Intermediate

Part:BBa_K1899009

Designed by: iGEM2016_HKUST   Group: iGEM16_Hong_Kong_HKUST   (2016-10-18)


B0032-LacI

LacI is conjugated to a medium strength RBS

Construct for Characterisation

Fig.1 lacI-lacp with reporter gene.

In order to characterise the efficiency of this construction intermediate, it was ligated with a construct containing lacp and GFP reporter gene, BBa_E0240. The expression of LacI was driven by a strong constitutive promoter, BBa_J23101, and terminated by BBa_B1006 terminator. The assembly processes were performed in BioBrick RFC10 standard.

Results

Experiments on comparing the GFP expression driven by lacp from constructs with and without BBa_B0032-lacI were performed. Negative control used was BBa_E0240. Results indicated that BBa_B0032-lacI driven by BBa_J23101 and terminated by BBa_B1006 could reduce the GFP expression by 2.9 times.

Fig 2. Comparison on the GFP expression of construct with and without BBa_B0032-lacI. BBa_E0240 was used as negative control. Characterisation was done using E. coli strain JW0336. Cells were first pre-cultured overnight and were subcultured to mid-log phase where GFP emission measurements were made using an EnVision® multilabel reader. This result was obtained by combining 3 characterisation data obtained in 3 different days. Error bar present SD from 3 biological replicates.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1166
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//chassis/prokaryote/ecoli
Parameters
None