings, we held discussions to refine our approach and have developed a set of future plans for advancing the project, focusing on improving biosensor performance and addressing the challenges encountered. For further details, please refer to our Engineering page, which provides a comprehensive overview of the processes, challenges, and troubleshooting steps we encountered throughout the development of our biosensor constructs.

![Summary Image](https://static.igem.wiki/teams/5152/results/2.webp)

{% endblock %}