Part:BBa_K4594004
LuxC-D-A-B-E-G-F
The part consists of the basic lux operon(luxC, luxD, luxA, luxB, luxE) and genes proposed to enhance the brightness(luxG, luxF). The whole part has been successfully expressed in E.coli BL21(DE3) within a pET-28A plasmid. After 16h induction by IPTG, the blue luminescence can be observed by naked eyes in dark environment. It's better to start the induction when OD600 is between 0.6-0.8, and the best concentration of IPTG is between 0.05-0.2 mM according to our experiments. Additionally, results clearly showed luxF and luxG strongly enhanced the brightness of designed BL21(DE3) (Figure 3). And luminescence assay excitingly showed the addition of luxF and luxG increased the luminescence by 58% (Figure 4). So, our composite part is marvellous.
Fig1 BL21(DE3) with BBa_K4594004 and induced for 16h under 23℃.
Fig2 Determination of the best IPTG concentration for induction.The first 8 groups were BL21(DE3) with BBa_K4594004 in pET-28A plasmid and the negative control was BL21(DE3) with empty pET-28A plasmid. The luminescence test was conducted after 16h-induction under 23℃. And the concentration is shown on the graph. The result showed the best concentraion was between 0.05mM and 0.2mM.
Fig3 Comparing luminescence of BL21(DE3) with pET28a-luxCDABE or pET28a-luxCDABEGF. Images were taken by an ordinary phone camera. The left three tubes were BL21(DE3) with pET28a-luxCDABE and the right three tubes were BL21(DE3) with pET28a-luxCDABEGF. It was clear that luminescence can be enhanced by additional luxF and luxG. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG.
Fig4 The result of luminescence assay for BL21(DE3) with pET28a-luxCDABE and BL21(DE3) with pET28a-luxCDABEGF (n=6). The result showed that the addition of luxF and luxG increased the luminescence by 58%. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG. And the assay was conducted by a plate reader.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 7071
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 7071
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 5280
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 7071
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 7071
Illegal AgeI site found at 2937 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 1317
Illegal SapI site found at 3456
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