NYMU Taipei/Help me/planning

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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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