iGEM 2020 — Team HK_HCY

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# Team:HK HCY/Notebook

# Notebook

|  |  |
| --- | --- |
| **Month** | **Activities** |
| **November** | **Learn how to use pipette and other equipment** |
| **January**  **mainly on 21st** | **First meeting (invite Kenneth to be the advisor)**   **learn the basic concept of synthetic biology and laboratory skills**   **gather some ideas to choose our topic** |
| **Febrary to March** | **the logos were designed and one of them was selected**   **small groups are formed to work at specific tasks**   **the topic was defined (*****GM plant or bacteria which can produce cell-wall-deconstructing enzymes in order to increase the production of biofuel*****)** |
| **April** | **skype meeting with Prof.Tsang**   **google meet with Dr.Heike** |
| **May** | **break down topic to smaller specific questions and identify possible biological pathway(or gene in enzyme)** |
| **June,July** | **Finding papers, eg:the ways and pathways of producing antioxidants, and how to put in E.coil etc .** |
| **Sep** | **Confirm and finalize our topic, meeting with other igem teams** |

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# Team:HK HCY/Engineering

### ★ ALERT!

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# Engineering Success

### Silver Medal Criterion #1

Demonstrate engineering success in at least one aspect of your project. This achievement should be distinct from your Contribution for Bronze.

Please see the [2020 Medals Page](https://2020.igem.org/Judging/Medals) for more information.

If you plan to show engineering success by creating a new Part that has been shown to work as expected, you must document your contribution on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page) for your team to be eligible for this criteria.

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# Team:HK HCY/Experiments

# Experiments

# 1st Approach: inhibit the activity of LuxS AI-2 synthase

### Equipment

* Shaking incubator at 37°C
* Stationary incubator at 37 °C
* Water bath at 42 °C
* Ice bucket filled with ice
* Microcentrifuge tubes
* Sterile spreading device

### Reagents

* LB agar plate (with appropriate antibiotic)
* LB or SOC media
* Competent cells
* Plasmid

### Procedure

1. Take competent cells out of -80°C and thaw on ice (approximately 20-30 mins).
2. Remove [agar plates](https://www.addgene.org/protocols/pouring-lb-agar-plates/) (containing the appropriate [antibiotic](https://www.addgene.org/mol-bio-reference/#antibiotics)) from storage at 4°C and let warm up to room temperature and then (optional) incubate in 37°C incubator.
3. Mix 1 - 5 μl of DNA (usually 10 pg - 100 ng) into 20-50 μL of competent cells in a microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few times.
4. Incubate the competent cell/DNA mixture on ice for 20-30 mins.
5. Heat shock each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 secs (45 secs is usually ideal, but this varies depending on the competent cells you are using).
6. Put the tubes back on ice for 2 min.
7. Add 250-1,000 μl LB or SOC media (without antibiotic) to the bacteria and grow in 37°C shaking incubator for 45 min.
8. Plate some or all of the transformation onto a 10 cm [LB agar plate](https://www.addgene.org/protocols/pouring-lb-agar-plates/) containing the appropriate antibiotic.
9. Incubate plates at 37°C overnight.

### Reference:

<https://www.addgene.org/protocols/bacterial-transformation/>

<https://youtu.be/1PqWnu2uBrY>

# 2nd approach: degrading the signalling molecules extracellularly using the kinase LsrK

### Materials and tools

* agar plate
* sample
* E. coli
* primers from E. coli
* the QIAquick gel extraction kit
* Ampicillin stock at 50 mg/ml
* LB agar (1.5%) plate (Should contain ampicillin at 50 μg/ml)
* LB media (20 ml) (Should be prepared and sterilized in advance. Before bacteria inoculation, ampicillin should be added to final concentration of 50 μg/ml.)
* TB media (1 liter) (Should be prepared and sterilized in advance. Before bacteria inoculation, ampicillin should be added to final concentration of 50 μg/ml.)
* 100 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) stock solution (Should be filtered using 0.2 μm filter)
* 0.1M dithiothreitol (DTT) stock solution (Should be filtered using 0.2 μm filter)
* S30 lysate buffer (1.5 liters) containing:
  1. 10mM Tris-acetate at pH = 7.4
  2. 14mM magnesium acetate
  3. 60 mM potassium acetate
  4. 1 mM DTT 0.5 ml/liter 2-mercaptoethanol
  5. (The S30 lysate buffer was prepared in advance without the addition of DTT and 2-mercaptoethanol, sterilized and stored at 4˚C. Prior to use, DTT and 2-mercaptoethanol were added.)
* Sterilized Erlenmeyer flasks (2 of 100 ml)
* Sterilized Erlenmeyer flasks with baffles (2 of 2 liters)
* Floor incubator shaker
* Centrifuge (Should enable at least 13,000 x g )
* High pressure homogenizer (Should be pre-cooled to 4˚C)
* -80˚C freezer
* Sterilized 1.5-ml plastic tubes
* A spectrophotometer
* Sterilized graduated cylinder
* Sterilized centrifuge tubes
* Sterilized pipette tips
* Cell-free reaction component Final concentration in solution HEPES-KOH (pH = 8) 55 mM
  1. Magnesium acetate 14 mM
  2. Potassium acetate 50 mM
  3. Ammonium acetate 155 mM
  4. Polyethylene glycol (PEG) 3% (v/v) 3-phosphoglycerate (3-PGA) 40 mM
  5. 20 amino acids 2.5 mM
  6. Adenine triphosphate (ATP) 1.2 mM
  7. Guanidine triphosphate (GTP) 1 mM
  8. Uridine triphosphate (UTP) 0.8 mM
  9. S30-T7 lysate 30% (v/v) DNA template 10 μg/mL
  10. DNase, RNase free H2O to reaction volume

### Steps

1. After transforming the plasmid into the e coil, cells were stored at -80 °C, and the enzyme (Mwas expressed and purified.
2. Streak the bacteria on an LB-agar plate.
3. Use a single colony to inoculate 10 ml LB media in 100 mL flask, and grow it overnight at 37˚C with shaking at 250 rpm on a floor incubator shaker. This step will obtain a starter solution (duplicate).
4. Inoculate each one of the 500 ml TB media inside a 2 liters flask using the 10 ml starters.
5. Grow the culture at 37˚C until it reaches OD6001, by monitoring the culture’s OD at 600 nm using a spectrophotometer. Once the required OD is achieved, add 0.4 mM IPTG.
6. Grow the culture at 37˚C until it reaches OD6004.
7. Centrifuge at 7,000 x g for 10 min at 4˚C.
8. Re-suspend each pellet in 500 ml of S30 lysate buffer, and centrifuge at 7,000 x g for 10 min at 4˚C.
9. Resuspend in 15 ml of S30 lysate buffer.
10. Break the cells by one pass through a high pressure homogenizer at a working pressure of 15,000 psi, with an air pressure of 4 bar.
11. Add 100 μL of 0.1 M DTT to each 10 ml of homogenized suspension.
12. Centrifuged the suspension at 24,700 x g or 13,000 x g for 30 min at 4˚C.
13. Divide the supernatant into aliquots of 200 μL into 1.5 ml tubes.
14. Freeze the tubes immediately by liquid nitrogen and store at -80˚C for further use.
15. Prepare the CFPS reaction mixtures according to the Cell-free reaction component list. Make sure to thaw and add the lysate just prior to the incubation and protein production step.
16. Incubate the reaction using a floor incubator shaker at 250 rpm, or a Thermomixer® at 1200 rpm at a constant temperature for 2 hours. When this protocol is first used, it is recommended to incubate the reaction at 37˚C. In addition, perform a parallel reaction without DNA, to obtain a negative control.
17. Evaluate the produced protein amount using a suitable method, according to the target protein properties.
18. Add the cell-free lysate of LsrK into the biofuel sample.
19. Record the results.

### Reference

A Simple and Rapid Method for Preparing a Cell-Free Bacterial Lysate for Protein Synthesis

<https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0165137&type=printable>

Incorporating LsrK AI-2 Quorum Quenching Capability in a Functionalized Biopolymer Capsule

<https://sci-hub.se/10.1002/bit.26397>

Cross Species Quorum Quenching Using a Native AI-2 Processing Enzyme

[https://sci-hub.se/https://pubmed.ncbi.nlm.nih.gov/20025244/](https://sci-hub.se/https%3A//pubmed.ncbi.nlm.nih.gov/20025244/)

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# Team:HK HCY/Results

# Results

### Predicted Result

Our predicted result is that a mass amount of lsrK can be delivered to the bacteria extracellularly, phosphorylating the AI-2 quorum sensing molecules to phospho-AI2. Also, we predict the small short 12-mer peptideT can be penetrated into the cell cytoplasm and hence inhibit the activity of AI-2 synthase, LuxS.

We expect the efficiency to reach up to 55%, which can significantly reduce the formation of biofilm in biofuel to improve the production efficiency. We expect the peptideT to reduce 25% of AI-2 synthesis by inhibiting LuxS and further 40% reduction after AI-2 is phosphorylated. The estimation is conservative as we are not able to access the laboratory.

### Improvement

Previous researches found that a deletion mutant strain ΔluxS and complemented strain C-LuxS participated in regulating and controlling stress resistance, biofilm formation and virulence in H. parasuis, a gram-negative bacterium. ΔluxS strain decreased the production of AI-2 molecules and the tolerance toward oxidative stress and heat shock, and it reduced the abilities of autoagglutination, hemagglutination, and adherence, whereas it increased the abilities to form biofilm in vitro.

Some research also used Streptococcus pneumoniae, which is gram-positive as the bacterium. D39 wild-type and an isogenic D39ΔluxS strain were utilized to evaluate in vitro biofilm formation in the experiment. The results showed that the biofilm biomass and density of D39ΔluxS were significantly lower than those of D39 wild-type. This showed that the S. pneumoniae LuxS/AI-2 quorum-sensing system is necessary for biofilm formation.

As several papers showed that LuxS/AI-2 quorum-sensing system is necessary for the biofilm formation, our team decided to inhibit the system to reduce AI-2 formed which is necessary for the biofilm formation.

From previous studies, we found that LsrK is a kinase which catalyzes the phosphorylation of autoinducer-2, a signaling molecule involved in quorum sensing. It subsequently inactivates the transcriptional regulator LsrR and leads to the transcription of the LsrR operon. As a result, quorum sensing will be inhibited. We also found that peptide T is a putative high-affinity peptide ligand that has been shown to specifically inhibit LuxS.

To test if LsrK and peptide T really works out for inhibiting the LuxS/AI-2 quorum-sensing system, our project aims to reduce biofilm formation of gram-positive bacterium L. Fermentum 2-1 by inhibiting its quorum sensing mechanism, which is to lessen the communication between the bacteria.

In one way, the first approach is to inhibit the signal molecule synthesis, which is the activity of LuxS(Al-2 synthase), using Peptide T. Peptide T will be put into the intracellular LuxS protein. Since we are uncertain about the ways for transferring peptide T into LuxS, we will perform experiments to find out if the peptide is small enough to diffuse over the cell membrane. If not, a signalling peptide may be needed. The inhibition of Al-2 biosynthesis can lead to the disruption of bacterial quorum sensing.

On the other hand, the second approach is to inhibit the signaling molecules itself, which is to degrade Al-2 molecules extracellularly using kinase LsrK. By adding LsrK into the bacterial culture, in which AI-2 is phosphorylated outside the cell, the bacterial cross talk controlled by AI-2 is then significantly reduced. By doing this, Lsrk is first expressed from E.coli to extract it’s cell free lysate, then it will be put into biofuel. Lactobacillus promoter and T7 promoter, Lactobacillus promoter and Lactobacillus opera on in a plasmid is inserted into the plasmid.

By inhibiting the activity of LuxS and degrading Al-2 molecules, the Al-2 concentration is greatly decreased. By lessening the amount of quorum sensing molecules AI-2, it interferes with the quorum sensing mechanism, hence reducing the biofilm formation.

Although peptide T was shown to specifically inhibit LuxS, the inhibition was only partial, which is only 25%. It does not completely bind to either the substrate-binding site or the metal-binding site. Moreover, The synthetic peptide T is linear and does not mimic the native conformation of the respective protein, which decreases the efficiency of inhibition. Furthermore, the LuxS expressed by E. coli lacks the native conformation normally conferred by post-translational modification; thus, the native conformation of the substrate- and metal-binding sites may have been altered, such that an effective inhibitor could not be tested.

To solve the above problem, we have thought of cyclizing the peptide T and methylating select amide bond nitrogens. These methods are proposed to increase membrane permeability and bioavailability. Furthermore, if peptide T is cyclized, it may be easier to bind either the substrate-binding site or the metal-binding site, so as to increase the inhibition. It will be more likely to mimic the native conformation of the protein.