Difference between revisions of "Part:BBa K2014005"

Revision as of 20:41, 20 October 2016

AraC-pBAD->sfGFP_B

Usage and Biology

We constructed AraC-pBAD->sfGFP_B as a fluorescent marker to measure the effect, codon optimization may have on heterologous protein production in E. coli.
It is believed that by codon optimization one can substantially increase the gene expression and that the optimized gene will more effectively compete for cell resources and will be more accurately translated [Kane JK, 1995]. We would like to check which approach to optimize a reading frame is the best and to what extent it can improve the expression of the optimized gene. We consider improvements of such traits like: codon usage, codon adaptation index, contexts of codons and secondary structures in coding sequences. We intentionally started our comparisons from implementing general optimization rules, which effects can be easily compared in simple induced expression experiments.
We have started from a simple optimization of sfGFP, in which we changed every codon of sfGFP [Pedelacq JD, 2006] to the most abundant synonymous codon in all reading frames of E.coli K12 orfeome, according to the codon usage table generated for us by Prof. W. Karłowski. At the N-terminus of coding sequence there is a stable 6-histidine tag (Fig. 1). The reporter gene is cloned under arabinose promoter (AraC-pBAD) a wild-type E. coli promoter that is tightly controlled by L-arabinose and is used in pBAD expression vectors (Invitrogen, Thermo-Fischer).

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 1144
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 979
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI site found at 961

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 __NOTOC__
 <partinfo>BBa_K2014005 short</partinfo>
-We were using standard AraC-pBAD promoter to compare the influence of different ORFs structures coding for the same protein sfGFP.
-<!-- Add more about the biology of this part here
 ===Usage and Biology===
+We constructed AraC-pBAD->sfGFP_B as a fluorescent marker to measure the effect, codon optimization may have on heterologous protein production in <i>E. coli</i>. <br>
+It is believed that by codon optimization one can substantially increase the gene expression and that the optimized gene will more effectively compete for cell resources and will be more accurately translated  [Kane JK, 1995]. We would like to check which approach to optimize a reading frame is the best and to what extent it can improve the expression of the optimized gene. We consider improvements of such traits like: codon usage, codon adaptation index, contexts of codons and secondary structures in coding sequences. We intentionally started our comparisons from implementing general optimization rules, which effects can be easily compared in simple induced expression experiments.  <br>
+We have started from a simple optimization of sfGFP, in which we changed every codon of sfGFP [Pedelacq JD, 2006] to the most abundant synonymous codon in all reading frames of E.coli K12 orfeome, according to the codon usage table generated for us by Prof. W. Karłowski.  At the N-terminus of coding sequence there is a stable 6-histidine tag (<b>Fig. 1</b>). The reporter gene is cloned under arabinose promoter (AraC-pBAD) a wild-type <i> E. coli </i> promoter that is tightly controlled by L-arabinose and is used in pBAD expression vectors (Invitrogen, Thermo-Fischer).
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