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== The 2015 UCLA iGEM Interlab/Measurements Study Notebook ==
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<!--
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<h2>The Interlab/Measurement Study Notebook</h2>
+
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<h1> Does promoter strength vary from lab to lab, city to city? </h1>
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<details>
+
==<u>Introduction</u>==
−
  <summary>5/10 - 5/16: Designing Protein Cage Protease Sequence Insertion Sites</summary>
+
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.  
−
  <details>
+
−
      <summary>5/14: Meeting with Dr. Yeates</summary>
+
−
      <p>Met with Sri and Todd to give feedback on our site</p>
+
−
      <p>Main points listed below (paraphrased from Sri's notes on Slack):</p>
+
−
      <ul>
+
−
        <li>Site 1/4 looked pretty good; but the major thing is to do insertions rather than replacements on the protein</li>
+
−
        <li>Try to have least one G on each side of insertion</li>
+
−
              <ul><li>so for site 1 (300-301), after S it would be SG<b>LVPRGS</b>G</li></ul>
+
−
        <li>Todd will ask in the lab that there might be a few variants that might be useful (nicer looking cages, easier to purify, etc.)</li>
+
−
        <li>Will likely do a combination of gene synthesis (~4 constructs) and site-directed mutagenesis (to allow for more sites and linker variations)</li>
+
−
        <li>Will attempt about 12 constructs</li>
+
−
      </ul>
+
−
      <br/>
+
−
      <p>Other ideas to think about:</p>
+
−
      <ul>
+
−
        <li>Using cysteines for chemical modifications (can use for some interesting assays to test cage disassociation)</li>
+
−
        <li>Could encapsulate a protease inside the cage to trigger a protease cascade (might be tough with current design)</li>
+
−
        <li>Would be interesting to see if we can replace all cysteines with serine or alanine</li>
+
−
      </ul>
+
−
  </details>
+
−
  <details>
+
−
      <summary>5/11: Preliminary Designs</summary>
+
−
      <p>Found ~8 potential sites using PyMol and DSSP figures for 3vdx 16nm 12-subunit cage</p>
+
−
      <br/>
+
−
      <p>Thrombin: Leu-Val-Pro-Arg-Gly-Ser (LVPRGS)</p>
+
−
      <p>Linker: 264-294 (by inspection)</p>
+
−
      <br/>
+
−
      <p>Potential Mutation Sites</p>
+
−
      <ul>
+
−
        <li>297-302 (IIPSGP → LVPRGS) *least aa change + near linker</li>
+
−
        <li>31-36 (GFPLSG → LVPRGS)</li>
+
−
        <li>125-130 (LEPFLL → LVPRGS)</li>
+
−
        <li>134-139 (DNPDGA → LVPRGS) *highly accessible to protease </li>
+
−
        <li>217-222 (DVPALI → LVPRGS)</li>
+
−
        <li>230-235 (TLPIEN → LVPRGS)</li>
+
−
        <li>370 - 375 (GDPNNM → LVPRGS)</li>
+
−
        <li>331-336(TRPILS→ LVPRGS)</li>
+
−
      </ul>
+
−
      <br/>
+
−
  </details>
+
−
</details>
+
−
-->
+
−
</html>
+
  
−
==<u>Goals</u>==
+
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.
−
The goal of this project is to produce various mutants of a 3-dimensional protein fusion capable of self-assembling into a tetrahedral cage structure (PDB: 3VDX). These variants will have thrombin protease sites (LVPRGS) introduced in selected locations, allowing for dissociation of the cage structure upon thrombin treatment. Ultimately, we aim to develop a controllable system allowing for both drug-loading and release using the protein cage scaffold.
+
  
−
==<u>Achievements</u>==
+
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.
−
As of 5/22, we have a detailed list of ~15 unique potential sites to insert into the protein cage. We will narrow this list to ~12 sites, and begin designing constructs beginning of next week. Four of our best sites will be synthesized through IDT, while the remaining will be produced through site directed mutagenesis.
+
−
===Design===
+
−
Following is a list of our insertion sites. Sites marked with asterisks are preferred sites. Sites were selected by examining the DSSP secondary structure to ensure minimal disruption of existing alpha-helices or beta sheets, ensuring sites were sandwiched by glycine residues, and using PyMOL to check if the site would be accessible to the protease. Top candidates are based on two main criteria:
+
  
−
1.  Geometric accessibility by protease
+
==<u>Experimental Design</u>==
  
−
2. Speculated extent of conformational changes by insertion (minimum preferred)
+
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:
  
−
PyMOL PSE file with sites highlighted: https://drive.google.com/file/d/0B7kb5ShZyyqVcU0xWlVYbUVfSW8/view?usp=sharing
 
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
−
! Site Number
+
! Device #
−
! Residues
+
! Benchling Link
−
! Original Sequence
+
! Spec Sheet & FASTA File
−
! Mutant Sequence
+
! Promoter
−
! Notes
+
! GFP Generator
 +
! Final Device Backbone
 
|-
 
|-
−
| Site 1*
+
| Device #1
−
| 298-305
+
| https://benchling.com/s/EnB0uD9g/edit
−
| IPSGPLKA
+
| [https://drive.google.com/open?id=0BztPXrXqzHY8WngyWHNwUTdIRmc Specs] and [https://drive.google.com/open?id=0BztPXrXqzHY8cWJYU3FRU0Fkb2c FASTA]
−
| IPSG'''LVPRGSG'''PLKA
+
| [http://parts.igem.org/Part:BBa_J23101 BBa_J23101]
−
| Satisfies 1 and 2, depending on oligomerization geometry
+
| [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]
 +
(B0034-E0040-B0015)
 +
|pSB1C3
 
|-
 
|-
−
| Site 2*
+
| Device #2
−
| 134-140
+
| https://benchling.com/s/hX68sjpy/edit
−
| DNPDGAA
+
| [https://drive.google.com/open?id=0BztPXrXqzHY8SW1PcXk5RERNeGs Specs] and [https://drive.google.com/open?id=0BztPXrXqzHY8U0lfZFJ6bWgzTWc FASTA]
−
| DNPD'''GLVPRGS'''GAA
+
| [http://parts.igem.org/Part:BBa_J23106 BBa_J23106]
−
| Satisfies 1 and 2
+
| [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]
 +
|pSB1C3
 
|-
 
|-
−
| Site 4*
+
| Device #3
−
| 252-258
+
| https://benchling.com/s/8q493PdY/edit
−
| VEGAPHG
+
| [https://drive.google.com/open?id=0BztPXrXqzHY8cUFVcE1QSHFhZzA Specs] and [https://drive.google.com/open?id=0BztPXrXqzHY8bUt2eDZOMjlSckk FASTA]
−
| VEG'''LVPRGSG'''APHG
+
| [http://parts.igem.org/Part:BBa_J23117 BBa_J23117]
−
| Satisfies 1 and 2; is close to linker region
+
| [http://parts.igem.org/Part:BBa_I3504 BBa_I3504]
 +
|pSB1C3
 
|-
 
|-
−
| Site 5*
+
| Positive Control [http://parts.igem.org/Part:BBa_I20270 BBa_I20270]
−
| 331-336
+
| N/A
−
| TRPILSP
+
| N/A
−
| TRP'''GLVPRGSG'''ILSP
+
| [http://parts.igem.org/Part:BBa_J23151 BBa_J23151]
−
| Satisfies 1 and 2, depending on oligomerization geometry
+
| GFPmut3b Promoter MeasKit
 +
(B0032-E0040-B0010-B0012)
 +
|pSB1C3
 
|-
 
|-
−
| Site 10*
+
| Negative Control [http://parts.igem.org/Part:BBa_R0040 BBa_R0040]
−
| 243-248
+
| N/A
−
| ALPSAE
+
| N/A
−
| ALP'''GLVPRGSG'''SAE
+
| TetR repressible promoter (BBa_R0040)
−
| Satisfies 1, possibly 2 also, very exposed for thrombin cleavage
+
| N/A
−
|-
+
| pSB1C3
−
| Site 13*
+
−
| 351-354
+
−
| VPSE
+
−
| '''GL'''VP'''RGSG'''E
+
−
| Satisfies 1; only 4 insertions needed.
+
−
|-
+
−
| Site 6
+
−
| 228-233
+
−
| DRTLPI
+
−
| DRTL'''GLVPRGSG'''PI
+
−
| Satisfies 1
+
−
|-
+
−
| Site 7
+
−
| 216-220
+
−
| IDVPA
+
−
| IDV'''GLVPRGSG'''PA
+
−
| Satisfies 1, possibly 2, easily accessible and within longer turn
+
−
|-
+
−
| Site 8
+
−
| 64-72
+
−
| SSQPTTGYD
+
−
| SSQPTT'''GLVPRGS'''GYD
+
−
| Satisfies 1, but not ideally oriented for access by thrombin
+
−
|-
+
−
| Site 9
+
−
| 417-422
+
−
| RMGAVT
+
−
| RMG'''LVPRGSG'''AVT
+
−
| Satisfies 1, depending on geometry, possible steric hindrance (inner portion of cage)
+
−
|-
+
−
| Site 11
+
−
| 123-129
+
−
| ASLEPFL
+
−
| ASL'''GVPRGSG'''EPGL
+
−
| Sterically hindered. 6 insertions needed.
+
−
|-
+
−
| Site 12
+
−
| 318-321
+
−
| KNTD
+
−
| KN'''GLVPRGSG'''TD
+
−
| Satisfies 1, 7 insertions needed.
+
−
|-
+
−
| Site 14
+
−
| 190-196
+
−
| AASGGFF
+
−
| AASG'''LVPRGS'''GFF
+
−
| Satisfies neither 1 nor 2, sterically hindered and close to secondary structures.  Probably not good.
+
 
|}
 
|}
  
−
===Cloning===
+
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.
−
Have not started yet. Will begin designing gBlocks and primers for site-directed mutagenesis early next week.
+
 
 +
==<u> Protocols </u>==
 +
 
 +
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 [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 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 [https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202 (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)]
 +
 
 +
==<u> Where to go from here </u>==
 +
 
 +
The following is the process for all 3 devices and the 2 controls (Promoter MeasKit and pTetR empty vector).
 +
*<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. 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]
 +
 
 +
* <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 for cloning into pSB1C3:
 +
 
 +
* <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]
 +
 
 +
* <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 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. 
 +
 
 +
*8. Digest miniprepped devices using EcoRI and PstI and run on a gel to verify size of the insert.  
  
−
===Protein Expression===
+
*9. Sequence the 3 devices and compared alignments for predicted sequences to verify proper device implementation.  
−
Long way to go till we get here.
+
  
−
==<u>What we are working on now</u>==
+
Following is the plan for fluorescence measurement and data analysis of the three devices, using + and - controls for baseline measurement.  
−
1. Transform the iGEM registry parts needed for the Interlab study (J23101, J23106, J2311, I13504, I20270 (+ control), and R0040 (- control).
+
[TBD - Fluorescence measurement and FACS analysis.]
−
2.  Test transformation efficiency of Kosuri Lab NEB Electrocompetent Cells (using J004450 as the control).
+
  
 
==<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
    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
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