Notebook



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

 

 

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

 

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)

 

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

 

 

  • 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)

 

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

 

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.

 

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)

 

PCR validation to check if our PCR set is available

 

 

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:

12hrs:

 

4, 8 shows no pellets.`

 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

 

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

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

 

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.

 

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

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.

 

 

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

 

 

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.

 

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…

 

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 ?

 

17/9/2024(Tue)

Carry out with M13 primer sets.

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

 

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.

left to right: Cd+, pUC19, -ve

 

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

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.

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.

 

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.

 

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.

 

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.

 

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.