Composite

Part:BBa_K2824008

Designed by: Jiang Qiaochu   Group: iGEM18_NEU_China_B   (2018-10-06)


Lldr-T7-lldPRD operon promoter-GFP

The aim of this part is using llDPRD promoter to activate downstream GFP reporter gene expression under different lactic acid concentration inductions and this operon is repressed without lactic acid induction. Briefly, LLdR repression protein specifically binds to llDPRD promoter and impedes downstream gene expression, while lactic acid enables to antagonize and replace LLdR protein to activate llDPRD promoter (Figure 1). In order to improve the GFP expression, we integrated a strong promoter, T7, into the upstream of lldPRD region.

This part is constructed based on the part lldRO1-plldR-lldRO2 (BBa_k82000). lldRO1-plldR-lldRO2 part is a lactic acid regulated promoter that is open to gene transcription in the presence of lactic acid. Comparing with the T7-lldPRD operon promoter-GFP part, the improvement of this part is that the LLDR is added to part and placed under the control of two promoters respectively to form a double expression regulatory system. This comparison with the two plasmids working together is more convenient for testing the success of the system.

Improve the Characterization of BBa_k82000

We can easily find that the Lldr-T7-lldPRD operon promoter-GFP part are in a higher level of expression comparing to the lldPRD operon promoter-GFP when the lactate concentration is lower than nearly 1.5 mM. Considering that the lactate concentration in yogurt is generally no more than 1 mM, the conclusion can give that the T7 promoter can enhance the signal intensity for our experiments (Figure 2).

T--NEU_China_B--8end0.png

Figure 1:Lldr-T7-lldPRD operon promoter-GFP

T--NEU_China_B--8end1.png

Figure 2:lldPRD operon promoter-GFP vs Lldr-T7-lldPRD operon promoter-GFP

To see more details about the construction and result, click the hyperlink below: lldPRD operon promoter + RBS from E. coli ( lldRO1-plldR-lldRO2 ):BBa_k82000(http://parts.igem.org/Part:BBa_K822000)


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2390
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 2555
    Illegal XhoI site found at 4356
    Illegal XhoI site found at 5248
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 567
    Illegal AgeI site found at 1608
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1792
    Illegal BsaI.rc site found at 5714
    Illegal BsaI.rc site found at 6373
    Illegal SapI.rc site found at 1236


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