Keithaiken (Talk | contribs) |
Keithaiken (Talk | contribs) |
||
Line 230: | Line 230: | ||
<center> | <center> | ||
<div class="segmentDiv"> | <div class="segmentDiv"> | ||
− | <div="contentDiv" | + | <div="contentDiv" id="text50"><p class="segment1"> content content content content content content content content content content content content content content content content content content content content content content content content content content</p></div> |
− | <div="contentDiv" | + | <div="contentDiv" id="image65"><img src="https://static.igem.org/mediawiki/2017/thumb/5/5f/Lethbridge_multioverexpression.tiff/800px-Lethbridge_multioverexpression.tiff.png" style="padding: 30px" width=487px height=362px /></div> |
</div> | </div> | ||
</center> | </center> |
Revision as of 23:39, 28 October 2017
Overview
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 X 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 | |
X (list #) parts submitted | ||
Participated in the InterLab study |
Part Collection - xxxx
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 |
As an example, Figure 1 showcases the overexpression of four individual TX-TL proteins.
content content content content content content content content content content content content content content content content content content content content content content content content content content
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.