Team:Lethbridge/Results
Overview
Provided simplified protocols and learning tools for the education system in collaboration with the Lethbridge High School Team
Addressed policy issues regarding cell-free systems
Assessed the biosecurity implications of our project
Developed and tested software tools for biocontainment and to mitigate dual-use
Improved 4 parts by optimizing coding sequences for optimal expression in E. coli and for simple purification
Demonstrated proof of concept for multi-protein purification to simplify the system
17 parts characterized and documented
Worked closely with user groups to inform our design
Assisted Lethbridge High School with wet lab work and received help with education interviews
Collaborated with Florida State University (add details)
Team is registered!
Project is showcased on wiki
Project attributions clearly detailed
Safety forms submitted
Judging form completed
Parts documented on the registry
9 parts submitted
Participated in the InterLab study
| Provided simplified protocols and learning tools for the education system in collaboration with the Lethbridge High School Team Addressed policy issues regarding cell-free systems Assessed the biosecurity implications of our project Developed and tested software tools for biocontainment and to mitigate dual-use Improved X parts by optimizing coding sequences for optimal expression in E. coli and for simple purification Demonstrated proof of concept for multi-protein purification to simplify the system |
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| Team is registered | X parts characterized and documented | Provided simplified protocols and learning tools for the education system in collaboration with the Lethbridge High School Team |
| Project is showcased on wiki | Worked closely with user groups to inform our design | Addressed policy issues regarding cell-free systems |
| Project attributions clearly detailed | Assisted Lethbridge High School with wet lab work and received help with education interviews | Assessed the biosecurity implications of our project |
| Safety forms submitted | Collaborated with Florida State University (add details) | Developed and tested software tools for biocontainment and to mitigate dual-use |
| Judging form completed | Improved 4 parts by optimizing coding sequences for optimal expression in E. coli and for simple purification | |
| Parts documented on the registry | Demonstrated proof of concept for multi-protein purification to simplify the system | |
| 9 parts submitted | ||
| Participated in the InterLab study |
We have provided a collection of parts for cell-free protein expression
Integrated Human Practices - xxxx
Public Engagement and Education - xxxx
Software - xxx
Proteins
Test Overexpressions
We conducted test overexpressions of our constructs in BL21 DE3 Gold, an E. coli strain capable of overexpressing T7 RNA polymerase by induction with IPTG. All overexpression characterization is documented on each individual parts page.
In total, we successfully overexpressed:
| Part | BioBrick |
|---|---|
| ArgRS | BBa_K2443001 |
| AspRS | BBa_K2443003 |
| GlyRSα | BBa_K2443007 |
| GlyRSβ | BBa_K2443008 |
| HisRS | BBa_K2443009 |
| MetRS | BBa_K2443013 |
| PheRSα | BBa_K2443014 |
| SerRS | BBa_K2443017 |
| TrpRS | BBa_K2443019 |
| MTF | BBa_K2443022 |
| EF-Tu | BBa_K2443027 |
| EF-Ts | BBa_K2443028 |
| RF3 | BBa_K2443031 |
| RRF | BBa_K2443031 | MK | BBa_K2443033 | CK | BBa_K2443034 | T7 RNA Polymerase | BBa_K2443037 |
As an example, Figure 1 showcases the overexpression of four individual TX-TL proteins.
Figure 1:Test over-expressions of proteins in E. coli BL21 GOLD (DE3) visualized on a 12% SDS-PAGE for 80 min at 200 V. Lanes are as follows: 1- Protein ladder; 2- HisRS; 3- HisRS Induced; 4- TrpRS; 5- TrpRS Induced; 6- RF3; 7- RF3 Induced; 8- RRF; 9- RRF Induced
Multi-Protein Purification
tRNA
tRNA Purification
Validation Construct
In vitro Transcription
To confirm our ability to detect successful transcription, the spinach aptamer was in vitro transcribed using T7 RNA polymerase (previously purified) and purified by phenol chloroform extraction. Following addition of the fluorophore, DFHBI, fluorescence was measured using a fluorimeter. The fluorimeter data illustrates that fluorescence was observed following addition of DFHBI, with an excitation of 447nm and emission of 497nm.
PURExpress
Statistics
