Regulatory

Part:BBa_K2201020

Designed by: Lennard Karsten   Group: iGEM17_Bielefeld-CeBiTec   (2017-10-20)


lacO_tight1 from E. coli BL21(DE3) and optimized

lacO_tight1 from E. coli BL21(DE3) and optimized

Usage and Biology

This lac Operon was designed for optimal repression. The design is described here.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters

The goal was to investigate repression and induction of our designed lacO_tight1. lacO_tight1 was compared to different constructs. All constructs contained lacO to negatively regulated mRFP1 expression. The DNA constructs that were compared to each other are listed in Figure 1.



Figure 1: Different mRFP-DNA constructs that were compared for a fluorescence measurement. All constructs were inserted in pSB1C3

The DNA-constructs for mRFP-expression were transformed in E. coli BL21(DE3) and cultivated in 96 well plates for 3 h in the Tecan Infinte M200. Over night preculture was inoculated at OD650=0.1 again for 3 h for better technical replicate. Shown in Figure 2 are two biological replicates that were performed (left and right). Incubation in the Tecan was performed at 37 degrees and 300 rpm orbital shaking. OD650 and fluorescence (ex. 570 nm; em. 615 nm; maxima were not chose to avoid overlay effects) was measured at indicated time points.



<b>Figure 2: A: lacO_tight1 shows very low expression. B: lacO_tight2 was designed for better expression. For better expression tac promoter and T7g10 RBS was used. All OD650 measurements were similar of the different cultivations.

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