iGEM 2021 — Team Hong_Kong_HKU

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# Team:Hong Kong HKU/Notebook

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* Project

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![](https://static.igem.org/mediawiki/2021/5/58/T--Hong_Kong_HKU--bannerm.png)

Notebook

Here is a daily account of what happened in wet lab for the last month of summer.

![](https://static.igem.org/mediawiki/2021/thumb/e/e3/T--Hong_Kong_HKU--nb1.png/1200px-T--Hong_Kong_HKU--nb1.png)
![](https://static.igem.org/mediawiki/2021/thumb/5/58/T--Hong_Kong_HKU--nb2.png/1200px-T--Hong_Kong_HKU--nb2.png)
![](https://static.igem.org/mediawiki/2021/thumb/a/a6/T--Hong_Kong_HKU--nb3.png/1200px-T--Hong_Kong_HKU--nb3.png)
![](https://static.igem.org/mediawiki/2021/thumb/1/18/T--Hong_Kong_HKU--nb4.png/1200px-T--Hong_Kong_HKU--nb4.png)
![](https://static.igem.org/mediawiki/2021/thumb/e/e4/T--Hong_Kong_HKU--nb5.png/1200px-T--Hong_Kong_HKU--nb5.png)

About us

HKU Evolve 2021 aims to engineer a clean pathway to degrade PET plastics.

Contact us

igemhku@hku.hk

Our Sponsors

[IDT](https://sg.idtdna.com/pages)

[iGEM](https://igem.org/Main_Page)

[Snapgene](https://www.snapgene.com/)

[Twist](https://www.twistbioscience.com/)

HKU EVOLVE

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

# Team:Hong Kong HKU/Engineering

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* Project

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  [Contribution](https://2021.igem.org/Team%3AHong_Kong_HKU/Contribution)
  [UN SDGs](https://2021.igem.org/Team%3AHong_Kong_HKU/Sustainable)
* Laboratory

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  [Experiments](https://2021.igem.org/Team%3AHong_Kong_HKU/Experiments)
  [Notebook](https://2021.igem.org/Team%3AHong_Kong_HKU/Notebook)
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* Dry Lab

  [Modelling](https://2021.igem.org/Team%3AHong_Kong_HKU/Model)
  [Hardware](https://2021.igem.org/Team%3AHong_Kong_HKU/Hardware)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHong_Kong_HKU/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AHong_Kong_HKU/Implementation)
  [Integrated Human Practices](https://2021.igem.org/Team%3AHong_Kong_HKU/Human_Practices#intro)
  [Education & Communication](https://2021.igem.org/Team%3AHong_Kong_HKU/Communication)
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# Engineering

![](https://static.igem.org/mediawiki/2021/5/58/T--Hong_Kong_HKU--bannerm.png)

[Rationale](#rationale)

---

[Design](#design)

---

[Co-culture of PET-degrading/sucrose-utilising E. coli and sucrose secreting cyanobacteria](#coculture)

---

[1. Co-culture](#1coculture)

---

[2. PET degradation](#2pet)

---

[3. Sucrose uptake of *E. coli*](#3sucrose)

---

[4. Sucrose secretion of cyanobacteria](#4sucrose)

---

# Rationale

Our project aims to develop a E. coli and S. elongatus co-culture system, where *S. elongatus* can photosynthetically metabolise sucrose, which fuels the production and secretion of PETase and MHETase in *E. coli*.

A plasmid that codes for PETase and MHETase is transformed into *E. coli*. PETase digests PET into monomeric MHET and trace amounts of BHET, while MHETase further digests MHET into terephthalic acid and ethylene glycol. While a few *E. coli* strains can readily uptake sucrose as an energy source (eg. *E. coli* W), we have engineered the BL21 strain we are using to incorporate sucrose-utilising genes, i.e. sucrose permease, hydrolase, and fructokinase, into the bacterium genome through the lambda Red system. This ensures the function would not be easily lost through the generations as with exogenous plasmids. Sucrose produced by *S. elongatus* can be used by *E. coli*. We would also like to maximise sucrose production by *S. elongatus* through expressing cscB, which codes for sucrose permease that allows more sucrose transport in and out of the cell.

Our project is based on previous literature that has demonstrated successful co-culturing of *E. coli* and *S. elongatus*, and PETase/MHETase expression in *E. coli* [1, 2, 3, 4]. With the hardware and each function working as intended, the system should be able to maintain a long-term equilibrium between the two organisms, and produce enough PETase and MHETase to digest PET plastic.

Due to limitations in obtaining lab consumables and access to equipment, we were unfortunately unable to follow through with our experimental design. Our complete experimental design is detailed below.

# Design

This project involves several components, which will first be tested and quantified individually, and then in a co-culture system.

Plasmids

Three [plasmids](https://2021.igem.org/Team%3AHong_Kong_HKU/Parts) will be synthesised and constructed for our system:

1. **Plastic plasmid**: this plasmid contains coding regions for PETase and MHETase, two enzymes that will digest PET plastics into monomeric terephthalic acid and ethylene glycol.
![](https://static.igem.org/mediawiki/2021/thumb/d/da/T--Hong_Kong_HKU--plastic.PNG/900px-T--Hong_Kong_HKU--plastic.PNG)
This plasmid is chemically transformed into BL21 (DE3), an *E. coli* strain that excels at recombinant protein production. For the sake of experimental verification, PETase and MHETase sequences are attached to red and cyan fluorescent protein signals respectively, so protein secretion can be visualised easily.

2. **Sucrose-uptake plasmid**: this plasmid contains coding regions for the cscA, cscB and cscK genes, which are sucrose-6-phosphate hydrolase, sucrose permease, and fructokinase respectively. cscA allows for the metabolism of sucrose; cscB is a permease that allows sucrose entry into the cell; and cscK further aids in the uptake and digestion of sucrose. This plasmid allows *E. coli* to utilise sucrose as an energy source in addition to glucose.
![](https://static.igem.org/mediawiki/2021/thumb/2/21/T--Hong_Kong_HKU--coli_sucrose.PNG/900px-T--Hong_Kong_HKU--coli_sucrose.PNG)
This plasmid is incorporated into the *E. coli* genome via the lambda Red system in order to ensure it is passed down to future generations. This way, the sucrose-uptake ability will be innate to the bacteria, and only viable cells receive the PETase/MHETase plasmid. This also minimises the chance of a cell losing either or both of the plasmids across generations. The method requires the pKD46 plasmid to be transformed into the target bacterial cell, followed by a second transformation (often electroporation) of a linearised piece of DNA (a PCR product of the above plasmid).

3. **Cyanobacteria plasmid**: this plasmid contains coding regions for the lacI gene and cscB, a sucrose permease that can allow sucrose to pass through the cell membrane. The lacI gene creates an IPTG-induced system that stimulates the cyanobacteria to secrete sucrose when needed.
![](https://static.igem.org/mediawiki/2021/thumb/6/65/T--Hong_Kong_HKU--cyano.PNG/900px-T--Hong_Kong_HKU--cyano.PNG)
This plasmid is transformed into PCC 7942 by natural transformation, as this strain is naturally competent and actively takes up exogenous DNA from the environment.

*These plasmids are assembled using standard biobrick assembly methods.

* All parts synthesised by IDT and Twist Bioscience contain biobrick suffix and prefix regions, which can be recognised by specific restriction enzymes such as EcoRI, PstI, XbaI and SpeI.
* A standard digestion protocol is followed to create sticky ends for each insert and the corresponding backbone gene fragment. This is followed by a standard ligation protocol and chemical transformation of plasmids into DH5a (DE3) competent cells.
* Plasmids are confirmed by colony PCR or restriction digestion, and isolated and purified with a standard minipreparation kit/protocol.*

![](https://static.igem.org/mediawiki/2021/4/4a/T--Hong_Kong_HKU--coc.png)

# Co-culture of PET-degrading/sucrose-utilising E. coli and sucrose secreting cyanobacteria

Experimental design is separated into 4 main stages: 1) co-culture of *E. coli* and *S. elongatus*, 2) PET-degrading abilities of *E. coli*, 3) sucrose-utilising abilities of *E. coli*, and 4) sucrose secretion by *S. elongatus*.

# 1. Co-culture

While direct of co-culture of *E. coli* and *S. elongatus* is possible and has been done by multiple research groups as well as iGEM teams, maintaining equilibrium requires a functional sucrose-utilising E. coli strain [5, 6, 7, 8] such as *E. coli* W, which we were unable to source. Furthermore, Hays et al. (2017) noted in their paper that wild type *E. coli* W grows most optimally when presented with more than 5g/L of sucrose, while cscB-enhanced *S. elongatus* produces merely 36 mg of sucrose per litre per hour. As a solution, the team had to further engineer *E. coli* W to adapt to a low-sucrose condition [9].

At a very initial stage of our project, we decided that compartmentalising the two organisms would serve a better purpose for our prototype model. This allows the easy separation and exchange of nutrients and organisms.

To allow the exchange of nutrients, i.e. sucrose from *S. elongatus* to *E. coli*, we design a compartment with dialysis membranes between the organisms. Dialysis membranes have pores that are generally 1 to 10 nm in diameter, whereas sucrose molecules are 1.08631 nm in diameter. As a result, two compartments with *E. coli* and *S. elongatus* separated by a dialysis membrane would theoretically function well. From previous literature, such as one by Hays et al. (2017), cyanobacteria have been experimentally co-cultured with multiple organisms, including *E. coli*. Such co-cultures require an alternate BG-11 recipe that can support *E. coli* growth, which was named (CoB)BG-11 [10]. In other words, both compartments would contain the same medium of (CoB)BG-11.

In the same study that looked at *S. elongatus* and *E. coli* co-culture, the researchers noted that the long-term stability between these two organisms could be due to *E. coli* developing the ability to live off other metabolites produced by the cyanobacteria, and that it is not solely dependent on sucrose as a carbon source. Therefore, precaution should be taken against any extra nutrients introduced into the system that can alter the co-culture. This especially applies to the system’s implementation, since the adaptability of *E. coli* to utilise any immediate energy source could cause trouble if it leaks into open waters.

![](https://static.igem.org/mediawiki/2021/3/3f/T--Hong_Kong_HKU--ideal.png)

Schematic representation of an ideal co-culture container.

1.1 Cyanobacteria

Cyanobacteria works as the energy factory in our system by converting sunlight into sucrose for our bioengineered *E. coli* (BL21(DE3)). For our system, the species and strain of cyanobacteria used is *S. elongatus* (PCC 7942). To enable the construction of an accurate [co-culture](https://2021.igem.org/Team%3AHong_Kong_HKU/Model) model, it is essential to know the growth conditions and growth rate of PCC 7942.

1.1.1 Growth conditions

PCC 7942 is a freshwater strain that can be cultured in BG-11 medium (freshwater). However, while liquid culture protocols are widely established, there are a lot of different solid medium recipes available online. Since solid cultures are more conveniently used in experiments and often utilised for preservation (of up to 2 months), we took different recipes and tested them with PCC 7942, i.e. different concentrations of agarose powder (1.5%, 1.25%, 1% and 0.75%), and using different types of agar like bacto agar. Colonies formed 2 weeks after streaking on 1.5% BG-11/agar gel. However, due to this slow growth rate of PCC 7942 and limited resources to accommodate our plates, we reverted to liquid cultures for further experiments.

![](https://static.igem.org/mediawiki/2021/0/01/T--Hong_Kong_HKU--conc.png)
PCC 7942 grows well under 30°C with bright light. We placed tubes of cyanobacteria in a shaking incubator (200 rpm) under a constant light source, and supplemented them with fresh BG-11 medium when cultures became opaque or too concentrated.

1.1.2 Growth rate: measurement of OD

To effectively quantify the number of cyanobacteria, we implemented microbial OD measurement. We [measured the OD](https://2021.igem.org/Team%3AHong_Kong_HKU/Results) of cyanobacteria tubes every day at a set time.

An alternative method of measurement would be to use counting chamber slides and manually count the number of cyanobacteria cells. A serial dilution would then be employed to obtain the data necessary for generating a growth curve.

# 2. PET degradation

To test the function of PETase plasmid and the produced PET enzyme, a digestion experiment would be conducted. For PETase secretion, we directly check the degradation rate of PET plastic strips in culture medium, as performed by previous iGEM teams as well [11]. We also test the efficiency of the plasmid in different strains of *E. coli*, i.e. DH5a(DE3), a strain optimised for cloning, and BL21(DE3), a strain optimised for protein expression.

After transforming the plasmid into each strain, we pick a colony and culture them in 5mL of LB medium in a 37°C shaking incubator. At the same time, we add 10 PET squares (2cm x 2cm) into the culture tube. Every 12-16 hours, we take out 1 square of PET and image the surface with scanning electron microscopy. We will also add 5mL of fresh LB medium into the culture during this time. After 20 days, all strips will be taken out for imaging. This will give us a time-dependent insight into PETase activity and provides us with a preliminary idea for the speed and efficiency of wild type PETase. Alternatively, we can take 1mL of the previous day’s culture and transfer it to a new tube along with all strips of PET, while adding 4mL of fresh LB medium. This ensures that *E. coli* does not overgrow.

This test will be adjusted based on experimental results. The aim of adding fresh LB medium is to ensure *E. coli* continues to grow and produce PETase at a steady rate. However, it is difficult to maintain bacteria and enzyme concentration at a constant. While it is possible to heat kill *E. coli* at 60°C for 5 minutes and leave PETase active (denaturing temperature at 69.4°C), the co-culture design does not allow for this method [12]. It is also advisable to extract PETase from *E. coli* and directly test with the enzyme instead of testing with the bacterial chassis as well. After extraction of PETase, as there is no need to switch out medium and plastic trips, we are able to check how long it takes for the enzyme to digest PET strips until there is a significant and measurable difference after imaging.

Should the resources be available, we can also perform high-performance liquid chromatography (HPLC) to check the presence of PET monomers in the supernatant of the enzyme-plastic mixture instead. This is a more direct way of making sure that PETase is functional, since it can detect trace amounts of terephthalic acid and ethylene glycol that might not be visible under a microscope.

# 3. Sucrose uptake of *E. coli*

We then test the sucrose uptake ability of *E. coli* by measuring its growth rate when presented with different concentrations of sucrose and glucose. After transforming *E. coli* with a sucrose plasmid (with csc A, csc B, and csc K genes that enables sucrose metabolism), we culture it in different concoctions, i.e. LB, LB with glucose, LB with sucrose, LB with both glucose and sucrose etc. We measure the OD of the solution after incubation overnight at 600 nm to determine how well *E. coli* can survive in each situation. The experimental procedures are adjusted according to results. With enough data, a growth curve is constructed to determine optimum sucrose concentration. This allows us to construct a co-culture ratio between *E. coli* and *S. elongatus* that can allow them to be sustainably maintained. According to literature, while *S. elongatus* is able to generate 36 mg sucrose L−1 hr−1, and *E. coli* takes in sucrose at 7mmolgCDW−1 hr−1, an equilibrium of 1:10 for *E. coli* and *S. elongatus* is possible [13, 14].
![](https://static.igem.org/mediawiki/2021/thumb/a/a5/T--Hong_Kong_HKU--hays.png/1200px-T--Hong_Kong_HKU--hays.png)

Co-cultures persist through time and perturbation (Hays et al., 2017).

# 4. Sucrose secretion of cyanobacteria

To test whether *S. elongatus* secretes more sucrose after being engineered, we use a sucrose kit to compare the sucrose content at set time points. We should also note that the over-expression of cscB lowers *S. elongatus* growth [15].

The literature we base our modelling off of uses a 48-hour constant light interval for cyanobacteria growth [16]. This can be tested against a 12-hour cycle, which better mimics the natural environment and would provide a better estimate of the model working at a real-life condition.

After we verify this, we circle back to Stage 1 and test the co-culture system with the now-enhanced sucrose secretion efficiency of *S. elongatus*. We can then establish an appropriate co-culture ratio, followed by verification of the PET degradation efficiency and application of this system in our proposed hardware prototype.

# References

[1] Hays, S. G., Yan, L. L., Silver, P. A., & Ducat, D. C. (2017). Synthetic photosynthetic consortia define interactions leading to robustness and photoproduction. *Journal of Biological Engineering, 11*(1). <https://doi.org/10.1186/s13036-017-0048-5>.

[2] Seo, H., Kim, S., Son, H. F., Sagong, H.-Y., Joo, S., & Kim, K.-J. (2019). Production of extracellular petase from Ideonella sakaiensis using SEC-dependent signal peptides in *E. coli*. *Biochemical and Biophysical Research Communications, 508*(1), 250–255. <https://doi.org/10.1016/j.bbrc.2018.11.087>.

[3] Shi, L., Liu, H., Gao, S., Weng, Y., & Zhu, L. (2021). Enhanced extracellular production of ispetase in *Escherichia coli* via engineering of the PELB signal peptide. *Journal of Agricultural and Food Chemistry, 69*(7), 2245–2252. <https://doi.org/10.1021/acs.jafc.0c07469>.

[4] Janatunaim, R. Z., & Fibriani, A. (2020). Construction and cloning of plastic-degrading recombinant enzymes (MHETase). *Recent Patents on Biotechnology, 14*(3), 229–234. <https://doi.org/10.2174/1872208314666200311104541>.

[5] Liu, H., Cao, Y., Guo, J., Xu, X., Long, Q., Song, L., & Xian, M. (2021). Study on the isoprene-producing co-culture system of *Synechococcus elongates–escherichia coli* through OMICS ANALYSIS. *Microbial Cell Factories, 20*(1). <https://doi.org/10.1186/s12934-020-01498-8>.

[6] Team:nevada/Project/Co-cult. (n.d.). Retrieved October 16, 2021, from [https://2011.igem.org/Team:Nevada/Project/Co-Cult](https://2011.igem.org/Team%3ANevada/Project/Co-Cult).

[7] Team:UCL/results. (n.d.). Retrieved October 16, 2021, from  [https://2017.igem.org/Team:UCL/Results](https://2017.igem.org/Team%3AUCL/Results)

[8] *Towards an Engineered Co-culture Toolbox*. Team:Duesseldorf. (n.d.). Retrieved October 12, 2021, from  [https://2018.igem.org/Team:Duesseldorf](https://2018.igem.org/Team%3ADuesseldorf).

[9] Ibid.

[10] Ibid.

[11] Team:ITB Indonesia/Results. (n.d.). Retrieved October 16, 2021, from [https://2017.igem.org/Team:ITB\_Indonesia/Results](https://2017.igem.org/Team%3AITB_Indonesia/Results).

[12] Zhong-Johnson, E. Z., Voigt, C. A., & Sinskey, A. J. (2021). An absorbance method for analysis of enzymatic degradation kinetics of poly(ethylene terephthalate) films. *Scientific Reports, 11*(1). <https://doi.org/10.1038/s41598-020-79031-5>.

[13] Ibid.

[14] Mohamed, E. T., Mundhada, H., Landberg, J., Cann, I., Mackie, R. I., Nielsen, A. T., Herrgård, M. J., & Feist, A. M. (2019). Generation of an *E. coli* platform strain for improved sucrose utilization using adaptive laboratory evolution. *Microbial Cell Factories, 18*(1). <https://doi.org/10.1186/s12934-019-1165-2>.

[15] Ibid.

[16] Ibid.

About us

HKU Evolve 2021 aims to engineer a clean pathway to degrade PET plastics.

Contact us

igemhku@hku.hk

Our Sponsors

[IDT](https://sg.idtdna.com/pages)

[iGEM](https://igem.org/Main_Page)

[Snapgene](https://www.snapgene.com/)

[Twist](https://www.twistbioscience.com/)

HKU EVOLVE

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

# Team:Hong Kong HKU/Experiments

* [Home (current)](https://2021.igem.org/Team%3AHong_Kong_HKU)
* Team

  [About Us](https://2021.igem.org/Team%3AHong_Kong_HKU/Team)
  [Attributions](https://2021.igem.org/Team%3AHong_Kong_HKU/Attributions)
  [Collaboration](https://2021.igem.org/Team%3AHong_Kong_HKU/Collaborations)
* Project

  [Description](https://2021.igem.org/Team%3AHong_Kong_HKU/Description)
  [Contribution](https://2021.igem.org/Team%3AHong_Kong_HKU/Contribution)
  [UN SDGs](https://2021.igem.org/Team%3AHong_Kong_HKU/Sustainable)
* Laboratory

  [Engineering](https://2021.igem.org/Team%3AHong_Kong_HKU/Engineering)
  [Experiments](https://2021.igem.org/Team%3AHong_Kong_HKU/Experiments)
  [Notebook](https://2021.igem.org/Team%3AHong_Kong_HKU/Notebook)
  [Results](https://2021.igem.org/Team%3AHong_Kong_HKU/Results)
  [Parts](https://2021.igem.org/Team%3AHong_Kong_HKU/Parts)
* Dry Lab

  [Modelling](https://2021.igem.org/Team%3AHong_Kong_HKU/Model)
  [Hardware](https://2021.igem.org/Team%3AHong_Kong_HKU/Hardware)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHong_Kong_HKU/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AHong_Kong_HKU/Implementation)
  [Integrated Human Practices](https://2021.igem.org/Team%3AHong_Kong_HKU/Human_Practices#intro)
  [Education & Communication](https://2021.igem.org/Team%3AHong_Kong_HKU/Communication)
  [Entrepreneurship](https://2021.igem.org/Team%3AHong_Kong_HKU/Entrepreneurship)
* [Meme Gallery (current)](https://2021.igem.org/Team%3AHong_Kong_HKU/Meme)

# Experiments

![](https://static.igem.org/mediawiki/2021/5/58/T--Hong_Kong_HKU--bannerm.png)

# Protocols

Here is a document for the protocols we used for our project, some of which are compiled from different sources after extensive research.

They include:

* Standard PCR
* Standard restriction digestion of biobricks
* Standard ligation of biobricks
* Chemical transformation
* Lambda Red Protocol with pKD46
* Natural transformation for cyanobacteria
* O.D. measurement for cyanobacteria
* Co-culture, growth conditions according to Liu et al.

About us

HKU Evolve 2021 aims to engineer a clean pathway to degrade PET plastics.

Contact us

igemhku@hku.hk

Our Sponsors

[IDT](https://sg.idtdna.com/pages)

[iGEM](https://igem.org/Main_Page)

[Snapgene](https://www.snapgene.com/)

[Twist](https://www.twistbioscience.com/)

HKU EVOLVE

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

# Team:Hong Kong HKU/Results

* [Home (current)](https://2021.igem.org/Team%3AHong_Kong_HKU)
* Team

  [About Us](https://2021.igem.org/Team%3AHong_Kong_HKU/Team)
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  [Collaboration](https://2021.igem.org/Team%3AHong_Kong_HKU/Collaborations)
* Project

  [Description](https://2021.igem.org/Team%3AHong_Kong_HKU/Description)
  [Contribution](https://2021.igem.org/Team%3AHong_Kong_HKU/Contribution)
  [UN SDGs](https://2021.igem.org/Team%3AHong_Kong_HKU/Sustainable)
* Laboratory

  [Engineering](https://2021.igem.org/Team%3AHong_Kong_HKU/Engineering)
  [Experiments](https://2021.igem.org/Team%3AHong_Kong_HKU/Experiments)
  [Notebook](https://2021.igem.org/Team%3AHong_Kong_HKU/Notebook)
  [Results](https://2021.igem.org/Team%3AHong_Kong_HKU/Results)
  [Parts](https://2021.igem.org/Team%3AHong_Kong_HKU/Parts)
* Dry Lab

  [Modelling](https://2021.igem.org/Team%3AHong_Kong_HKU/Model)
  [Hardware](https://2021.igem.org/Team%3AHong_Kong_HKU/Hardware)
* Human Practices

  [Overview](https://2021.igem.org/Team%3AHong_Kong_HKU/Human_Practices)
  [Implementation](https://2021.igem.org/Team%3AHong_Kong_HKU/Implementation)
  [Integrated Human Practices](https://2021.igem.org/Team%3AHong_Kong_HKU/Human_Practices#intro)
  [Education & Communication](https://2021.igem.org/Team%3AHong_Kong_HKU/Communication)
  [Entrepreneurship](https://2021.igem.org/Team%3AHong_Kong_HKU/Entrepreneurship)
* [Meme Gallery (current)](https://2021.igem.org/Team%3AHong_Kong_HKU/Meme)

# Results

![](https://static.igem.org/mediawiki/2021/5/58/T--Hong_Kong_HKU--bannerm.png)

[Results](#results)

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[Cloning](#cloning)

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[Cyanobacteria Growth](#cyano)

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[General Troubleshooting](#general)

# Results

Cloning

The initial step of the project was to clone our desired genes into 3 plasmids, i.e. plastic-degrading PETase/MHETase plasmid for *E. coli*, sucrose-uptake plasmid also for *E. coli*, and sucrose-secretion plasmid for *S. elongatus*.

![](https://static.igem.org/mediawiki/2021/0/01/T--Hong_Kong_HKU--lac_csc_2.png)

Colony PCR of lacI and cscB: lanes 1 and 2 show a size of ~1.1 kb, while lanes 3-6 are ~1.2 kb, and lanes 7-10 ~1.5 kb. LacI is ~1150 bp, and with added flanked regions by primers, it should be ~1.3 kb. **Lanes 3-6 show the correct lacI fragment. Lanes 7-10 show the correct cscB fragment.**

![](https://static.igem.org/mediawiki/2021/2/23/T--Hong_Kong_HKU--lac_csc_6.png)

Restriction digestion analysis of lacI and cscB plasmids: Lane 1 with 4A5 and lacI plasmid and a single RE cut shows an expected ~4 kb (4.5 kb) band, while lane 2 shows cscB (1.5 kb) and 1A3 (2.1 kb). **Lanes 1 and 2 both show the successful cloning of lacI and cscB gene fragments respectively.**

![](https://static.igem.org/mediawiki/2021/8/87/T--Hong_Kong_HKU--cscABK_11.png)

Restriction digestion analysis: lanes 1-5 show RE digestion of cscA with ampicillin backbone, while lanes 6-10 show that of cscB. Lanes 3, 4 and 8 seem to show correct bands, but sizes of DNA were too close to be separated in gel. After running for additional time to separate them, **lanes 3, 4 show the correct band size of 1.8 kb (cscA) and 2.1 kb (1A3 backbone), and lane 8 with 2.7 kb (cscB + K) and 2.1 kb (1A3 backbone).** Lane 5 shows a RFP contamination.

![](https://static.igem.org/mediawiki/2021/thumb/a/ad/T--Hong_Kong_HKU--cscABK_13.png/1200px-T--Hong_Kong_HKU--cscABK_13.png)

Colony PCR and restriction digestion analysis: (Left) lanes 1, and 2 show colony PCR of cscA single fragment plasmid, while lanes 3 shows that of cscB+K. Lane 5 and 6 are cscA X/P digestion and cscB+K S/P digestion respectively. Lane 1 showed no bands, while lane 2 showed the expected size of 1.8 kb (cscA). Lane 3 showed an expected size of 2.7 kb. **Lanes 5 and 6 both showed correct digestion bands, indicating successful clones.** With a successful clone proven by lane 5, lane 1 is thus possibly PCR error. (Right) The experiment was repeated and positive results were observed for lanes 1-5, but S/P digestion band was larger than expected. Troubleshooting identified a problematic backbone.

![](https://static.igem.org/mediawiki/2021/4/4a/T--Hong_Kong_HKU--cscABK_16.png)

Restriction digestion analysis of standard Biobrick assembly: lanes 1-8 show an E/P restriction digestion of a cscA and B+K plasmid. While the expected size should be 2 and 4.5 kb, all lanes except for lane 6 show bands of ~2.5 kb and ~4 kb. **Lane 6 shows bands that are closest to the expected sizes, indicating a correct clone and thus a complete BL21 sucrose plasmid.**

Cyanobacteria Growth

While PCC 7942 grows relatively slowly, we have acquired UTEX 2973 and plotted its growth in a 30°C illuminated shaking incubator. The optical density (O.D.) is measured daily, and its log(OD) value is plotted against time.

![](https://static.igem.org/mediawiki/2021/thumb/e/e7/T--Hong_Kong_HKU--curve.png/1200px-T--Hong_Kong_HKU--curve.png)
While data is limited and we did not observe clear lag/log/stationary stages, it shows healthy growth and is a positive test for our protocols.

![](https://static.igem.org/mediawiki/2021/thumb/a/a9/T--Hong_Kong_HKU--setup.png/1200px-T--Hong_Kong_HKU--setup.png)

# General Troubleshooting

While cloning is successful, our team encountered a number of caveats along the way, one of which is a constant and persistent RFP contamination. We tried our best to maintain lab hygiene and obtain clean backbones by repeating gel purification processes, but most of the time we were met with the surprising contamination of red cultures and colonies.

![](https://static.igem.org/mediawiki/2021/thumb/1/13/T--Hong_Kong_HKU--rfp.png/1200px-T--Hong_Kong_HKU--rfp.png)
![](https://static.igem.org/mediawiki/2021/thumb/1/1d/T--Hong_Kong_HKU--red_plates.png/1200px-T--Hong_Kong_HKU--red_plates.png)

Moreover, we would often get yeast colonies on our bacterial plates. Yeasts generally grow slower than *E. coli*, but an easier differentiation is that yeasts normally appear more domed, white, and opaque than *E. coli*, as seen on the image on the right-most.

![](https://static.igem.org/mediawiki/2021/thumb/9/99/T--Hong_Kong_HKU--yeast.png/1200px-T--Hong_Kong_HKU--yeast.png)

We speculated that the competent cells, incubator, or lab environment was/were contaminated with yeast spores. Working without a bunsen burner until August, transformation used to be done with a simple alcohol lamp. Moreover, with a lack of working fridges and freezers, we were required to transport of reagents from different labs, which increased chances of contamination. Plates were kept at a fridge, but it might not be cold enough to ensure the antibiotics' integrity on agar plates. These along with limited access to facilities such as Nanodrop, ice machine, a -80C fridge for competent cells), or stable access to shaking incubator (with light) led to our long troubleshooting and iteration cycles.

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HKU Evolve 2021 aims to engineer a clean pathway to degrade PET plastics.

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