Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk/21 May 2015"

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(→‎Designing Protease Sites)
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=Inoculation of colonies from 5/19 Tranformation=
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This was my approach to designing the protease sites -Phillip
  
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Four colonies from the 1:1 kanamycin plate from 5/19 were suspended in 100uL ddH2O.
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Using the DSSP algorithm from PDB, the parts of the protein with no alpha helices, beta sheets, or turns and five or greater amino acids are the following. 
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30-35, 64-72, 123-129, 131-139, 216-220, 228-233, 298-302, 331-336, 417-422
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Revised sites in terms of location. Bold indicates insertion
  
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Each sample was inoculated in four different culture tubes containing 5mL LB broth and 5uL 1000X kanamycin and incubated overnight at 37C.
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Top candidates are based on two criteria:
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1. Exposure at the surface for protease accessibility
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2. Speculated to have minimal conformational changes by insertion
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In general, original proline residues on the side were kept, in case they were involved in transitioning between secondary structural elements.  Also, insertions were flanked  with glycines in order to allow extra flexibility. 
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*131-139:  (KTDDNPDGA) to KTDDNPDGLVPRGSGA.  Satisfies 1 and 2.
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228-233:  (DRTLPI) to DRTLGLVPRGSGIPI.  Satisfies 1.
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*331-336:  (TRPILS) to TRPGLVPRGSGILS.  Satisfies 1 and 2, depending on oligomerization geometry.
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*298-302:  (IPSGP) to IPSGLVPRGSGP.  Satisfies 1 and 2, depending on oligomerization geometry.
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216-220:  (IDVPA) to IDVGLVPRGSGPA. 
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64-72:  (SSQPTTGYD) to SSQPTTGLVPRGSGYD
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417-422:  (RMGAVT) to RMGGLVPRGSGAVT.  Satisfies 1, depending on geometry.

Revision as of 23:03, 22 May 2015

iGEM UCLA




This was my approach to designing the protease sites -Phillip

Using the DSSP algorithm from PDB, the parts of the protein with no alpha helices, beta sheets, or turns and five or greater amino acids are the following. 30-35, 64-72, 123-129, 131-139, 216-220, 228-233, 298-302, 331-336, 417-422 Revised sites in terms of location. Bold indicates insertion

Top candidates are based on two criteria: 1. Exposure at the surface for protease accessibility 2. Speculated to have minimal conformational changes by insertion In general, original proline residues on the side were kept, in case they were involved in transitioning between secondary structural elements. Also, insertions were flanked with glycines in order to allow extra flexibility.

  • 131-139: (KTDDNPDGA) to KTDDNPDGLVPRGSGA. Satisfies 1 and 2.

228-233: (DRTLPI) to DRTLGLVPRGSGIPI. Satisfies 1.

  • 331-336: (TRPILS) to TRPGLVPRGSGILS. Satisfies 1 and 2, depending on oligomerization geometry.
  • 298-302: (IPSGP) to IPSGLVPRGSGP. Satisfies 1 and 2, depending on oligomerization geometry.

216-220: (IDVPA) to IDVGLVPRGSGPA. 64-72: (SSQPTTGYD) to SSQPTTGLVPRGSGYD 417-422: (RMGAVT) to RMGGLVPRGSGAVT. Satisfies 1, depending on geometry.