Team:UCLA/Notebook/Interlab Study
Contents
The 2015 UCLA iGEM Interlab/Measurements Study Notebook
Introduction
The 2015 UCLA iGEM Team is proud to participate in the Second International InterLab Measurement Study in synthetic biology. As members of the synthetic biology community, we are committed to providing robust data for development of novel characterization methods in the rapidly growing biological design fields of synthetic biology.
The purpose of the 2015 InterLab study is to "measure and characterize fluorescence data for three specific genetic devices" expressing GFPmut3b (SwissProt: P42212) from active iGEM teams participating around the world. By collecting fluorescence data from multiple teams in absolute units, variability in measurement and consistency of data collected from instrumentation following a uniform procedure can be determined within a high degree of accuracy.
This notebook will record all protocols, daily experiments, basic parameters and images, as well as the raw data used to prepare the Interlab Worksheet, Protocol, and Wiki page for submission at the 2015 Giant Jamboree.
Experimental Design
Three separate genetic "devices" were constructed using Biobricks Standard Assembly 10, in addition to positive control BBa_I20270 (Constitutive Family Promoter J23151 inserted upstream of the promoter MeasKit) and negative control BBa_R0040 (pTetR - empty control plasmid). All devices were subcloned in the standard pSB1C3 (Chloramphenicol resistant) backbone and transformed into NEB 5-alpha Electrocompetent Escherichia coli (C2989K#). As such, E. coli K-12 DH5-alpha laboratory strains were used as the chassis for fluorescent measurement. Details as to the location of the registry pieces used to construct the devices are below:
| Device # | Promoter | Registry Location | Backbone | GFP Generator | Registry Location | Backbone | Final Device Backbone |
|---|---|---|---|---|---|---|---|
| Device #1 | BBa_J23101 | 2015 1-20K | pSB1C3 | BBa_I3504
(B0034-E0040-B0015) |
2015 4-21J | pSB1A2 | pSB1C3 |
| Device #2 | BBa_J23106 | 2015 1-22A | pSB1C3 | BBa_I3504
(B0034-E0040-B0015) |
2015 4-21J | pSB1A2 | pSB1C3 |
| Device #3 | BBa_J23117 | 2015 1-22K | pSB1C3 | BBa_I3504
(B0034-E0040-B0015) |
2015 4-21J | pSB1A2 | pSB1C3 |
| Postive Control (BBa_I20270) | J23151 | 2015 3-8P | pSB1C3 | GFPmut3b
(B0032-E0040-B0010-B0012) |
2015 3-8P | pSB1C3 | pSB1C3 |
| Negative Control | TetR repressible promoter (BBa_R0040) | 2015 2-6F | pSB1C3 | N/A | N/A | N/A | pSB1C3 |
All devices constructed using the BioBricks Standard Assembly 10 method used digestion and ligation to assemble the final vector. For Devices 1-3, the GFP generator I13504 was digested using XbaI and PstI restriction endonucleases, and ligated downstream of the constitutive family promoters at vector digested SpeI and PstI sites, generating a BioBricks scar at the SpeI/XbaI ligation junction.
Raw lab notebook entries
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