NYMU Taipei/Help me/planning

From 2007.igem.org

(Difference between revisions)
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<ol>
<ol>
     <li>把含[[Image:800px-PSB2K3 map.jpg|50px]]的plasmid transform E. coli，拿到plasmid DNA。（需要：protocol, reagent）</li>
     <li>把含[[Image:800px-PSB2K3 map.jpg|50px]]的plasmid transform E. coli，拿到plasmid DNA。（需要：protocol, reagent）</li>
-
     <li>把<a href="http://partsregistry.org/Part:BBa_C0082">tar-Envz</a>中的Envz切下（[[RE site: NdeI, ?]])，接到cloning vector上（需要：protocol, 實驗室有的vector）<a href="http://partsregistry.org/Part:BBa_M0040">LVA tag</a></li>
+
     <li>把[http://partsregistry.org/Part:BBa_C0082 tar-Envz]中的Envz切下（[[RE site: NdeI, ?]])，接到cloning vector上（需要：protocol, 實驗室有的vector）<a href="http://partsregistry.org/Part:BBa_M0040">LVA tag</a></li>
-
     <li>把<a href="http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=48994873&amp;from=2315049&amp;to=2317850&amp;view=gbwithparts">RcsC</a>用PCR分離出來 (內含AflII)（Reverse primer RE site須設計: NdeI），接到cloning vector上 (需要：<a href="http://us.expasy.org/cgi-bin/ft_viewer.pl?P14376">把每個domain的DNA sequence搞清楚</a>)</li>
+
     <li>把[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=48994873&amp;from=2315049&amp;to=2317850&amp;view=gbwithparts RcsC]用PCR分離出來 (內含AflII)（Reverse primer RE site須設計: NdeI），接到cloning vector上 (需要[http://us.expasy.org/cgi-bin/ft_viewer.pl?P14376 把每個domain的DNA sequence搞清楚])</li>
     <li>設計primer時須考慮ribosome binding site的問題</li>
     <li>設計primer時須考慮ribosome binding site的問題</li>
</ol>
</ol>
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<p>RcsC uses <a href="http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&amp;qty=1&amp;c_start=1&amp;list_uids=48994873&amp;uids=&amp;dopt=gbwithparts&amp;dispmax=5&amp;sendto=&amp;fmt_mask=0&amp;from=2315049&amp;to=2318000&amp;extrafeatpresent=1&amp;ef_CDD=8&amp;ef_MGC=16&amp;ef_HPRD=32&amp;ef_STS=64&amp;ef_tRNA=128&amp;ef_microRNA=256&amp;ef_Exon=512#sequence_48994873">UUG</a> as start codon!!</p>
<p>RcsC uses <a href="http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&amp;qty=1&amp;c_start=1&amp;list_uids=48994873&amp;uids=&amp;dopt=gbwithparts&amp;dispmax=5&amp;sendto=&amp;fmt_mask=0&amp;from=2315049&amp;to=2318000&amp;extrafeatpresent=1&amp;ef_CDD=8&amp;ef_MGC=16&amp;ef_HPRD=32&amp;ef_STS=64&amp;ef_tRNA=128&amp;ef_microRNA=256&amp;ef_Exon=512#sequence_48994873">UUG</a> as start codon!!</p>
<p>Location of transmembrane signal</p>
<p>Location of transmembrane signal</p>
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<p><a href="http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Quick_reference">BioBrick standard</a></p>
+
<p>[http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Quick_reference BioBrick standard]</p>
<p>IGFBP3</p>
<p>IGFBP3</p>
<p>IGFBP7</p>
<p>IGFBP7</p>
<p>RcsC:</p>
<p>RcsC:</p>
<p>&nbsp;</p>
<p>&nbsp;</p>
-
<p>OLIGO <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_START">start</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_LEN">len</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_TM">tm</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_GC">gc%</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_ANY">any</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_REPEAT">3'</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_OLIGO_SEQ">seq</a> <br />
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<p>OLIGO [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_START start] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_LEN len] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_TM tm] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_GC gc%] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_ANY any] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_REPEAT 3'] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_OLIGO_SEQ seq] <br />
LEFT PRIMER 1 21 55.92 38.10 4.00 2.00 GAAATACCTTGCTTCTTTTCG<br />
LEFT PRIMER 1 21 55.92 38.10 4.00 2.00 GAAATACCTTGCTTCTTTTCG<br />
RIGHT PRIMER 1417 20 58.14 45.00 8.00 2.00 GAACATCGATTTTGACTGGC</p>
RIGHT PRIMER 1417 20 58.14 45.00 8.00 2.00 GAACATCGATTTTGACTGGC</p>
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</ol>
</ol>
<p>RcsC primer:</p>
<p>RcsC primer:</p>
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<p>OLIGO <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_START">start</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_LEN">len</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_TM">tm</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_GC">gc%</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_ANY">any</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_REPEAT">3'</a> <a href="http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_OLIGO_SEQ">seq</a> <br />
+
<p>OLIGO [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_START start] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_LEN len] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_TM tm] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_GC gc%] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_ANY any] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_REPEAT 3'] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_OLIGO_SEQ seq] <br />
LEFT PRIMER 1 21 55.92 38.10 4.00 2.00 GAAATACCTTGCTTCTTTTCG<br />
LEFT PRIMER 1 21 55.92 38.10 4.00 2.00 GAAATACCTTGCTTCTTTTCG<br />
RIGHT PRIMER 1417 20 58.14 45.00 8.00 2.00 GAACATCGATTTTGACTGGC</p>
RIGHT PRIMER 1417 20 58.14 45.00 8.00 2.00 GAACATCGATTTTGACTGGC</p>
<p>&nbsp;</p>
<p>&nbsp;</p>

Revision as of 14:41, 18 October 2007

Phase 1

  1. 把含50px的plasmid transform E. coli，拿到plasmid DNA。（需要：protocol, reagent）
  2. 把[http://partsregistry.org/Part:BBa_C0082 tar-Envz]中的Envz切下（RE site: NdeI, ?)，接到cloning vector上（需要：protocol, 實驗室有的vector）<a href="http://partsregistry.org/Part:BBa_M0040">LVA tag</a>
  3. 把[http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?val=48994873&from=2315049&to=2317850&view=gbwithparts RcsC]用PCR分離出來 (內含AflII)（Reverse primer RE site須設計: NdeI），接到cloning vector上 (需要[http://us.expasy.org/cgi-bin/ft_viewer.pl?P14376 把每個domain的DNA sequence搞清楚])
  4. 設計primer時須考慮ribosome binding site的問題

需要人力；每一任務需要一人全力投入。

C3CCBKRB016

 

Primer design

RcsC uses <a href="http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&qty=1&c_start=1&list_uids=48994873&uids=&dopt=gbwithparts&dispmax=5&sendto=&fmt_mask=0&from=2315049&to=2318000&extrafeatpresent=1&ef_CDD=8&ef_MGC=16&ef_HPRD=32&ef_STS=64&ef_tRNA=128&ef_microRNA=256&ef_Exon=512#sequence_48994873">UUG</a> as start codon!!

Location of transmembrane signal

[http://openwetware.org/wiki/Synthetic_Biology:BioBricks/Part_fabrication#Quick_reference BioBrick standard]

IGFBP3

IGFBP7

RcsC:

 

OLIGO [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_START start] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_LEN len] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_TM tm] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_GC gc%] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_ANY any] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_REPEAT 3'] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_OLIGO_SEQ seq]
LEFT PRIMER 1 21 55.92 38.10 4.00 2.00 GAAATACCTTGCTTCTTTTCG
RIGHT PRIMER 1417 20 58.14 45.00 8.00 2.00 GAACATCGATTTTGACTGGC

Phase 2

  1. 把RcsC和Envz接在一起。
  2. 把Reporter接到OmpR binding site後面。
  3.  Quantitative characterization of promotor efficiency (basal transcription level)

RcsC primer:

OLIGO [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_START start] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_LEN len] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_TM tm] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_GC gc%] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_ANY any] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_REPEAT 3'] [http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www_results_help.cgi#PRIMER_OLIGO_SEQ seq]
LEFT PRIMER 1 21 55.92 38.10 4.00 2.00 GAAATACCTTGCTTCTTTTCG
RIGHT PRIMER 1417 20 58.14 45.00 8.00 2.00 GAACATCGATTTTGACTGGC

 

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