Difference between revisions of "Part:BBa K2350012"
 
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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.
 
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.
  
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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, this Indigoidine gene comes from Photorhabdus luminescens laumondii TT01 (DSM15139). BBa_K1152008 at first can only express in E. coli, and we furthermore improve it by constructing this pigment gene sequence with a backbone pPIGBACK (BBa_K2350012), which can express in cyanobacteria.
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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 to see whether Indigoidine would express in S. elongatus PCC 7942.
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Figure 1.
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Figure 1 is PrbcL-IndC fusion PCR electrophoresis result. M represents 1 kb marker, and PrbcL-IndC is 4287 bp.
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[[Image:PrbcL-IndC Parts.jpg]]
  
  
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Indigoidine C synthetase
 
  
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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).
 
  
  
−
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 to see whether Indigoidine would express in S. elongatus PCC 7942.
 
  
  

Latest revision as of 12:24, 26 October 2017


PrbcL-IndC

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, this Indigoidine gene comes from Photorhabdus luminescens laumondii TT01 (DSM15139). BBa_K1152008 at first can only express in E. coli, and we furthermore improve it by constructing this pigment gene sequence with a backbone pPIGBACK (BBa_K2350012), which can express in cyanobacteria.


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 to see whether Indigoidine would express in S. elongatus PCC 7942.




Figure 1.

Figure 1 is PrbcL-IndC fusion PCR electrophoresis result. M represents 1 kb marker, and PrbcL-IndC is 4287 bp.



PrbcL-IndC Parts.jpg





Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1641
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 2904