Composite

Part:BBa_K2350025:Design

Designed by: JO-NING HUNG   Group: iGEM17_NYMU-Taipei   (2017-10-22)
Revision as of 14:02, 22 October 2017 by Lilyhung (Talk | contribs) (→‎Design Notes)


pPIGBACK-PrbcL-IndC

The vector, pPIGBACK (BBa_K2350009), is used to transform into S. elongatus PCC 7942 with the inserted pigment gene in our project. pPIGBACK contains 5’- and 3’-ends of the neutral site II (NSII), replication origin of pBR322, ampicillin resistance gene, and double terminator BBa_B0015. We fuse IndC with the intrinsic promoter of Rubisco large subunit (PrbcL), which is proved to be functional in cyanobacteria, and cloned into our pigment vector, pPIGBACK,to see whether Indigoidine would express in S. elongatus PCC 7942. All Pst1 cutting sites in pPIGBACK-PrbcL-IndC are site-mutated using site-directed mutagnesis.


In nowadays studies, an Indigoidine synthetase Sc-IndC and an associated helper protein Sc-IndB were identified from Streptomyces chromofuscus ATCC 49982 and successfully expressed in Escherichia coli BAP1 to produce the blue pigment. The IndB gene codes for a putative phosphatase and the IndC gene codes for Indigoidine synthase. Together, these enzymes convert L-glutamine into Indigoidine. Recently, it has been shown that IndC alone can produce Indogoidine, and the inclusion of IndB expression in the system will increase yields significantly.


As we know, L-Glutamine is the direct biosynthetic precursor of Indigoidine, and it is a key amino acid in primary metabolism and thus naturally exists in S. elongatus PCC7942. Because glutamine related products are already existed in S. elongatus PCC7942, we only need to activate the expression of Sc-IndC in S. elongatus PCC7942 which leads to the production of Indigoidine. However, due to the access difficulties of Streptomyces chromofuscus ATCC 49982, we decided to use the previous part for IndC, which has been submitted to the iGEM Parts Registry (BBa_K1152008). According to the part design, our Indigoidine gene comes from Photorhabdus luminescens laumondii TT01 (DSM15139).




Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 5564
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1641
    Illegal BglII site found at 6126
    Illegal BamHI site found at 6423
    Illegal XhoI site found at 5361
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6344
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 5215
    Illegal BsaI site found at 6026
    Illegal SapI.rc site found at 2904



Design Notes

1. All Pst1 cutting sites in pPIGBACK-PrbcL-IndC are site-mutated using site-directed mutagnesis.


2. EcoR1 and Xma1 are the cutting sites using to ligate PrbcL-IndC with pPIGBACK.

Source

IndC comes from BBa_K1152008, and pPIGBACK is one of our submitted parts, BBa_K2350009.


References

Pei-Hong Chen, Hsien-Lin Liu, Yin-Ju Chen, Yi-Hsiang Cheng, Wei-Ling Lin, Chien-Hung Yeh and Chuan-Hsiung Chang (2012). Enhancing CO2 bio-mitigation by genetic engineering of cyanobacteria