Team:Harvard/flavor/notebook

From 2010.igem.org

(Difference between revisions)
(06-15-2010 [ top ])
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==<html><a class="labnotebook" name="06-15-2010">06-15-2010</a></html> [ [[#top|top]] ]==
==<html><a class="labnotebook" name="06-15-2010">06-15-2010</a></html> [ [[#top|top]] ]==
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===Primer Designs for Agrobacterium Vector===
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*Primers were designed to amplify from the Expression Series pORE Agrobacterium plasmid (e3) 1)the pENTcup2 promoter, 2) the tNOS stop sequence, 3) the tNOS stop sequence + additional stop codon and 4) the pHLP promoter.<br>
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Note: the NOSterm_BB_R primer works for both tNOS and tNOS+STOP codon sequences.
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    pENTcup2_BB_F
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    CCTTTCTAGAGGGATCTTCTGCAAGCATCT
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    pENTcup2_BB_R
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    AAGGCTGCAGCGGCCGCTACTAGTTCCGGTGGGTTTTGAGGT
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    STOP_NOSterm_BB_F
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    CCTTTCTAGATGAGATCGTTCAAACATTTGG
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 +
    NOSterm_BB_F
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    CCTTTCTAGAGATCGTTCAAACATTTGGCA
 +
 +
    NOSterm_BB_R
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    AAGGCTGCAGCGGCCGCTACTAGTGATCTAGTAACATAGATGACA
 +
 +
    pHPL_BB_F
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    CCTTTCTAGAAACGTGGATACTTGGCAGTG
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 +
    pHPL_BB_R
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    AAGGCTGCAGCGGCCGCTACTAGTCTTTTGAGCTTAGAGGTTTTT
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===BioBrick Transformation===
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*Colonies were observed after overnight culture growth, but in low quantities.
 +
**J45700 and J45004 (both Wintergreen Pathway) showed minimal number of colonies on both 10μL and 100μL cultures.
 +
**J45250 and J45014 (both Banana Pathway) showed no colonies on either the 10μL or 100μL cultures.
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===Codon Usage in Arabidopsis===
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Using http://gcua.schoedl.de/ <br><br>
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'''Wintergreen Pathway:''' <br>
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BioBrick Part #: BBa_J45700 <br>
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Original Organism: ''Pseudomonas aeruginosa'' <br>
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[[Image:Wintergreen Pathway (full) Compare_Page_1.jpg|150px|BBa_J45700]]
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[[Image:Wintergreen Pathway (full) Compare_Page_2.jpg|150px|BBa_J45700]]
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[[Image:Wintergreen Pathway (full) Compare_Page_3.jpg|150px|BBa_J45700]]
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[[Image:Wintergreen Pathway (full) Compare_Page_4.jpg|150px|BBa_J45700]]
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'''Banana Pathway:''' <br>
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BioBrick Part #: BBa_J45014 <br>
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Original Organism: ''Saccharomyces cerevisiae'' <br>
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[[Image:codon_table_BBa_J45014.jpg|150px|BBa_J45014]]
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[[Image:codon_table_BBa_J45014_pg_2.jpg|150px|BBa_J45014]]
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[[Image:Codon_table_BBa_J45014_pg_3.jpg|150px|BBa_J45014]]
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BioBrick Part #: BBa_J45250 <br>
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[[Image:codon_table_BBa_J45250_pg1.jpg|150px|BBa_J45250]]
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[[Image:codon_table_BBa_J45250_pg2.jpg|150px|BBa_J45250]]
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[[Image:codon_table_BBa_J45250_pg3.jpg|150px|BBa_J45250]]
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[[Image:codon_table_BBa_J45250_pg4.jpg|150px|BBa_J45250]]
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'''Valencene Pathway:'''
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 +
[[Image:Citrus_sinesis_valencene_synthase_Page_1.jpg|150px|Valencene]]
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[[Image:Citrus_sinesis_valencene_synthase_Page_2.jpg|150px|Valencene]]
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[[Image:Citrus_sinesis_valencene_synthase_Page_3.jpg|150px|Valencene]]
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===Valencene Extraction===
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Used RNeasy Plant Mini Kit to extract RNA from the <u>flavedo</u> of an Organic Valencia Orange.
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[[Image:Flavedo.png|500px|Flavedo]]
==<html><a class="labnotebook" name="06-16-2010">06-16-2010</a></html> [ [[#top|top]] ]==
==<html><a class="labnotebook" name="06-16-2010">06-16-2010</a></html> [ [[#top|top]] ]==

Revision as of 00:19, 25 October 2010



notebook calendar [ top ]

Week 1 06-14-2010 06-15-2010 06-16-2010 06-17-2010 06-18-2010
Week 2 06-21-2010 06-22-2010 06-23-2010 06-24-2010 06-25-2010
Week 3 06-28-2010 06-29-2010 06-30-2010 07-01-2010 07-02-2010
Week 4 07-05-2010 07-06-2010 07-07-2010 07-08-2010 07-09-2010
Week 5 07-12-2010 07-13-2010 07-14-2010 07-15-2010 07-16-2010
Week 6 07-19-2010 07-20-2010 07-21-2010 07-22-2010 07-23-2010
Week 7 07-26-2010 07-27-2010 07-28-2010 07-29-2010 07-30-2010
Week 8 08-02-2010 08-03-2010 08-04-2010 08-05-2010 08-06-2010
Week 9 08-09-2010 08-10-2010 08-11-2010 08-12-2010 08-13-2010

06-14-2010 [ top ]

  • Miraculin and brazzein constructs due to arrive on Wednesday from Mr. Gene

BioBrick Transformation

BioBrick parts from the 2010 iGEM kit were transformed and grown in highly competent TURBO bacteria.

  • Wintergreen Scent Pathway:

BBa_J45700 - entire pathway, Ampicillin
BBa_J45004 - BSMT1 only, Ampicillin
(Not in 2010 BB Kit: BBa_J45017 - PchB, PchA)

  • Banana Scent Pathway:

BBa_J45250 - ATF3 + Promoter, Ampicillin?
BBa_J45014 - ATF3 only, Ampicillin
(Not in BB 2010 Kit: BBa_J45400 - BAT2 and THI3)

06-15-2010 [ top ]

Primer Designs for Agrobacterium Vector

  • Primers were designed to amplify from the Expression Series pORE Agrobacterium plasmid (e3) 1)the pENTcup2 promoter, 2) the tNOS stop sequence, 3) the tNOS stop sequence + additional stop codon and 4) the pHLP promoter.

Note: the NOSterm_BB_R primer works for both tNOS and tNOS+STOP codon sequences.

   pENTcup2_BB_F
   CCTTTCTAGAGGGATCTTCTGCAAGCATCT
   pENTcup2_BB_R
   AAGGCTGCAGCGGCCGCTACTAGTTCCGGTGGGTTTTGAGGT
   STOP_NOSterm_BB_F
   CCTTTCTAGATGAGATCGTTCAAACATTTGG
   NOSterm_BB_F
   CCTTTCTAGAGATCGTTCAAACATTTGGCA
   NOSterm_BB_R
   AAGGCTGCAGCGGCCGCTACTAGTGATCTAGTAACATAGATGACA
   pHPL_BB_F
   CCTTTCTAGAAACGTGGATACTTGGCAGTG
   pHPL_BB_R
   AAGGCTGCAGCGGCCGCTACTAGTCTTTTGAGCTTAGAGGTTTTT

BioBrick Transformation

  • Colonies were observed after overnight culture growth, but in low quantities.
    • J45700 and J45004 (both Wintergreen Pathway) showed minimal number of colonies on both 10μL and 100μL cultures.
    • J45250 and J45014 (both Banana Pathway) showed no colonies on either the 10μL or 100μL cultures.

Codon Usage in Arabidopsis

Using http://gcua.schoedl.de/

Wintergreen Pathway:
BioBrick Part #: BBa_J45700
Original Organism: Pseudomonas aeruginosa

BBa_J45700 BBa_J45700 BBa_J45700 BBa_J45700

Banana Pathway:
BioBrick Part #: BBa_J45014
Original Organism: Saccharomyces cerevisiae
BBa_J45014 BBa_J45014 BBa_J45014

BioBrick Part #: BBa_J45250
BBa_J45250 BBa_J45250 BBa_J45250 BBa_J45250

Valencene Pathway:

Valencene Valencene Valencene

Valencene Extraction

Used RNeasy Plant Mini Kit to extract RNA from the flavedo of an Organic Valencia Orange.

Flavedo

06-16-2010 [ top ]

06-17-2010 [ top ]

06-18-2010 [ top ]

06-21-2010 [ top ]

06-22-2010 [ top ]

06-23-2010 [ top ]

06-24-2010 [ top ]

06-25-2010 [ top ]

06-28-2010 [ top ]

06-29-2010 [ top ]

06-30-2010 [ top ]

07-01-2010 [ top ]

07-02-2010 [ top ]

07-05-2010 [ top ]

07-06-2010 [ top ]

07-07-2010 [ top ]

07-08-2010 [ top ]

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07-15-2010 [ top ]

07-16-2010 [ top ]

07-19-2010 [ top ]

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07-21-2010 [ top ]

07-22-2010 [ top ]

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07-26-2010 [ top ]

07-27-2010 [ top ]

07-28-2010 [ top ]

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07-30-2010 [ top ]

08-02-2010 [ top ]

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