New Parts:
A total of 9 Basic parts have been added to the registry. 5 Composite parts have been designed based on multiple combinations of the new basic parts and the previously used parts. The new basis parts that were designed are compatible with the RCF1000 assembly strategy making them suitable for Golden Gate and MoClo assembly techniques.
New Basic Parts
| Biobrick ID | Part Type | Length(bp) | Description |
|---|---|---|---|
| http://parts.igem.org/Part:BBa_K4836000 | CDS | 1497 | CUW_0748: Bacterial analogue of Human Serotonin Transporter |
| http://parts.igem.org/Part:BBa_K4836003 | CDS | 623 | The Coding sequence for Serotonin N-Acetyl Transferase (SNAT) |
| http://parts.igem.org/Part:BBa_K4836004 | CDS | 1094 | The coding sequence for Caffeic acid O-Methyl Transferase (COMT) |
| http://parts.igem.org/Part:BBa_K4836005 | CDS | 711 | mCherry Reporter CDS |
| http://parts.igem.org/Part:BBa_K4836006 | CDS | 702 | TagBFP CDS |
| http://parts.igem.org/Part:BBa_K4836008 | CDS | 720 | LasR Transcription Activator CDS |
| http://parts.igem.org/Part:BBa_K4836009 | Promoter | 131 | pLasI inducible promoter |
| http://parts.igem.org/Part:BBa_K4836010 | CDS | 1320 | Serotonin N-Acetyl Transferase (SNAT)+ TagBFP fusion protein |
| http://parts.igem.org/Part:BBa_K4836011 | CDS | 1794 | Caffeic acid O-Methyl Transferase (COMT)+ mCherry fusion protein |
New Composite parts
| Biobrick ID | Length(bp) | Description |
|---|---|---|
| http://parts.igem.org/Part:BBa_K4836007 | 897 | mCherry under pLasI Inducibe promoter |
| http://parts.igem.org/Part:BBa_K4836012 | 3274 | SNAT and COMT expression cassette with Fluorescent reporter proteins |
| http://parts.igem.org/Part:BBa_K4836015 | 146 | The composite part consists of Inducible promoter pLasI and RBS |
LasR Mutants
The quorum-sensing molecule employed in the probiotic system is very sensitive to serotonin by the standards of serotonin levels in the gut lumen. The LasR variant activates plasI promoter at around , while lumen serotonin levels might vary anywhere between 10uM to 100uM. Thus, a less sensitive lasR variant would be a great addition to the project. To achieve this goal, we did a mutational impact analysis on contact residues of Serotonin in the LasR-Serotonin complex.
The “wild type” LasR molecule and serotonin were set for docking. 16 contact residues of Serotonin with LasR were identified. They are
LEU36 TYR56 TRP60 ARG61 TYR64 ASP73 THR75 VAL76 TRP88 TYR93 PHE101 ALA105 LEU110 VAL111 THR115 SER129
Next, point mutations were performed in Contact Residues. The respective amino acids (for eg. L in the case of LEU36) were replaced with Alanine (A). Alanine is the common substitute residue for mutation studies as it lacks a functional side chain.
Each of the 16 mutants was docked to Serotonin, and their respective Vina scores are as follows
| Mutant | Vina Score | exp(Vina Score) |
|---|---|---|
| wild-type | -7.2 | 0.00074659 |
| LEU36 | -7.1 | 0.0008251 |
| TYR56 | -6.7 | 0.00123091 |
| TRP60 | -7.2 | 0.00074659 |
| ARG61 | -7.3 | 0.00067554 |
| TYR64 | -6.9 | 0.00100779 |
| ASP73 | -7.4 | 0.00061125 |
| THR75 | -7.2 | 0.00074659 |
| VAL76 | -7.3 | 0.00067554 |
| TRP88 | -7.4 | 0.00061125 |
| TYR93 | -7.4 | 0.00061125 |
| PHE101 | -7.1 | 0.0008251 |
| ALA105 | -7.2 | 0.00074659 |
| LEU110 | -7.3 | 0.00067554 |
| VAL111 | -7.4 | 0.00061125 |
| THR115 | -7.2 | 0.00074659 |
| SER129 | -7.2 | 0.00074659 |
Mutants vs exp(Vina Score) is as follows.
_vs.Mutants(1).png)
The results show that when certain residues in the LasR protein are mutated from Tyrosine (Y) to Alanine (A), there is a significant decrease in binding affinity with serotonin. These residues play a crucial role in the binding of LasR to serotonin, likely due to their aromatic nature.
For more details on how there these mutations were performed, visit Model.
Synbio Game
We have developed the “Synbio Spark” educational game to educate students under the age of 10 about the concepts of genetics, molecular biology, and biotechnology. This app was developed in Collaboration with the Indian Institute of Technology Tirupati (IIT Tirupati). This app's fun interactive games effectively introduce synthetic biology concepts to school students.
For more details about the games in the app, visit Synbio Game.
Posters:
Poster for the Synbio Spark event:
We made a poster for the Synbio Spark event and shared it with school students. The first part of the poster explains cool things about Genetic Engineering and how it's used in different ways. The second part lists websites and tools online where students can learn more about synthetic biology. The poster is meant to encourage students to do self-learning and individual research.
Pamphlet distributed at Regional Science Centre, Tirupati.
We made a pamphlet for people visiting the Regional Science Centre in Tirupati. The pamphlet provides information about Irritable Bowel Syndrome (IBS), including what causes it, its symptoms, and how to prevent it. The pamphlet's purpose is to raise awareness about IBS among the public.
