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 | + | <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 | + | <li>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])</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&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!!</p> | <p>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!!</p> | ||
<p>Location of transmembrane signal</p> | <p>Location of transmembrane signal</p> | ||
| - | <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> </p> | <p> </p> | ||
| - | <p>OLIGO | + | <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> | ||
| - | <p>OLIGO | + | <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> </p> | <p> </p> | ||
Revision as of 14:41, 18 October 2007
Phase 1
- 把含50px的plasmid transform E. coli，拿到plasmid DNA。（需要：protocol, reagent）
- 把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>
- 把RcsC用PCR分離出來 (內含AflII)（Reverse primer RE site須設計: NdeI），接到cloning vector上 (需要把每個domain的DNA sequence搞清楚)
- 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
IGFBP3
IGFBP7
RcsC:
OLIGO start len tm gc% any 3' 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
- 把RcsC和Envz接在一起。
- 把Reporter接到OmpR binding site後面。
- Quantitative characterization of promotor efficiency (basal transcription level)
RcsC primer:
OLIGO start len tm gc% any 3' 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
