Difference between revisions of "Part:BBa K3279001"

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Firstly, the CrtB gene was amplified from Rhodobacter sphaeroides's genomic sequence by PCR (see Figure 1a) and was cloned into pET-30a(+) plasmid. Then, colony PCR was run to check the success of ligation (see Figure 1b).
 
Firstly, the CrtB gene was amplified from Rhodobacter sphaeroides's genomic sequence by PCR (see Figure 1a) and was cloned into pET-30a(+) plasmid. Then, colony PCR was run to check the success of ligation (see Figure 1b).
 
[[File:CAU CrtB Fig1.png|500px|thumb|center|'''Fig. 1''' pET-CrtB plasmid construction. (a) Amplified CrtB from Rhodobacter sphaeroides's genomic sequence; (b) Colony PCR verified that CrtB was successfully assembled into the pET-30a (+) vector.]]
 
[[File:CAU CrtB Fig1.png|500px|thumb|center|'''Fig. 1''' pET-CrtB plasmid construction. (a) Amplified CrtB from Rhodobacter sphaeroides's genomic sequence; (b) Colony PCR verified that CrtB was successfully assembled into the pET-30a (+) vector.]]
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In order to determine the optimal induction condition of CrtB, we transferred the constructed plasmid pET-30a-CrtB into E. coli BL21 (DE3) and induced it under gradient IPTG concentration and different temperature conditions (see Figure 2). We found that CrtB could be induced at both 25℃ and 30℃, and the minimum IPTG concentration to induced CrtB was 0.02 mM.
 +
[[File:CAU CrtB Fig2.png|700px|thumb|center|'''Fig. 2''' SDS-PAGE assay for CrtB protein. (a) Induce CrtB at 25℃; (b) Induce CrtB at 30℃.]]
  
 
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Revision as of 05:17, 19 October 2019


Phytoene Synthase (CrtB) from Rhodobacter sphaeroides

This part is a phytoene synthase which transforms geranylgeranyl diphosphate into 15-cis-phytoene. It is a crucial enzyme in lycopene synthesis pathway and always works together with geranylgeranyl diphosphate synthase (CrtE) and phytoene desaturase (CrtI). This sequence is optimized for use in E.coli.

Usage and Biology

Firstly, the CrtB gene was amplified from Rhodobacter sphaeroides's genomic sequence by PCR (see Figure 1a) and was cloned into pET-30a(+) plasmid. Then, colony PCR was run to check the success of ligation (see Figure 1b).

Fig. 1 pET-CrtB plasmid construction. (a) Amplified CrtB from Rhodobacter sphaeroides's genomic sequence; (b) Colony PCR verified that CrtB was successfully assembled into the pET-30a (+) vector.

In order to determine the optimal induction condition of CrtB, we transferred the constructed plasmid pET-30a-CrtB into E. coli BL21 (DE3) and induced it under gradient IPTG concentration and different temperature conditions (see Figure 2). We found that CrtB could be induced at both 25℃ and 30℃, and the minimum IPTG concentration to induced CrtB was 0.02 mM.

Fig. 2 SDS-PAGE assay for CrtB protein. (a) Induce CrtB at 25℃; (b) Induce CrtB at 30℃.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 245
    Illegal NotI site found at 968
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1025
    Illegal XhoI site found at 334
    Illegal XhoI site found at 1006
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 264
    Illegal NgoMIV site found at 722
  • 1000
    COMPATIBLE WITH RFC[1000]