iGEM 2021 — Team Hong_Kong_UCCKE

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

# Team:Hong Kong UCCKE/Notebook

[UCCKE 2021](https://2021.igem.org/Team%3AHong_Kong_UCCKE)

* About
  + [Team](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Team)
  + [Collaboration](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Description)
  + [Engineering](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Experiments)
  + [Protocols](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Protocols)
  + [Results](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Results)
  + [Modeling](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Model)
  + [Contribution](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Implementation)
  + [Notebook](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Notebook)
* Parts
  + [Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Parts)
  + [Basic Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/BasicParts)
  + [Composite Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/CompositeParts)
* [Safety](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Safety)
* [Human Practice](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Human_Practices)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3718)

# Notebook

The process of our project by days

![](#)

2 Lee On Lane, Kwun Tong, Kowloon, Hong Kong

+852 2343 6677

uccke.brt

brt@uccke.edu.hk

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

# Team:Hong Kong UCCKE/Engineering

[UCCKE 2021](https://2021.igem.org/Team%3AHong_Kong_UCCKE)

* About
  + [Team](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Team)
  + [Collaboration](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Description)
  + [Engineering](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Experiments)
  + [Modeling](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Model)
  + [Contribution](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Implementation)
* Parts
  + [Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Parts)
  + [Basic Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/BasicParts)
  + [Composite Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/CompositeParts)
* [Safety](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Safety)
* [Human Practice](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Human_Practices)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3718)

# Engineering Success

Our Team has planned several experiments to confirm each parts we designed, the our design and engineering process is illustrated by the flowchart below:

![](https://static.igem.org/mediawiki/2021/1/1b/T--Hong_Kong_UCCKE--Experimental_flowchart.png)

### Design sgRNA

We have designed the CRISPR Cas9 single guided RNA for knocking out *arcA* and *iclR* genes in the *E. coli* genome. Their sequence and part design can be found in K3718003 & K3718004.

### Transform sgRNA and Cas9 into wild type *E. coli*

sgRNAs for *arcA* and *iclR* will be contained in a plasmid with CRISPR Cas9 gene. The plasmid will then be transformed into a wild type *E. coli* by heat shock transformation.

### SDS PAGE/ Diagnostic PCR

After transformation, SDS PAGE or colony PCR will be done to confirm whether the *E. coli* is successfully knocked out. Forward and reverse PCR primers will be designed for *arcA* and *iclR*.

If the *E. coli* is not knocked out, we may have to redesign the sgRNA or repeat the transformation of the plasmids.

### Cell growth assay

After the *E. coli* is sure to be knocked out, we will wait for the plasmids to be lost after generations of cells. To check if the cell growth is reduced after the genes are knocked out, a cell growth assay will be done to compare the growth rate of WT *E. coli* and K.O. *E. coli*. We will design the sgRNA again if the cell growth of K.O. *E. coli* is not lower than that of WT *E. coli*.

### Transform MRP into wild type E. coli

Plasmids containing MRP gene will be transformed into normal strain E. coli before directly transforming them into the K.O. strains. The sequence of MRP can be found in K3425100.

### SDS PAGE

SDS PAGE will be used to test if the MRP is expressed. The transformation will be redo if the bends are not shown in the SDS PAGE.

### Transform MRP into K.O. strain

If the protein expression is successful, the MRP gene will be transformed into the K.O. strain to make sure the MRP protein can also be expressed in the K.O. *E. coli*.

### SDS PAGE

SDS PAGE will be used to test if the MRP is also expressed in the K.O. *E. coli*. The transformation will be redo if the bends are not shown in the SDS PAGE.

### Transform rescue genes into K.O. strain

While the transformation of MRP is doing, plasmids with *iclR* and *arcA* gene will be transformed into *arcA-iclR- E. coli* as rescue genes.

### Cell growth assay

Cell growth assay will be done to compare the growth rate of wild type *E. coli*, *iclR- E. coli*, *arcA- E. coli*, *arcA-iclR- E. coli* and rescued KO *E. coli*. If the growth rate of the rescued *E.coli* is lower than that of KO strains, we may need to redo the transformation step.

### Caffeine as model

After the experiments of MRP expression and rescue KO *E. coli* have proven to be successful, we will use caffeine-sensitive MRP (K3718010) as a testing part to model the system. Caffeine will be used to homodimerize the receptors, and is expected to induce the expression of downstream genes. This model will be used to conduct three experiments to confirm the functionality of the tool. By cloning in an antibody VHH gene against caffeine and *gfp* as a desired gene to be expressed, we expect the platform is activated in the presence of caffeine to express *gfp*, *arcA* and *iclR*, which will result in an increase in growth rate and a visible green color under UV.

#### Cell growth assay

We will conduct a cell growth assay to compare the growth rate of wild type *E. coli*, *iclR- E. coli*, *arcA- E. coli*, *arcA-iclR- E. coli*, rescued *E. coli* and the *E. coli* with the MRP (caffeine) and rescue genes.

#### Leak expression

We will measure the leak expression of MRP’s downstream genes before adding caffeine to let MRP bind to the CadC region.

#### Dose response

We will measure how much concentration of caffeine is needed for MRP to detect and express the downstream genes effectively. This is tested by adding different concentrations of caffeine to our final kit and find the optimum concentration to induce MRP.

## Modeling

The growth rate of *arcA-iclR- E. coli* is what we would be model for, since our lab does not allow us to knock out the *E. coli* genome by ourselves. We will use the growth parameters of *arcA-* and *iclR- E. coli* to predict the growth parameters of *arcA-iclR- E. coli*.

For the details of modelling, please go to [[modeling]](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Model).

Contacts

2 Lee On Lane, Kwun Tong, Kowloon, Hong Kong

+852 2343 6677

uccke.brt

brt@uccke.edu.hk

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

# Team:Hong Kong UCCKE/Experiments

[UCCKE 2021](https://2021.igem.org/Team%3AHong_Kong_UCCKE)

* About
  + [Team](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Team)
  + [Collaboration](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Description)
  + [Engineering](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Experiments)
  + [Modeling](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Model)
  + [Contribution](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Implementation)
* Parts
  + [Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Parts)
  + [Basic Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/BasicParts)
  + [Composite Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/CompositeParts)
* [Safety](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Safety)
* [Human Practice](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Human_Practices)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3718)

# Experiments

## Analyzing growth dynamics of *E. coli* strains - Bacterial growth assay

A schematic diagram is shown below to illustrate the flow of this experiment:

![](https://static.igem.org/mediawiki/2021/6/64/T--Hong_Kong_UCCKE--cell_growth_assay.png)

### Obtaining of *E. coli* strains

*E. coli* strains *arcA-*knockout (*arcA-*) and *iclR-*knockout (*iclR-*), and their mother strain K-12 BW25113 (WT) are ordered from the National Institute of Genetics, as a part of the National BioResource Project (NIG, Japan). The Keio collection is a bacterial strain collection of a “set of precisely defined, single-gene deletions of all nonessential genes”. The bacterial strains are shipped to our lab in the form of filter disc and the knockout strains are Kanamycin-resistant.

### Recovering Keio *E. coli*

To recover the cells, the three bacterial discs were suspended in antibiotic-free LB media. The cultures were incubated overnight in a shaking incubator at 37C. The WT culture is then plated on an antibiotic-free LB agar plate while the knockout strains *arcA-* and *iclR-* are plated in Kanamycin agar plates. The plates were incubated at 37C overnight to obtain single colonies on each plate.

### Transforming iclR gene into *iclR-*

The recombinant plasmid containing BBa\_K3718007, with Ampicillin resistance, is purchased from IDT. The composite part is shown below:

![](https://static.igem.org/mediawiki/2021/thumb/6/61/T--Hong_Kong_UCCKE--iclR_Exp_part.png/800px-T--Hong_Kong_UCCKE--iclR_Exp_part.png)

Since *iclR- E. coli* is not a competent cell, a selected colony on the Kanamycin agar plate is suspended in 250μl of calcium chloride transformation solution in preparation of the transformation. 400-600ng of the above plasmid is added to the suspended colony. The following steps are the same as a general transformation procedure.

Transformed *iclR- E. coli* are plated on an Ampicillin plate. Colonies on this plate after overnight incubation should have obtained the plasmid thus the iclR gene. To verify the transformation, colony PCR is done and the results are visualized using gel electrophoresis.

![](https://static.igem.org/mediawiki/2021/thumb/c/c6/T--Hong_Kong_UCCKE--cPCR_iclR.jpeg/800px-T--Hong_Kong_UCCKE--cPCR_iclR.jpeg)

The amplified region is approximately 1.1kb. The band sizes of all colonies are between 1 and 1.5 kb, which reassures the desired plasmid has been successfully transformed into the *iclR- E. coli* to become the *iclR-*rescue *E. coli*.

### Overnight (O/N) culture

Overnight cultures of the four *iclR- E. coli* strains are made in preparation for the cell growth assay. 3 single colonies of each strain are selected to be inoculated in 5ml antibiotic-free LB media so that there are 3 biological replicates. (WT on antibiotic-free plate, *arcA-* on Kanamycin plate, *iclR-* on Kanamycin plate, and *iclR-*rescue on Ampicillin plate.)

### Starter culture

For each of 12 O/N cultures, 50μl of O/N culture is transferred to 5ml of fresh antibiotic-free LB medium. These starter cultures are allowed to grow until their exponential phases at 2 hours (a pilot experiment was done). 200μl of each starter culture is transferred orderly into a 96-well microplate. The OD at 600nm of each starter culture is determined by the plate reader.

### Starting the experimental culture

On a new sterilized 96-well Sarstedt clear flat bottom microplate, 200μl of fresh antibiotic-free LB medium was transferred into each well. Using the measured OD of the starter cultures, the volume required to achieve OD = 0.05 in each well was calculated. The calculated amount of fresh culture was transferred out from the well, and the same amount of starter culture was transferred into that well, so that the total volume of all occupied wells is 200μl. There are two technical replicates for each biological replicate. That multiplies to become six replicates for each strain. Fresh LB medium is transferred into some wells as blank.

Every step above was done in a sterile environment. We discovered in a trial run that a lid is necessary to avoid water loss to ensure accurate readings. Since we did not have one, we carefully covered the plate with a sterilized cling wrap over the top of the plate, ensuring there is no folding nor wrinkle on the wrap and it is evenly distributed. The experimental cultures, carried in the microplate, are placed into the plate reader. The FLUOstar® Omega plate reader has the following settings:

* Temperature: 37C
* Microplate: SARSTEDT 96
* 1 discrete wavelength at 600nm
* Pathlength correction on
* No well scan
* 288 cycles with 300s interval
* 6 flashes per reading
* 0.5s settling time before reading
* Orbital shaking at 200rpm while non-reading time

### Data analysis using GroFit

The following procedures are done to obtain the box plots below:

![](https://static.igem.org/mediawiki/2021/thumb/8/84/T--Hong_Kong_UCCKE--analysis_flow.png/800px-T--Hong_Kong_UCCKE--analysis_flow.png)

The detailed procedures can be found in [[modeling]](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Model).

![](https://static.igem.org/mediawiki/2021/c/c5/T--Hong_Kong_UCCKE--box_mu.png)![](https://static.igem.org/mediawiki/2021/0/0d/T--Hong_Kong_UCCKE--box_A.png)

### Results

Box plots of the maximum growth rates and maximum cell densities are plotted by R using the parameters obtained from GroFit. Compared to WT, *iclR- E. coli* showed reduced growth while *iclR-rescue* *E. coli* showed the same to a slightly higher maximum growth rate. *arcA-* has a very large data range and no outliers can be excluded. However, using ANOVA and post-hoc test, there was no significant difference between strains in either maximum growth rates and maximum cell density. Despite not having significant results, the comparison between WT, *iclR-*, and *iclR-rescue E. coli* provide a sound foundation for us to carry on with growth assays using double-knockout strains.

### Reference

M. Kahm, G. Hasenbrink, H. Lichtenberg-Fraté, J. Ludwig, and M. Kschischo, “Grofit: Fitting biological growth curves with R,” *Journal of Statistical Software*, vol. 33, no. 7, Feb. 2010.

Contacts

2 Lee On Lane, Kwun Tong, Kowloon, Hong Kong

+852 2343 6677

uccke.brt

brt@uccke.edu.hk

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

# Team:Hong Kong UCCKE/Results

[UCCKE 2021](https://2021.igem.org/Team%3AHong_Kong_UCCKE)

* About
  + [Team](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Team)
  + [Collaboration](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Collaborations)
  + [Attributions](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Attributions)
* Project
  + [Description](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Description)
  + [Engineering](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Engineering)
  + [Experiments](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Experiments)
  + [Protocols](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Protocols)
  + [Results](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Results)
  + [Modeling](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Model)
  + [Contribution](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Contribution)
  + [Implementation](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Implementation)
  + [Notebook](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Notebook)
* Parts
  + [Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Parts)
  + [Basic Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/BasicParts)
  + [Composite Parts](https://2021.igem.org/Team%3AHong_Kong_UCCKE/CompositeParts)
* [Safety](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Safety)
* [Human Practice](https://2021.igem.org/Team%3AHong_Kong_UCCKE/Human_Practices)
* [Judging Form](https://igem.org/2021_Judging_Form?id=3718)

# Results

The experimental results

![](#)

2 Lee On Lane, Kwun Tong, Kowloon, Hong Kong

+852 2343 6677

uccke.brt

brt@uccke.edu.hk