Difference between revisions of "Part:BBa K3022003"

Revision as of 08:56, 3 October 2019

lac-CFppk1 Cassette

This part will be sub cloned into pSB1C3, a plasmid of high copy number, to yield pSB1C3-lac-CFppk1. Polyphosphate synthesis capability of C. freundii transformed with this plasmid will be subjected to compare with C. freundii that harbored medium-copy expressing vector pBBR1MCS2-CFppk1. In this way, we can demonstrate that why medium copy number is a better option of vector choice.

In addition of CFppk1, this part also includes the promoter and terminator sequences of the lac expression system, thus composing a CFppk1 expression cassette. As such, when it was sub cloned into a plasmid of certain copy number, we can get a relative precise result regarding plasmid copy-number’s effects on ppk1 expressing level and polyphosphate yields, rather than a promiscuous conclusion drawn from the differences of promoting and/or terminating strength imposed by differed plasmid expressing system.

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
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 <partinfo>BBa_K3022003 short</partinfo>
-The  polyphosphate kinase in Citrobacter freundii ATCC 8090 is responsible for its intracelluar inorganic polyphosphate (polyP) production via reversibly catalyzing the transfer of terminal phosphate from ATP to a growing polyP chain. This organism is our chasiss, in which its native PPK1 will be overexpressed with a plasmid of medium-copy numbers.
+This part will be sub cloned into pSB1C3, a plasmid of high copy number, to yield pSB1C3-lac-CFppk1. Polyphosphate synthesis capability of C. freundii transformed with this plasmid will be subjected to compare with C. freundii that harbored medium-copy expressing vector pBBR1MCS2-CFppk1. In this way, we can demonstrate that why medium copy number is a better option of vector choice.
-Although PPKs from E. coli and C. freundii shares 96% identity, the C. freundii PPK1 has a glutamate and a lysine residue in positions 327 and 328, where E. coli PPK1 has much less strongly charged alanine and glutamine residues. These natural mutations of C. freundii PPK1 are distant from the PPK1 active site and found in interfaces between monomers of the PPK1 tetramer.This leads to a dramaticl increase of intracellular polyP accumulation.
+In addition of CFppk1, this part also includes the promoter and terminator sequences of the lac expression system, thus composing a CFppk1 expression cassette. As such, when it was sub cloned into a plasmid of certain copy number, we can get a relative precise result regarding plasmid copy-number’s effects on ppk1 expressing level and polyphosphate yields, rather than a promiscuous conclusion drawn from the differences of promoting and/or terminating strength imposed by differed plasmid expressing system.
 <!-- Add more about the biology of this part here