Difference between revisions of "Team:UCLA/Notebook/Protein Cages"

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| IPSGPLKA
 
| IPSGPLKA
 
| IPSG'''LVPRGSG'''PLKA
 
| IPSG'''LVPRGSG'''PLKA
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|-
 
|-
 
| Site 2
 
| Site 2
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| DNPDGAA
 
| DNPDGAA
 
| DNPD'''GLVPRGS'''GAA
 
| DNPD'''GLVPRGS'''GAA
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|
 
|-
 
|-
 
| Site 3
 
| Site 3
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| AASGGFF
 
| AASGGFF
 
| AASG'''LVPRGS'''GFF
 
| AASG'''LVPRGS'''GFF
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|
 
|-
 
|-
 
| Site 4*
 
| Site 4*
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| VEGAPHG
 
| VEGAPHG
 
| VEG'''LVPRGSG'''APHG
 
| VEG'''LVPRGSG'''APHG
 +
|
 
|-
 
|-
 
| Site 5
 
| Site 5
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| TRPILSP
 
| TRPILSP
 
| TRP'''GLVPRGSG'''ILSP
 
| TRP'''GLVPRGSG'''ILSP
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|
 
|}
 
|}
 
===Cloning===
 
===Cloning===
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We have successfully cloned and sequence verified '''two''' honeybee silk constructs into the igem psb1c3 plasmid backbone, in the correct biobrick format. A comprehensive list of our honeybee parts can be found [https://2015.igem.org/Team:UCLA/Parts here]
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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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*The first sequence is just the honeybee silk protein #3 in the psb1c3 backbone.  The annotated sequence, along with further details on how we designed and cloned this biobrick can be found here. [http://parts.igem.org/Part:BBa_K1763000 BBa_K1763000]
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*The second sequence is the same silk coding sequence, but with regulatory elements upstream for protein expression, including [http://parts.igem.org/Part:BBa_R0010 BBa_R0010 Promoter] and [http://parts.igem.org/Part:BBa_B0034 RBS BBa_B0034 ].  The sequence can be found here. [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001  ] along with Further details on the design and cloning of this biobrick.
+
  
 
===Protein Expression===
 
===Protein Expression===
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Haven't accomplished anything yet :P
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Long way to go till we get here.
  
 
==<u>What we are working on now</u>==
 
==<u>What we are working on now</u>==
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We are currently at the starting phases of honeybee silk protein expression.  We will be expressing [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001  ] and purifying the protein with BugBuster lysozyme kit.  We will then run our purified protein on an SDS PAGE gel to ascertain the purity of our purified silk protein. For our progress so far, see our lab notebook entries from [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/18_May_2015 5/18] and [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/19_May_2015 5/19].
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We are currently narrowing and refining our list of selected mutation sites.
  
 
==<u>Raw lab notebook entries</u>==
 
==<u>Raw lab notebook entries</u>==

Revision as of 21:05, 22 May 2015

iGEM UCLA




Goals

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.

Achievements

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.

Site Number Residues Original Sequence Mutant Sequence Notes
Site 1* 298-305 IPSGPLKA IPSGLVPRGSGPLKA
Site 2 134-140 DNPDGAA DNPDGLVPRGSGAA
Site 3 190-196 AASGGFF AASGLVPRGSGFF
Site 4* 252-258 VEGAPHG VEGLVPRGSGAPHG
Site 5 331-336 TRPILSP TRPGLVPRGSGILSP

Cloning

Have not started yet. Will begin designing gBlocks and primers for site-directed mutagenesis early next week.

Protein Expression

Long way to go till we get here.

What we are working on now

We are currently narrowing and refining our list of selected mutation sites.

Raw lab notebook entries

April
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
May
M T W T F S S
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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
June
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
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
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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
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7 8 9 10 11 12 13
14 15 16 17 18 19 20
21 22 23 24 25 26 27
28 29 30