Difference between revisions of "Team:UCLA/Notebook/Interlab Study"

(Introduction)
(Where to go from here)
 
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==<u>Introduction</u>==
 
==<u>Introduction</u>==
The 2015 UCLA iGEM Team is proud to participate in the [[https://2015.igem.org/Tracks/Measurement/Interlab_study|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 2015 UCLA iGEM Team is proud to participate in the [https://2015.igem.org/Tracks/Measurement/Interlab_study 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.   
+
The purpose of the 2015 InterLab study is to "measure and characterize fluorescence data for three specific genetic devices" expressing [http://www.uniprot.org/uniprot/P42212 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.
 
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.
Line 13: Line 13:
 
==<u>Experimental Design</u>==
 
==<u>Experimental Design</u>==
  
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 <i>Escherichia coli </i> (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:
+
Three separate genetic "devices" were constructed using IDT gBlocks Gene Fragments synthesis, in addition to positive control [http://parts.igem.org/Part:BBa_I20270 BBa_I20270] (Constitutive Family Promoter [http://parts.igem.org/wiki/index.php?title=Part:BBa_J23151 J23151] inserted upstream of the promoter MeasKit) and negative control [http://parts.igem.org/Part:BBa_R0040 BBa_R0040] (pTetR - empty control plasmid). All gBlocks were designed on Benching to simulate the sequence of the BioBricks standard assembly product, for uniformity in measurement with teams that opted for RFC10 standard assembly.  All devices were subcloned in the standard [http://parts.igem.org/Part:pSB1C3 pSB1C3] (chloramphenicol resistance marker) backbone and transformed into BL21(DE3) <i>Escherichia coli </i>. As such, E. coli BL21(DE3) 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:
  
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
 
! Device #
 
! Device #
 +
! Benchling Link
 +
! Spec Sheet & FASTA File
 
! Promoter
 
! Promoter
! Registry Location
 
! Backbone
 
 
! GFP Generator
 
! GFP Generator
! Registry Location
 
! Backbone
 
 
! Final Device Backbone
 
! Final Device Backbone
 
|-
 
|-
 
| Device #1
 
| Device #1
| BBa_J23101
+
| https://benchling.com/s/EnB0uD9g/edit
| 2015 1-20K
+
| [https://drive.google.com/open?id=0BztPXrXqzHY8WngyWHNwUTdIRmc Specs] and [https://drive.google.com/open?id=0BztPXrXqzHY8cWJYU3FRU0Fkb2c FASTA]
| pSB1C3
+
| [http://parts.igem.org/Part:BBa_J23101 BBa_J23101]
| BBa_I3504  
+
| [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]
 
(B0034-E0040-B0015)
 
(B0034-E0040-B0015)
| 2015 4-21J
 
| pSB1A2
 
 
|pSB1C3
 
|pSB1C3
 
|-
 
|-
 
| Device #2
 
| Device #2
| BBa_J23106
+
| https://benchling.com/s/hX68sjpy/edit
| 2015 1-22A
+
| [https://drive.google.com/open?id=0BztPXrXqzHY8SW1PcXk5RERNeGs Specs] and [https://drive.google.com/open?id=0BztPXrXqzHY8U0lfZFJ6bWgzTWc FASTA]
| pSB1C3
+
| [http://parts.igem.org/Part:BBa_J23106 BBa_J23106]
| BBa_I3504  
+
| [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]
(B0034-E0040-B0015)
+
| 2015 4-21J
+
| pSB1A2
+
 
|pSB1C3
 
|pSB1C3
 
|-
 
|-
 
| Device #3
 
| Device #3
| BBa_J23117
+
| https://benchling.com/s/8q493PdY/edit
| 2015 1-22K
+
| [https://drive.google.com/open?id=0BztPXrXqzHY8cUFVcE1QSHFhZzA Specs] and [https://drive.google.com/open?id=0BztPXrXqzHY8bUt2eDZOMjlSckk FASTA]
| pSB1C3
+
| [http://parts.igem.org/Part:BBa_J23117 BBa_J23117]
| BBa_I3504  
+
| [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]
(B0034-E0040-B0015)
+
| 2015 4-21J
+
| pSB1A2
+
 
|pSB1C3
 
|pSB1C3
 
|-
 
|-
| Postive Control (BBa_I20270)
+
| Positive Control [http://parts.igem.org/Part:BBa_I20270 BBa_I20270]
| BBa_J23151
+
| N/A
| 2015 3-8P
+
| N/A
| pSB1C3
+
| [http://parts.igem.org/Part:BBa_J23151 BBa_J23151]
| GFPmut3b  
+
| GFPmut3b Promoter MeasKit
 
(B0032-E0040-B0010-B0012)
 
(B0032-E0040-B0010-B0012)
| 2015 3-8P
 
| pSB1C3
 
 
|pSB1C3
 
|pSB1C3
 
|-
 
|-
| Negative Control
+
| Negative Control [http://parts.igem.org/Part:BBa_R0040 BBa_R0040]
| TetR repressible promoter (BBa_R0040)
+
| 2015 2-6F
+
| pSB1C3
+
 
| N/A
 
| N/A
 
| N/A
 
| N/A
 +
| TetR repressible promoter (BBa_R0040)
 
| N/A
 
| N/A
 
| pSB1C3
 
| pSB1C3
 
|}
 
|}
  
All devices constructed using the BioBricks Standard Assembly 10 method used digestion and ligation to assemble the final vectorFor 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.
+
All devices constructed using the IDT gBlocks gene fragment synthesis schemeBiobrick scar sites between the promoter and GFP generator sequence were preserved to simulate traditional Biobricks standard assembly.
  
 
==<u> Protocols </u>==
 
==<u> Protocols </u>==
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The following are a list of protocols designed for the InterLab Study.  
 
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)
+
* Preparation of chemically competent E. coli BL21(DE3) cells using [http://www.zymoresearch.com/downloads/dl/file/id/166/t3001i.pdf Zymo Mix & Go Transformation Kit and Buffer Set]
* Rapid isolation of plasmid DNA (pSB1A2, pSB1C3) using the Promega PureYield MiniPrep System (Miniprep)
+
* Rapid isolation of plasmid DNA (pSB1C3) using the [https://www.promega.com/~/media/files/resources/protcards/pureyield%20plasmid%20miniprep%20system%20quick%20protocol.pdf 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 EcoRI and PstI restriction exonucleases [http://nebcloner.neb.com/#!/protocol/re/double/EcoRI,PstI (NEB EcoRI/PstI Digest)]
* Double digestion of plasmid DNA using SpeI and PstI restriction exunucleases (SpeI/PstI Digest)
+
* Rapid ligation and subcloning of gBlocks double digests into pSB1C3 vector backbone using T4 ligase [https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202 (NEB T4 Ligation)]
* Rapid ligation and subcloning of Standard Assembly 10 DNA digests using T7 ligase (T7 Ligation)
+
* Zymoclean Gel DNA recovery and purification of plasmid/digested DNA following gel electrophoresis [http://www.zymoresearch.com/downloads/dl/file/id/34/d4001i.pdf (Gel Extraction)]
* QiaQuick gel extraction and purification of plasmid/digested DNA following gel electrophoresis (Gel Extraction)
+
* Zymo DNA Clean and Concentrator-5 for rapid cleanup of PCR, Digestion, and Ligation products [http://www.zymoresearch.com/downloads/dl/file/id/35/d4003i.pdf (PCR Cleanup)]
  
 
==<u> Where to go from here </u>==
 
==<u> Where to go from here </u>==
  
The following is the process for all 4 device parts (promoter and GFP generator) and the 2 controls (Promoter MeasKit and pTetR empty).   
+
The following is the process for all 3 devices and the 2 controls (Promoter MeasKit and pTetR empty vector).   
*<s> 1. Resuspend dried plasmid in each of the registry wells in 10uL ddH2O. </s> <u> Refer here for details.</u>
+
*<s> 1. Order gBlocks gene fragments for all three devices </s> [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/12_August_2015 HERE]
  
*<s> 2.Transform plasmids using the standard NEB Electroporation method. </s> <u> 5/6 transformations completed, refer HERE. </u>
+
*<s> 2. Design and order primer pair for amplification of all three devices. </s> [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/12_August_2015 HERE] and [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/13_August_2015 HERE]
  
*3. Pick transformants and prepare cultures for miniprep and glycerol stocking.  
+
* <s> 3. Amplify all three devices, and PCR cleanup/gel extract each reaction. </s> [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/14_August_2015 HERE]
  
Following is for preparation of the 3 devices using the 4 device parts from the registry.
+
Following is for preparation of the 3 devices for cloning into pSB1C3:
  
*4. Digest miniprepped promoter plasmids with SpeI and PstI, and digest miniprepped GFP generator using XbaI and PstI.
+
* <s> 4. Digest devices using EcoRI and PstI </s> [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/17_August_2015#Double_Digestion_of_Devices_and_pSB1C3_Plasmid_Using_EcoRI_and_PstI HERE]
  
*5. Run digests on gels and gel purify/DNA clean and concentrate all digested pieces.  
+
* <s> 5. Run digests on gels and DNA clean and concentrate all digested pieces. </s> [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/17_August_2015#Double_Digestion_of_Devices_and_pSB1C3_Plasmid_Using_EcoRI_and_PstI HERE]
  
*6. Ligate digested pieces for final device construction, and transform using electroporation.  
+
*6. Ligate digested pieces in empty pSB1C3 vector backbone, and transform using chemical competency. </s> [https://2015.igem.org/Team:UCLA/Notebook/Interlab_Study/17_August_2015#Ligation_of_digested_Devices_in_digested_pSB1C3_using_T4_Ligase] and HERE
  
 
*7. Pick transformants and prepare cultures for miniprep and glycerol stocking.   
 
*7. Pick transformants and prepare cultures for miniprep and glycerol stocking.   
Line 113: Line 99:
  
 
Following is the plan for fluorescence measurement and data analysis of the three devices, using + and - controls for baseline measurement.  
 
Following is the plan for fluorescence measurement and data analysis of the three devices, using + and - controls for baseline measurement.  
[TBD]
+
[TBD - Fluorescence measurement and FACS analysis.]
  
 
==<u>Raw lab notebook entries</u>==
 
==<u>Raw lab notebook entries</u>==
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Latest revision as of 04:56, 21 August 2015

iGEM UCLA




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 [http://www.uniprot.org/uniprot/P42212 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 IDT gBlocks Gene Fragments synthesis, in addition to positive control [http://parts.igem.org/Part:BBa_I20270 BBa_I20270] (Constitutive Family Promoter [http://parts.igem.org/wiki/index.php?title=Part:BBa_J23151 J23151] inserted upstream of the promoter MeasKit) and negative control [http://parts.igem.org/Part:BBa_R0040 BBa_R0040] (pTetR - empty control plasmid). All gBlocks were designed on Benching to simulate the sequence of the BioBricks standard assembly product, for uniformity in measurement with teams that opted for RFC10 standard assembly. All devices were subcloned in the standard [http://parts.igem.org/Part:pSB1C3 pSB1C3] (chloramphenicol resistance marker) backbone and transformed into BL21(DE3) Escherichia coli . As such, E. coli BL21(DE3) 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 # Benchling Link Spec Sheet & FASTA File Promoter GFP Generator Final Device Backbone
Device #1 https://benchling.com/s/EnB0uD9g/edit Specs and FASTA [http://parts.igem.org/Part:BBa_J23101 BBa_J23101] [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]

(B0034-E0040-B0015)

pSB1C3
Device #2 https://benchling.com/s/hX68sjpy/edit Specs and FASTA [http://parts.igem.org/Part:BBa_J23106 BBa_J23106] [http://parts.igem.org/Part:BBa_I3504 BBa_I3504] pSB1C3
Device #3 https://benchling.com/s/8q493PdY/edit Specs and FASTA [http://parts.igem.org/Part:BBa_J23117 BBa_J23117] [http://parts.igem.org/Part:BBa_I3504 BBa_I3504] pSB1C3
Positive Control [http://parts.igem.org/Part:BBa_I20270 BBa_I20270] N/A N/A [http://parts.igem.org/Part:BBa_J23151 BBa_J23151] GFPmut3b Promoter MeasKit

(B0032-E0040-B0010-B0012)

pSB1C3
Negative Control [http://parts.igem.org/Part:BBa_R0040 BBa_R0040] N/A N/A TetR repressible promoter (BBa_R0040) N/A pSB1C3

All devices constructed using the IDT gBlocks gene fragment synthesis scheme. Biobrick scar sites between the promoter and GFP generator sequence were preserved to simulate traditional Biobricks standard assembly.

Protocols

The following are a list of protocols designed for the InterLab Study.

  • Preparation of chemically competent E. coli BL21(DE3) cells using [http://www.zymoresearch.com/downloads/dl/file/id/166/t3001i.pdf Zymo Mix & Go Transformation Kit and Buffer Set]
  • Rapid isolation of plasmid DNA (pSB1C3) using the Promega PureYield MiniPrep System (Miniprep)
  • Double digestion of plasmid DNA using EcoRI and PstI restriction exonucleases [http://nebcloner.neb.com/#!/protocol/re/double/EcoRI,PstI (NEB EcoRI/PstI Digest)]
  • Rapid ligation and subcloning of gBlocks double digests into pSB1C3 vector backbone using T4 ligase (NEB T4 Ligation)
  • Zymoclean Gel DNA recovery and purification of plasmid/digested DNA following gel electrophoresis [http://www.zymoresearch.com/downloads/dl/file/id/34/d4001i.pdf (Gel Extraction)]
  • Zymo DNA Clean and Concentrator-5 for rapid cleanup of PCR, Digestion, and Ligation products [http://www.zymoresearch.com/downloads/dl/file/id/35/d4003i.pdf (PCR Cleanup)]

Where to go from here

The following is the process for all 3 devices and the 2 controls (Promoter MeasKit and pTetR empty vector).

  • 1. Order gBlocks gene fragments for all three devices HERE
  • 2. Design and order primer pair for amplification of all three devices. HERE and HERE
  • 3. Amplify all three devices, and PCR cleanup/gel extract each reaction. HERE

Following is for preparation of the 3 devices for cloning into pSB1C3:

  • 4. Digest devices using EcoRI and PstI HERE
  • 5. Run digests on gels and DNA clean and concentrate all digested pieces. HERE
  • 6. Ligate digested pieces in empty pSB1C3 vector backbone, and transform using chemical competency. </s> [1] and HERE
  • 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 - Fluorescence measurement and FACS analysis.]

Raw lab notebook entries

July
M T W T F S S
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6 7 8 9 10 11 12
13 14 15 16 17 18 19
20 21 22 23 24 25 26
27 28 29 30 31
August
M T W T F S S
          1 2
3 4 5 6 7 8 9
10 11 12 13 14 15 16
17 18 19 20 21 22 23
24 25 26 27 28 29 30
31
September
M T W T F S S
  1 2 3 4 5 6
7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30