Difference between revisions of "Team:UCLA/Notebook/Interlab Study"
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==<u> Protocols </u>== | ==<u> Protocols </u>== | ||
| − | The following are a list of protocols designed | + | The following are a list of protocols designed for the InterLab Study. |
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| + | * Transformation of NEB Electrocompetent 5-alpha E. coli using the Bio-Rad Micropulser (Transformations) | ||
| + | * Rapid isolation of plasmid DNA (pSB1A2, pSB1C3) using the Promega PureYield MiniPrep System (Miniprep) | ||
| + | * Double digestion of plasmid DNA using XbaI and PstI restriction exonucleases (XbaI/PstI Digest) | ||
| + | * Double digestion of plasmid DNA using SpeI and PstI restriction exunucleases (SpeI/PstI Digest) | ||
| + | * Rapid ligation and subcloning of Standard Assembly 10 DNA digests using T7 ligase (T7 Ligation) | ||
| + | * QiaQuick gel extraction and purification of plasmid/digested DNA following gel electrophoresis (Gel Extraction) | ||
==<u> Where to go from here </u>== | ==<u> Where to go from here </u>== | ||
Revision as of 00:59, 6 July 2015
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) | BBa_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.
Protocols
The following are a list of protocols designed for the InterLab Study.
- Transformation of NEB Electrocompetent 5-alpha E. coli using the Bio-Rad Micropulser (Transformations)
- Rapid isolation of plasmid DNA (pSB1A2, pSB1C3) using the Promega PureYield MiniPrep System (Miniprep)
- Double digestion of plasmid DNA using XbaI and PstI restriction exonucleases (XbaI/PstI Digest)
- Double digestion of plasmid DNA using SpeI and PstI restriction exunucleases (SpeI/PstI Digest)
- Rapid ligation and subcloning of Standard Assembly 10 DNA digests using T7 ligase (T7 Ligation)
- QiaQuick gel extraction and purification of plasmid/digested DNA following gel electrophoresis (Gel Extraction)
Where to go from here
The following is the process for all 4 device parts (promoter and GFP generator) and the 2 controls (Promoter MeasKit and pTetR empty).
1. Resuspend dried plasmid in each of the registry wells in 10uL ddH2O.Refer here for details.
2.Transform plasmids using the standard NEB Electroporation method.5/6 transformations completed, refer HERE.
- 3. Pick transformants and prepare cultures for miniprep and glycerol stocking.
Following is for preparation of the 3 devices using the 4 device parts from the registry.
- 4. Digest miniprepped promoter plasmids with SpeI and PstI, and digest miniprepped GFP generator using XbaI and PstI.
- 5. Run digests on gels and gel purify/DNA clean and concentrate all digested pieces.
- 6. Ligate digested pieces for final device construction, and transform using electroporation.
- 7. Pick transformants and prepare cultures for miniprep and glycerol stocking.
- 8. Digest miniprepped devices using EcoRI and PstI and run on a gel to verify size of the insert.
- 9. Sequence the 3 devices and compared alignments for predicted sequences to verify proper device implementation.
Following is the plan for fluorescence measurement and data analysis of the three devices, using + and - controls for baseline measurement. [TBD]
Raw lab notebook entries
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