Team:NU Kazakhstan/Contribution
Background
Parts contribution
Basic parts
pRGPDuo2 plasmid provided and engineered by Rahul Gauttam, Aindrila Mukhopadhyay, and Steven W. Singer (2020). The plasmid was sent to us by Rahul Gauttam who is currently working as a postdoctoral fellow at Berkeley Lab (BBa_K4083000).
Moreover, our team also added three new basic parts into the Registry. There was no nadE synthetase gene from P. aeruginosa that was added to Registry.
Composite part
We created composite part where pRGPDuo2 plasmid was incorporated with nadE, rhlA, rhlB genes into it. Firstly, we inserted the nadE gene into the pRGPDuo2 plasmid for overexpression in P. aeruginosa to compare the yield of rhamnolipids with the wild type of P. aeruginosa. Secondly, we inserted rhlA, rhlB, nadE genes into the pRGPDuo2 plasmid and introduced it into P. putida for rhamnolipids production under electrofermentative conditions. (BBa_K4083008).
Contribution to parts of other iGEM teams
Apart from adding new parts into Registry, we also considered to make a informational contribution to some parts pages:
Informational contribution
Documentation and Troubleshooting
We adjusted and optimized conditions and parameters for PCR amplification to obtain nadE, rhlA, and rhlB genes from Pseudomonas aeruginosa. These results were obtained after numerous trials with different conditions and polymerases. We used Q5 high fidelity DNA polymerase from NEB. It is important to adjust conditions based on primers sequence, however, the following data can help other teams to obtain genes.
Preparation of PCR samples:
| Composition | Volume, ul |
|---|---|
| Q5 buffer | 5 |
| Q5 enhancer | 5 |
| 10 mM DNTP mix | 1 |
| 10 mM forward primer | 1.5 |
| 10 mM reverse primer | 1.5 |
| Q5 polymerase | 0.25 |
| Genomic DNA (75 ng) | - |
| Nuclease free water | Up to 25 |
PCR conditions for rhlB:
| Steps | t (°C) | time | |
|---|---|---|---|
| Initial Denaturation | 98 | 3 min | |
| Denaturation | 98 | 10 sec | >35 cycles |
| Annealing | 72 | 30 sec | |
| Elongation | 72 | 93 sec | |
| Final elongation | 72 | 5 min | |
| Hold | 4 |
PCR conditions for nadE and rhlA:
| Steps | t (°C) | time | |
|---|---|---|---|
| Initial Denaturation | 98 | 3 min | |
| Denaturation | 98 | 10 sec | >35 cycles |
| Annealing | 72 | 30 sec | |
| Elongation | 72 | 40 sec | |
| Final elongation | 72 | 2 min | |
| Hold | 4 |
We developed a new protocol for insertion of plasmid into Pseudomonas putida based on our observations, combining previous papers, and by switching different parameters and conditions.
While treatment of bacteria with CaCl2 is not performed before electroporation, we decided to do so because we were unable to conduct transformation of P. putida solely via electroporation.
Part 1. Preparation of competent cells
- Transfer one colony of P. putida from plate to 30 ml LB broth and leave in the +30 shaking incubator overnight
- Measure Optical density (OD) 600 nm. Preferable to use bacteria with OD of 1.0
- Pour 5 ml of bacterial culture into pre-chilled 15ml centrifuge tube
- Centrifuge at 4700 rpm at 4 °C, for 10 min
- Discard the supernatant
- Resuspend the pellet in 5 ml of 0.1M CaCl2 solution
- Put on ice for 30 minutes
- Centrifuge at 4700 rpm at 4 °C, for 10 min. Discard the supernatant
- Resuspend the pellet in 5 ml of 0.1M CaCl2 20% glycerol solution
- Put on ice for 10 minutes
- Centrifuge at 4700 rpm at 4 °C, for 10 min. Discard the supernatant
- Resuspend the pellet in 1 ml of sterile, ice-cold DI water
- Centrifuge at 4700 rpm at 4 °C, for 10 min to remove residues of LB broth, CaCl2 solutions. Discard the supernatant
- Resuspend the pellet in 50 µL of ice-cold DI water
Part 2. Electroporation of P. putida
- Measure concentration of plasmids with NanoDrop. Add 500 ng of plasmid DNA into competent cells. Mix gently
- Transfer bacteria to chilled electroporation cuvette
- Cap the cuvette and tap it lightly on the bench to settle the bacteria/ DNA mix.
- Put the cuvette back on ice and carry it to the electroporator.
- Turn on the electroporator and set it to 2.5 kV, 25 mF, 200 Ω. This is a standard setting for most P. putida strains. Other bacterial strains may require an adjustment of the electroporation conditions.
- Wipe the cuvette briefly with a Kimwipe to remove any residual water or ice, and then place it in the electroporation chamber.
- Push the pulse button. The time constant displayed should be around 53 ms.
- Immediately after the pulse has been delivered, add 1 ml of LB to the cuvette and pipette quickly but gently up and down. Avoid introducing air bubbles.
- Transfer the mixture to a fresh 1.5-ml microcentrifuge tube.
- Incubate at 30 °C for 1 h with shaking.
- Evenly spread 100 µL of your transformation onto a selective plate. Perform positive and negative control
- Incubate plates upside down overnight at 30 °C
Guidebooks
The pandemic situation that continued in 2021 significantly affected the work processes of iGEM teams and fostered us to look for new ways to integrate our projects. Online format prevalence of the activities opened the door for more cooperation. We believe that by sharing our experience, we can help other iGEM teams in their endeavors. We are happy to have an opportunity to spread knowledge. And getting relevant knowledge and guidance from other iGEM teams is equally important. Creating such co-guiding opportunities helps to go beyond the project and find new development opportunities. In the frames of our sharing experience activities, we have created two guidebooks. First one is about creating Summer Camp, and second guidebook is about how to make iGEM teams more inclusive.
Software contribution
Our team organized the first Biohackathon called “Code-On” in Central Asia. We developed tasks to allow them to solve them to help our project. Two of the tasks had great solutions which we want to use as a contribution to other iGEM teams.
MakeItEasy
As our team focused on considering the needs of visually impaired people, we developed a task to create a library or extension to modify websites for those people. One team created programming code for this purpose which can be used in IGEM Wiki pages. This code is written in JavaScript. It has 4 functions: changing font sizes, choosing color styles, removing images from the site, and enabling the text-to-speech feature. The choosing color style is made specifically for colorblind people. Moreover, this code can be easily implemented to the wiki page: button and script lines should be imported.
We hope that this code can be used by all other IGEM teams in the future to make information wiki more accessible.
EasyParts
Our team manually removed illegal restriction sites according to Biobricks RFC[10] and Type IIS standards. However, it created some difficulties in coding regions as it required not to alter the amino acid sequence. Therefore, our team developed this as a task for Biohackathon, and one of the teams was able to solve it better. Their program is Python-based, and it allows users to make silent mutations on the coding sites without altering the amino acid sequence. This program has a comfortable and accessible interface. Users just need to insert the genetic code of the coding region in the form of text or FASTA. Moreover, since some organisms may have different codons for different organisms, they implemented a library into this program where users can choose the type of organism they are using.
We believe that this program can help other IGEM teams to remove some restriction sites more comfortably.
Reference List:
This protocol was created by adjusting the following papers:
Lessard, J. (2013). Transformation of E.coli via electroporation. Methods in enzymology, 529, 321-327. https://doi.org/10.1016/B978-0-12-418687-3.00027-6
Soltabayeva, A. (2020). Competent cell preparation protocol
Gauttam, R., Mukhopadhyay, A., & Singer, S. (2020). Construction of a novel dual-inducible duet-expression system for gene (over)expression in Pseudomonas putida. Plasmid, 110, p.2. https://doi.org/10.1016/j.plasmid.2020.102514
CONTACT
igem@nu.edu.kz
Kabanbay batyr av., 53, Nur-Sultan, Kazakhstan
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