Difference between revisions of "Team:Lethbridge/Results"
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Revision as of 06:38, 31 October 2017
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
Part Collection: We have provided a collection of open-source parts for cell-free protein expression
Integrated Human Practices: The design of Next vivo was informed by the needs of three user groups, resulting in the development of an educational tool and an assessment of the biosecurity implications of cell-free systems
Public Engagement and Education: We have developed curriculum aligned lesson plans and a safe learning tool for the education system
Software:
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:
| BioBrick | Protein |
|---|---|
| BBa_K2443001 | ArgRS |
| BBa_K2443003 | AspRS |
| BBa_K2443007 | GlyRSα |
| BBa_K2443008 | GlyRSβ |
| BBa_K2443009 | HisRS |
| BBa_K2443013 | MetRS |
| BBa_K2443014 | PheRSα |
| BBa_K2443017 | SerRS |
| BBa_K2443019 | TrpRS |
| BBa_K2443022 | MTF |
| BBa_K2443027 | EF-Tu |
| BBa_K2443028 | EF-Ts |
| BBa_K2443031 | RF3 |
| BBa_K2443032 | RRF | BBa_K2443033 | MK | BBa_K2443034 | CK | BBa_K2443037 | T7 RNA Polymerase |
As an example, Figure 1 showcases the overexpression of four individual TX-TL proteins.
Figure 1 - Test overexpressions of TX-TL components. Test overexpressions 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
One of the key features of Next vivo is the ability to purify all of the components in a single step purification. As a proof of concept we expressed four of the TX-TL components and co-purified them all using a Nickel Sepharose chromatography column (see figure below). The four proteins used in this initial test were selected based on their molecular weights relative to each other for visualization purposes.
Figure 2 - Representative overexpression and multi-protein purification of TX-TL components. Each TX-TL component was expressed from E. coli cells carrying the plasmid encoding the specified component and samples three hours post induction were collected (Lane 2-5). The expressing cells of each component were pooled and lysed before applying the lysate to a Nickel Sepharose affinity column for isolation of just the hexahistidine tagged TX-TL components. After washing away the unwanted cellular proteins and debris, the TX-TL components were eluted from the Nickel Sepharose to a high level of purity (Lane 6).
tRNA Purification
The biggest issue we initially faced in developing Next vivo was determining how we could purify tRNA quickly and efficiently. The solution we decided upon was an adapted MS2 purification combined with a subsequent incubation with RNase H and a DNA oligo that would selectively cleave and release a tRNA of the proper size. For more information on the design, see the tRNA purification section here. (link)
Both the tRNAPhe-MS2 construct and MS2BP were expressed individually in E. coli BL21 DE3 cells. Upon which time the cells were lysed, the lysate combined, and applied to a nickel Sepharose affinity column. The MS2BP is able to bridge the Nickel Sepharose column and the tRNA-MS2 allowing the tRNA to be isolated from the cell lysate.
Figure 2 - 12% 8M urea PAGE run for 45 mins at 300 V. Lane one and two contain MS2 fraction phenol/chloro extracted and tRNA elution with 25 units RNaseH respectively. Lane three and four contain MS2 fraction phenol/chloro extracted and tRNA elution 50 units RNaseH respectively. Lane five and six contain MS2 fraction phenol/chloro extracted and tRNA elution with 100 units RNaseH respectively. LAnes seven and eight contain MS2 fraction Phenol/chloro extracted and tRNA elution with 10 units RNaseH respectively. Lane nine contains a tRNA standard as a size marker.
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.
Figure 3 -
PURExpress
Text Text Text
Figure 4 -
Statistics
