iGEM 2022 — Team shym-bil-nis

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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 %}
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![](https://static.igem.wiki/teams/4521/wiki/incl-title.svg)
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01.09.2022

Goal: To increase the copy number of pSB1C3 plasmid with IsPETase-W159H-S238F gene.
Experiment: Resuspension of pSB1C3 plasmid with IsPETase-W159H-S238F and C-terminal Hexahistidine Tag. Transformation of competent E. coli DH5α with pSB1C3 plasmid.
Result: We did not observe any E. Coli DH5α colonies on the medium, meaning transofrmation was not successful.

02.09.2022

Goal: To increase the copy number of pSB1C3 plasmid with IsPETase-W159H-S238F gene.
Experiment: Resuspension of pSB1C3 plasmid with IsPETase-W159H-S238F and C-terminal Hexahistidine Tag and transformation of competent E. coli DH5α with pSB1C3 plasmid.
Result: We did not observe any E. Coli DH5α colonies on the medium, meaning transofrmation was not successful.

05.09.2022

Goal:To increase the copy number of pSB1C3 plasmid with IsPETase-W159H-S238F gene by using E. coli BL21 and rosetta (DE3) strains.
Experiment:Perform transformation of competent E. coli BL21 and rosetta (DE3) with pSB1C3 plasmid.
Result: The growth of E. coli rosetta (DE3) was not observed, but E. coli BL21(DE3) showed many colonies, indicating that BL21 strain was transformed.

06.09.2022

Goal:Perform large seed to increase the
Experiment: We selected one bacterial colony and incubated in LB broth medium containing Chloramphenicol overnight.
Result: The E. coli BL21(DE3) bacteria colony with pSB1C3 plasmid appeared on the plate.

07.09.2022

Goal:Extract the pSB1C3 plasmid with the target gene.
Experiment: Miniprep and extraction of the plasmid from E. coli BL21(DE3)
Result: The pSB1C3 plasmid was successfully extracted.

08.09.2022

Goal: Transform competent Pseudomonas putida KT2440 with the pSB1C3 plasmid.
Experiment: Carry out transformation of competent Pseudomonas putida KT2440 with the pSB1C3 plasmid.
Result: The gene with a specific DNA site synthesizing the enzyme IsPETase-W159H-S238F was successfully incorporated into Pseudomonas putida KT2440 bacterium.

![](https://static.igem.wiki/teams/4521/wiki/image-12.svg)
![](https://static.igem.wiki/teams/4521/wiki/image-9.svg)
![](https://static.igem.wiki/teams/4521/wiki/image-7.svg)
![](https://static.igem.wiki/teams/4521/wiki/image-10.svg)

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{% block title %}Engineering{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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### Overview

## Overview

Since our mission is to

create a bacteria

that is

decomposing plastic

, and at the same time adapted to the

harsh
continental climate of Kazakhstan,

we need:

The genes of

Ideonella sakaiensis

bacteria that synthesize the

PETase enzyme.

When we have this gene, we

transform

and

insert this gene into the bacteria

of
the genus Pseudomonas putida, which is able to survive in our climate.

![](https://static.igem.wiki/teams/4521/wiki/eng-1.png)

![](https://static.igem.wiki/teams/4521/wiki/image-10.png)

pSB1C3

is a high copy number plasmid (RFC [10])

carrying chloramphenicol resistance.

The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).

pSB1C3 has

terminators bracketing its MCS

which are designed
to prevent transcription from \*inside\* the MCS from reading out into the vector. The efficiency of
these terminators is known to be < 100%. Ideally we would

construct a future set of terminators

for
bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.

### DesignDesign

![](https://static.igem.wiki/teams/4521/wiki/image-11-1.png)

### Future DesignFuture Design

![](https://static.igem.wiki/teams/4521/wiki/image-31.png)

In the future, our team plans to

continue

working on the project. To do this, we need to carry out a number of mutations on the bacterium P. putida KT2440.
At the moment, the DNA responsible for the synthesis of the PETase enzyme has been

successfully inserted

into the P. putida bacterium, but in the future

we will supplement

it with the genome responsible for the production of the

MHETase enzyme.

Thus, this part was created, which in the future will help us prove our project and create a unique organism with the ability to safely decompose plastic in the southern regions of our country.

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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![](https://static.igem.wiki/teams/4521/wiki/group-77.svg)
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1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part.

2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended.

3. The resuspension will be red, as the dried DNA has cresol red dye.

![](https://static.igem.wiki/teams/4521/wiki/group-107.svg)

1. Prepare the required materials:* 0.25-0.5 g of Chloramphenicol;
* 10 ml of 70% EtOH;

2. Weigh 0.25-0.5 g of Chloramphenicol;

3. Add 10 ml of 70% EtOH;

4. Dissolve completely;

5. Stock may be kept at -20°C for 1 year.

![](https://static.igem.wiki/teams/4521/wiki/group-96.png)

1. Fill the ice bucket with ice, and pre-chill 1.5ml tubes.

2. Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.

3. Pipette 50µl of competent cells into 1.5ml tube: 50µl in a 1.5ml tube per transformation.

4. Pipette 1µl of resuspended DNA into 1.5ml tube. Gently pipette up and down a few times. Keep all tubes on ice.

5. Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.

6. Close 1.5ml tubes, incubate on ice for 30min.

7. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Timing is critical.

8. Incubate on ice for 5min.

9. Pipette 950µl SOC media to each transformation: SOC should be stored at 4°C, but can be warmed to room temperature before use.

10. Incubate at 37°C for 1 hour, shaking at 200-300rpm.

11. Pipette 100µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.

12. Weigh 0.25g of Chloramphenicol and add 10 ml of 70% EtOH. Dissolve completely.

13. Pipette 25 mL of LB broth onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.

14. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up).

![](https://static.igem.wiki/teams/4521/wiki/miniprep.svg)

1. Spin the cell culture in a centrifuge to pellet the cells, empty the supernatant (media) into a waste collection container.

2. Resuspend pelleted bacterial cells in 250 µl Buffer P1 (kept at 4 °C) and transfer to a microcentrifuge tube. No cell clumps should be visible after resuspension of the pellet. Important: Ensure that RNase A has been added to Buffer P1.

3. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix. Do not vortex, as this will result in shearing of genomic DNA. If necessary, continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.

4. Add 350 μl Buffer N3 and invert the tube immediately and gently 4–6 times. To avoid localized precipitation, mix the solution gently but thoroughly, immediately after addition of Buffer N3. The solution should become cloudy.

5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A white pellet will form.

6. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.

7. Centrifuge for 30–60 s. Discard the flow-through.

8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60s.
Spinning for 60 seconds produces good results.

9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.* IMPORTANT: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions. They are right about this.

10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

![](https://static.igem.wiki/teams/4521/wiki/image-12.svg)
![](https://static.igem.wiki/teams/4521/wiki/image-9.svg)
![](https://static.igem.wiki/teams/4521/wiki/image-10.svg)

![](https://static.igem.wiki/teams/4521/wiki/future-experiments.svg)

1. If the protein is expressed

a. Induce the cells containing IsPETase gene controlled by T7 promoter with 0.4M, 0.1M, or 0M IPTG for either 6 hours or overnight, at either 15- or 30-degrees C.

b. Expression of T7 Cells

c. Lysis of cells according to our ultrasonication protocol.

d. Validate protein expression further with SDS Page (An SDS Page will confirm by showing that a protein of the correct weight is being expressed)

e. Agarose gel electrophoresis.

f. Histag purification.

g. Western blotting (A Western blot will show that our histag is functioning);

h. P-nitrophenyl butyrate (pNPB) Assay

2. NO – reperform the transformation.

3. Transformation of MHETase to Pseudomonas putida KT2440 with terminator T7 and promoter J23106.

4. Validate protein expression further with SDS Page.

5. PCR (to show contained in the cells by band size (1813 bp) in electrophoresis gel);

![](https://static.igem.wiki/teams/4521/wiki/results.svg)

Having had restricted access to the lab space and materials, we could not accomplish everything we planned. Despite our setbacks, we made a huge effort to carry out as many experiments as possible as mentioned in our lab notebook.

Our primary goal was to show that P. putida KT2440 could gain a new function by incorporating a foreign gene coding for PETase enzyme. We performed experiments according to the plan, and we were able to show that P. putida KT2440 strain is able to get transformed (Picture 1). The next experimental steps were halted due to timing and lab space constraints, but we developed a plan of future experiments to prove our theory. According to our plan we will continue our work with SDS Page and Western blotting, showing that a protein of the correct weight will be expressed. After we planned to transform MHETase enzyme to Pseudomonas putida KT2440 with terminator T7 and promoter J23106. So, in the end these enzymes, by working in pairs form SUPER ENZYME, which is able to accelerate the decomposition of the plastic process several times and convert it efficiently into its two environmentally benign monomers.

![](https://static.igem.wiki/teams/4521/wiki/image-7.svg)

References:

Biosafety Guidelines for Handling Microorganisms in the Teaching Laboratory: Development and Rationale by: Elizabeth A. B. Emmert and the ASM Task Committee on Laboratory Biosafety Department of Biological Sciences, Salisbury University, Salisbury. JOURNAL OF MICROBIOLOGY & BIOLOGY EDUCATION, May 2013, p. 78-83 Safety

Pseudomonas putida KT2440 (ID 139665) - BioProject - NCBI (nih.gov)

Structural studies reveal the molecular mechanism of PETase - PubMed (nih.gov)

Antibiotic Usage | Herman Lab | Nebraska (unl.edu)

https://www.protocols.io/view/How-to-make-a-25-50-mg-ml-Chloramphenicol-Stock-So-k3byl4rjgo5d/v1

Part:BBa K2010000 - parts.igem.org

Team:Harvard BioDesign/Experiments - 2016.igem.org

Конструирование и клонирование разлагающих пластик рекомбинантных ферментов (MHETase) - PubMed (nih.gov)

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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