Part:BBa_K2555003

pBAD-RBS-sfGFP

BBa_K2555003 contains pBAD promoter, RBS, and superfolder GFP. The construct is used to indirectly reflect the expression of cueR of BBa_K2555002 under different concentration of L-arabinose by measuring green fluorescent intensity with plate reader. BBa_K2555002 contains pBAD (L-arabinose inducing promoter) with RBS and CueR ( a transcription factor that can bind on pcopA of BBa_K2555000). CueR behaves as a net activator or a net repressor under different copper concentrations(1). BBa_K2555002 and BBa_K2555000 are used for construction of BBa_K2555004.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 1311
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1250
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1085
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 1067
  Illegal SapI.rc site found at 1355
  

Method

In order to show that our synthetic bacteria have a fully functional part that works, expression measurements were made. The construct was linked to sf GFP. For the parts plate reader was done. A plate reader is able to take optical density (OD600nm) and fluorescent measurements over time. OD is a measure of bacterial growth over time and fluorescence is a measure of protein expression over time.

Results

<img src="">

The results of a plate reader experiment with different arabinose concentrations after five hours. Error bars show standard deviation of three repeats

With the increase of Arabinose concentration, the relative fluorescence value of the construct increased, which indirectly reflected the CueR protein expression of BBa_K2555002 increased with the increase of Arabinose concentration.

References

(1)Danya J. Martell, Chandra P. Joshi, Ahmed Gaballa, Ace George Santiago, Tai-Yen Chen, Won Jung, John D. Helmann, and Peng Chen (2015) “Metalloregulator CueR biases RNA polymerase’s kinetic sampling of dead-end or open complex to repress or activate transcription” Proc Natl Acad Sci U S A. 2015 Nov 3; 112(44): 13467–13472.