Team:UPMC PARIS/Experiments
UPMC PARIS
Experiments
Part IV - Determination of the best purification system
Introduction
The goal is to test and compare the Gluthation purification system and the hisTrap one to systems to see which one would be the most effective for our box. To do so, we have purified GST-10XHIS proteins with both of the purification system. We have determined the GST enzymatic activity before and after the purifications to obtain the purification yields. Finally, we have done SDS-PAGE chromatographies to semi-quantify the proteins and compare the results with those obtained with the enzymatic activity.
Results
We cultured DH5α containing plasmid pet21 GST-10XHIS. We measured the OD every hour in order to follow the turbidity and thus determine the time corresponding to the exponential phase of growth(fig.11).
Figure.11: Growth curve of the DH5α containing the PET 21 GST-10HIS
All the points data come from a mean done by data collected from 5 cultures. Cultures were incubated in LB at37°C. The DO(600nm) was calculated every hour.
Analysis of GST enzymatic activity:
We calculated the GST activity of the sample taken before and after the purification. We took 5µL of the aliquotes to do the reaction. We then calculated the total reaction for the all volume before and after lysis that can be found in table.1.
The table.2 presents the the activities and purification yields obtained for both of the purification.
For the HisTrap column, we observe a purification yield equal to 72.1 %.
For the Gluthation Agarose Resin we observe a purification yield equal to 32%.
| Fraction | Enzymatic activity From 5 µL (Δabs/min) | Total enzymatic activity (Δabs/min) | Purification Yield |
|---|---|---|---|
| F1 HisTrap column | 0.13 | 208 | 72.1% |
| F2 HisTrap column | 0.15 | 150 | |
| F1 Gluthation Agarose Resin | 0.75 | 120 | 32% |
| F2 Gluthation Agarose Resin | 0.77 | 38.5 |
Collaboration with Team Harvard
Harvard team had the idea to gather ethical, academic and political issues concerning projects of 6 teams: Harvard, Linkoping, AQA_Unesp, Aalto-Helsinki, Hamburg and ours. Indeed all the teams’ projects have relevance to biomanufacturing and to creating biologically synthesized useful substances at a commercial scale. Thus, gathering perspectives from people involved in these fields will enable us to better understand our subject as it is complex and controversial.
To do so, they created a document where the 5 teams could add their questions, complete others issues and had their opinion. From all these questions, they created a Global Perspective Outreach survey that we shared on our Facebook page. Finally, we were sent the collected data which we used for our Human Practices.
Global Perspective Outreach Question Sheet:
https://docs.google.com/document/d/1rtxv2tz-lPitS1TWYdmaYDiCWiK-a4v5lEd77xOydEA/edit?usp=sharing
Global Perspective Outreach Survey:
https://docs.google.com/forms/d/1hdlfDTzIzjLxdI5Obn35zn1U2sPNzRSJan_CCWNGmao/edit?usp=sharing
Collected Data:
https://docs.google.com/spreadsheets/d/1cTS0aEbsyruC1wJYp0EqW3nwyheSbChfm_HPTcNLbQY/edit?usp=sharing
Collaboration with Team Dusseldorf-Köln
Both our teams met at the meet-up organized by Delft team in Delft in The Netherland. We talked together about our projects and realized they had something in common: they both include an optogenetic system. After that we kept being in touch through e-mails and skype sessions.
As our project went on, we had a hard time finding a way to emit the specific wave length we needed. Therefore, we asked Düsselforf-Köhln team for help and asked them how they managed to do it. They generously gave us the schematics for 3D-printing their lightning box so that we could 3D print our lightning system.
