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

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== The 2015 UCLA iGEM Interlab/Measurements Study Notebook ==
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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>
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==<u>Introduction</u>==
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  <summary>5/10 - 5/16: Designing Protein Cage Protease Sequence Insertion Sites</summary>
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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.  
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  <details>
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      <summary>5/14: Meeting with Dr. Yeates</summary>
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      <p>Met with Sri and Todd to give feedback on our site</p>
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      <p>Main points listed below (paraphrased from Sri's notes on Slack):</p>
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      <ul>
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        <li>Site 1/4 looked pretty good; but the major thing is to do insertions rather than replacements on the protein</li>
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        <li>Try to have least one G on each side of insertion</li>
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              <ul><li>so for site 1 (300-301), after S it would be SG<b>LVPRGS</b>G</li></ul>
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        <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>
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        <li>Will likely do a combination of gene synthesis (~4 constructs) and site-directed mutagenesis (to allow for more sites and linker variations)</li>
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        <li>Will attempt about 12 constructs</li>
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      </ul>
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      <br/>
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      <p>Other ideas to think about:</p>
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      <ul>
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        <li>Using cysteines for chemical modifications (can use for some interesting assays to test cage disassociation)</li>
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        <li>Could encapsulate a protease inside the cage to trigger a protease cascade (might be tough with current design)</li>
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        <li>Would be interesting to see if we can replace all cysteines with serine or alanine</li>
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      </ul>
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  </details>
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  <details>
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      <summary>5/11: Preliminary Designs</summary>
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      <p>Found ~8 potential sites using PyMol and DSSP figures for 3vdx 16nm 12-subunit cage</p>
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      <br/>
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      <p>Thrombin: Leu-Val-Pro-Arg-Gly-Ser (LVPRGS)</p>
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      <p>Linker: 264-294 (by inspection)</p>
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      <br/>
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      <p>Potential Mutation Sites</p>
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      <ul>
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        <li>297-302 (IIPSGP → LVPRGS) *least aa change + near linker</li>
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        <li>31-36 (GFPLSG → LVPRGS)</li>
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        <li>125-130 (LEPFLL → LVPRGS)</li>
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        <li>134-139 (DNPDGA → LVPRGS) *highly accessible to protease </li>
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        <li>217-222 (DVPALI → LVPRGS)</li>
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        <li>230-235 (TLPIEN → LVPRGS)</li>
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        <li>370 - 375 (GDPNNM → LVPRGS)</li>
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        <li>331-336(TRPILS→ LVPRGS)</li>
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      </ul>
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      <br/>
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  </details>
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</details>
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</html>
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==<u>Goals</u>==
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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.
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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.
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==<u>Achievements</u>==
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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.
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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.
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===Design===
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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:
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1.  Geometric accessibility by protease
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==<u>Experimental Design</u>==
  
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2. Speculated extent of conformational changes by insertion (minimum preferred)
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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:
  
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PyMOL PSE file with sites highlighted: https://drive.google.com/file/d/0B7kb5ShZyyqVcU0xWlVYbUVfSW8/view?usp=sharing
 
 
{| class="wikitable"
 
{| class="wikitable"
 
|-
 
|-
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! Site Number
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! Device #
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! Residues
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! Promoter
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! Original Sequence
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! Registry Location
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! Mutant Sequence
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! Backbone
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! Notes
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! GFP Generator
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! Registry Location
 +
! Backbone
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! Final Device Backbone
 
|-
 
|-
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| Site 1*
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| Device #1
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| 298-305
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| BBa_J23101
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| IPSGPLKA
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| 2015 1-20K
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| IPSG'''LVPRGSG'''PLKA
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| pSB1C3
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| Satisfies 1 and 2, depending on oligomerization geometry
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| BBa_I3504
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(B0034-E0040-B0015)
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| 2015 4-21J
 +
| pSB1A2
 +
|pSB1C3
 
|-
 
|-
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| Site 2*
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| Device #2
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| 134-140
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| BBa_J23106
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| DNPDGAA
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| 2015 1-22A
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| DNPD'''GLVPRGS'''GAA
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| pSB1C3
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| Satisfies 1 and 2
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| BBa_I3504
 +
(B0034-E0040-B0015)
 +
| 2015 4-21J
 +
| pSB1A2
 +
|pSB1C3
 
|-
 
|-
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| Site 4*
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| Device #3
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| 252-258
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| BBa_J23117
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| VEGAPHG
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| 2015 1-22K
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| VEG'''LVPRGSG'''APHG
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| pSB1C3
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| Satisfies 1 and 2; is close to linker region
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| BBa_I3504
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(B0034-E0040-B0015)
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| 2015 4-21J
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| pSB1A2
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|pSB1C3
 
|-
 
|-
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| Site 5*
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| Postive Control (BBa_I20270)
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| 331-336
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| J23151
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| TRPILSP
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| 2015 3-8P
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| TRP'''GLVPRGSG'''ILSP
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| pSB1C3
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| Satisfies 1 and 2, depending on oligomerization geometry
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| GFPmut3b
 +
(B0032-E0040-B0010-B0012)
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| 2015 3-8P
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| pSB1C3
 +
|pSB1C3
 
|-
 
|-
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| Site 10*
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| Negative Control
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| 243-248
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| TetR repressible promoter (BBa_R0040)
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| ALPSAE
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| 2015 2-6F
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| ALP'''GLVPRGSG'''SAE
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| pSB1C3
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| Satisfies 1, possibly 2 also, very exposed for thrombin cleavage
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| N/A
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|-
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| N/A
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| Site 13*
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| N/A
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| 351-354
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| pSB1C3
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| VPSE
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| '''GL'''VP'''RGSG'''E
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| Satisfies 1; only 4 insertions needed.
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−
|-
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| Site 6
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| 228-233
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| DRTLPI
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| DRTL'''GLVPRGSG'''PI
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| Satisfies 1
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−
|-
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| Site 7
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| 216-220
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| IDVPA
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| IDV'''GLVPRGSG'''PA
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| Satisfies 1, possibly 2, easily accessible and within longer turn
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−
|-
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| Site 8
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| 64-72
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| SSQPTTGYD
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| SSQPTT'''GLVPRGS'''GYD
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| Satisfies 1, but not ideally oriented for access by thrombin
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−
|-
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| Site 9
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| 417-422
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| RMGAVT
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| RMG'''LVPRGSG'''AVT
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| Satisfies 1, depending on geometry, possible steric hindrance (inner portion of cage)
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−
|-
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| Site 11
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| 123-129
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| ASLEPFL
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| ASL'''GVPRGSG'''EPGL
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| Sterically hindered. 6 insertions needed.
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−
|-
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| Site 12
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| 318-321
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| KNTD
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| KN'''GLVPRGSG'''TD
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| Satisfies 1, 7 insertions needed.
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−
|-
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| Site 14
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| 190-196
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| AASGGFF
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| AASG'''LVPRGS'''GFF
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| Satisfies neither 1 nor 2, sterically hindered and close to secondary structures.  Probably not good.
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|}
 
|}
  
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===Cloning===
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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.  
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Have not started yet. Will begin designing gBlocks and primers for site-directed mutagenesis early next week.
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===Protein Expression===
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Long way to go till we get here.
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==<u>What we are working on now</u>==
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1. Transform the iGEM registry parts needed for the Interlab study (J23101, J23106, J2311, I13504, I20270 (+ control), and R0040 (- control). 
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2.  Test transformation efficiency of Kosuri Lab NEB Electrocompetent Cells (using J004450 as the control).
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==<u>Raw lab notebook entries</u>==
 
==<u>Raw lab notebook entries</u>==
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Revision as of 23:18, 3 July 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 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

May
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June
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29 30
July
M T W T F S S
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August
M T W T F S S
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31
September
M T W T F S S
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