Difference between revisions of "Part:BBa K1362100"

Latest revision as of 12:08, 24 October 2014

RBS + NpuDnaE N-intein RFC[105] assembly construct (with His6)

This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting this intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the Nostoc punctiforme DnaE N-terminal split intein. The corresponding C-terminal construct is BBa_K1362101. Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond.

This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective parts in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on!

Usage and Biology

This Part is an N-intein assembly part according to [http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_105:_The_Intein_standard_-_an_universal_way_to_modify_proteins_after_translation RFC 105]. Specifically, it contains a T7 Ribosome binding site, an mRFP selection marker enclosed by overhangs A and NN followed by the N-intein Sub-part for Npu DnaE N-Intein with a hexahistidine-tag fused to the C-terminus.

Figure 2: N-terminal intein assembly construct with His6

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal AgeI site found at 826
Illegal AgeI site found at 938
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI site found at 1115
Illegal BsaI.rc site found at 40

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1362100 short</partinfo>
-This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting those intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the <i>Nostoc punctiforme</i> DnaE N-terminal split intein. The corresponding C-terminal construct is <partinfo>BBa_K1362101</partinfo>.
+This intein assembly construct is part of the iGEM team Heidelberg 2014's strategy for cloning with split inteins. Inteins are naturally occuring peptide sequences that splice out of a precursor protein and attach the remaining ends together to form a new protein. When splitting this intein sequence into an N-terminal and a C-terminal split intein one is left with a powerful tool to post-translationally modify whole proteins on the amino-acid sequence level. This construct was designed to express any protein of interest fused to the <i>Nostoc punctiforme</i> [[Part:BBa_K1362400|DnaE N-terminal]] split intein. The corresponding C-terminal construct is <partinfo>BBa_K1362101</partinfo>.
 Upon coexpression or mixture of the N- and C-constructs protein splicing takes place and the N- and C-terminal proteins of interest are irreversibly assembled via a newly formed peptide bond.
 This mechanism can be applied for a variety of different uses such as the activation of a protein through reconstitution of individually expressed split halves. See our [http://2014.igem.org/Team:Heidelberg split sfGFP experiment] and the respective [[Part:BBa_K1362170|parts]] in the registry for more information. Protein splicing offers many new possibilities and we hope to have set a foundation that you guys can build on!
-===<i>Nostoc punctiforme</i> DnaE split intein (<i>Npu</i>DnaE)===
+===Usage and Biology===
-Inteins can be found in all kingdoms of life, however their use for the organism remains yet unknown. In the past years many intein sequences were found in the genomes of cyanobacteria.
-<i>Npu</i>DnaE is a naturally split intein from the alpha subunit of Dna Polymerase III in Nostoc punctiforme bacteria which shows [[Part:BBa_K1362100:Design#References|[2]]] extraordinarly high splicing activity and therefore is on of the gold-standards for inteins since many years.
-<pre>
+This Part is an N-intein assembly part according to [http://openwetware.org/wiki/The_BioBricks_Foundation:RFC#BBF_RFC_105:_The_Intein_standard_-_an_universal_way_to_modify_proteins_after_translation RFC 105]. Specifically, it contains a T7 Ribosome binding site, an mRFP selection marker enclosed by overhangs A and NN followed by the N-intein Sub-part for Npu DnaE N-Intein with a hexahistidine-tag fused to the C-terminus.
-Amino Acid Sequence:
-N-Intein: AEY | CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN
-C-Intein: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN | CFN
-</pre>
+[[File:Heidelberg_orig_AssemblyConstructforN-Intein(1).png|thumb|width="400px|align=right|Figure 2''': N-terminal intein assembly construct with His6]]
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