Part:BBa_K2971004

crtEBI under pBad promotor

This composite part contains the genes crtE, crtB and crtI combined into a single reading frame to produce lycopene when expressed in Escherichia coli. The genes express geranylgeranyl diphosphate synthase (GGPPS), phytoene synthase (CrtB) and phytoene desaturase (CrtI) respectively. GGPPS and CrtB produce the saturated compound phytoene, which is then desaturated by CrtI into the conjugated compound lycopene [1]. When the part was expressed in E.coli, the pellet of the spun down expression culture acquired a pink color. The color validate that the part functions as intended.

Results

Figure 1: Expression of pBAD-crtEBI in Escherichia coli (BL21)
Expression was induced in 2% arabinose
From left to right :Uninduced E. coli with empty expression vector, Induced E.coli with empty expression vector, induced E.coli with crtEBI expression vector, uninduced E.coli with crtEBI expression vector.

The composite part was made by fusing crtEB (BBa_K2971010) and crtI (BBa_K2971001) via Gibson cloning. The part was then transformed into competent cells of E. coli. The presence of the insert was screened with colony pcr (figure 2). The colony with the insert was analyzed by sequencing to check for potential mutations or frame-shifts.

Figure 2: Colony PCR of cells transformed with crtEBI
Only one colony (8) showed the presence of the correct insert. 8 was then further investigated.

We used thin layer chromatography (TLC) to confirm that the red color we observed in the pellet was a caused by lycopene (figure 3). Pellets from 50ml cultures were lysed in a 1:9 mixture of acetone and benzene. After centrifugation the organic phase was applied to the TLC plate. The mobile phase was pure acetone.

Figure 3: TLC on lycopene extract of induced cell cultures
The right image shows the TLC plate under UV light. The left image shows the same plate in normal light. Extracts were taken from induced BBa K274120 (furthest left on the plate), induced empty vector (middle of plate) and induced Ba K2971004 (right on the plate). There is a clear red spot at the top of the mobile phase from the sample isolated from a K2971004 confirming the presence of lycopene.

References 1. Tian, B., & Hua, Y. (2010). Carotenoid biosynthesis in extremophilic Deinococcus–Thermus bacteria. Trends in Microbiology, 18(11), 512-520. doi:10.1016/j.tim.2010.07.007

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  Illegal NheI site found at 4181
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2854
  Illegal BamHI site found at 1144
  Illegal BamHI site found at 2093
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1295
  Illegal SapI site found at 961