Difference between revisions of "Team:Lethbridge/InterLab"
| Line 57: | Line 57: | ||
<tr> | <tr> | ||
<th>Construct</th> | <th>Construct</th> | ||
| + | <th>BioBrick</th> | ||
<th>Promoter</th> | <th>Promoter</th> | ||
<th>RBS</th> | <th>RBS</th> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Positive Control</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | </tr> | ||
| + | <tr> | ||
| + | <td>Negative Control</td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
| + | <td></td> | ||
</tr> | </tr> | ||
<tr> | <tr> | ||
<td>Test Device 1 </td> | <td>Test Device 1 </td> | ||
| + | <td></td> | ||
<td>J23101</td> | <td>J23101</td> | ||
<td>B0034</td> | <td>B0034</td> | ||
| Line 67: | Line 81: | ||
<tr> | <tr> | ||
<td>Test Device 2</td> | <td>Test Device 2</td> | ||
| + | <td></td> | ||
<td>J23106</td> | <td>J23106</td> | ||
<td>B0034</td> | <td>B0034</td> | ||
| Line 72: | Line 87: | ||
<tr> | <tr> | ||
<td>Test Device 3</td> | <td>Test Device 3</td> | ||
| + | <td></td> | ||
<td>J23117</td> | <td>J23117</td> | ||
<td>B0034</td> | <td>B0034</td> | ||
| Line 77: | Line 93: | ||
<tr> | <tr> | ||
<td>Test Device 4</td> | <td>Test Device 4</td> | ||
| + | <td></td> | ||
<td>J23101</td> | <td>J23101</td> | ||
<td>J364100</td> | <td>J364100</td> | ||
| Line 82: | Line 99: | ||
<tr> | <tr> | ||
<td>Test Device 5</td> | <td>Test Device 5</td> | ||
| + | <td></td> | ||
<td>J23106</td> | <td>J23106</td> | ||
<td>J364100</td> | <td>J364100</td> | ||
| Line 87: | Line 105: | ||
<tr> | <tr> | ||
<td>Test Device 6</td> | <td>Test Device 6</td> | ||
| + | <td></td> | ||
<td>J23117</td> | <td>J23117</td> | ||
<td>J364100</td> | <td>J364100</td> | ||
Revision as of 17:18, 28 October 2017
Overview
The ability for parts to provide repeatable outputs is key to engineering applications, including synthetic biology. The purpose of the 2017 interlab study was to address how reproducible and at what levels protein expression could be driven by standardized promoters and ribosomal binding sites. Constructs were provided by the iGEM measurement committee that utilized green fluorescent protein as a reporter. This allowed interlab study contributors to easily follow protein expression driven by the standardized promoters and ribosomal binding sites.
In order to better compare expression levels between research groups, the 2017 interlab study incorporated calibration protocols to allow for reporting of fluorescence levels in absolute levels. Specifically, fluorescence levels from biological samples were calibrated against fluorescence from a fluorescein standard curve, allowing for fluorescence results to be reported in terms of the amount of fluorescence produced by a certain concentration of fluorescein.
Test Devices
The negative control for the interlab study did not contain DNA coding for recombinant fluorescent-protein expression , thus was well-suited for examining background fluorescence of cellular components. The remaining constructs contained GFP (E0040) under the control of various promoters and ribosomal binding sites. The promoters and ribosomal binding sites utilized for Test Devices 1-6 are outlined below. All constructs contained E0040, B0010, and B0012 following the promoter and ribosomal binding site.
| Construct | BioBrick | Promoter | RBS |
|---|---|---|---|
| Positive Control | |||
| Negative Control | |||
| Test Device 1 | J23101 | B0034 | |
| Test Device 2 | J23106 | B0034 | |
| Test Device 3 | J23117 | B0034 | |
| Test Device 4 | J23101 | J364100 | |
| Test Device 5 | J23106 | J364100 | |
| Test Device 6 | J23117 | J364100 |
Methods
All methods were based on the iGEM InterLab 2017 Plate Reader Protocol found here:
https://2017.igem.org/Competition/InterLab_Study/Plate_Reader
Methods: Calibration
The calibration protocol for the OD 600 point was performed, followed by the generation of a
fluorescein fluorescence standard curve. More details of calibration protocols can be found here: https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf
Methods: Day 1
Following the iGEM InterLab protocol, the eight plasmids, Positive Control, Negative Control
and Test Device 1-6, were transformed into E. coli DH5-alpha cells.
Methods: Day 2
Two colonies were picked from each plate and grown overnight in 5 mL of LB +
Chloramphenicol at 37 ºC and 220 rpm.
Methods: Day 3
According to the provided interlab study protocol, OD600 and fluorescence were measured (excitation 485 nm, emission 530 nm) by our instrument at 0, 2, 4 and 6 hours. Full details of the protocol can be found here: https://static.igem.org/mediawiki/2017/8/85/InterLab_2017_Plate_Reader_Protocol.pdf
