Part:BBa_K2556032

The lac repressor controled by arabinose binds to the lac operator to inhibit transcription The lac repressor controlled by arabinose binds to the lac operator to inhibit transcription.

Usage and Biology

This part is mainly composed of three elements: 
(1)Arabinose induced promoter PBAD to express the gene of downstream. This part contains the promoter as well as the coding sequence for the repressor AraC which is transcribed in the opposite direction. (“upstream”) By binding to L (+)-arabinose, AraC changes its conformation. This causes the protein to diffuse from the DNA thereby inducing transcription.
(2)This part contains the lac operator as well as the coding sequence for the repressor lacI. The lacI repressor binds to the lac operator to inhibit transcription in E.coli. This inhibition can be relieved by adding lactose or isopropyl -beta -D-thiogalactopyranoside (IPTG).
(3)GFP, as the report gene, can verify the degree of repression by repressor.

Characterize

Different concentrations of arabinose will induce these parts with different concentration of repressor proteins. By adding different concentration of arabinose, we can measure the fluorescence intensity of GFP and find out the relationship between the concentration of arabinose and RFU (Relative fluorescence unit) intensity. And by adding IPTG，the LacI protein is able to be released from the lac operator .It is a characteristic that can be utilized to further verify the function of the lacI-Plac by adding IPTG or not.

Experimental Results

Strain was cultured in the test tubes with liquid minimal medium. Different conditions are set in each test tube and the conditions can be seen clearly from the chart below.(Parallel three groups)

Table1.

OD600 and GFP values of the bacterial culture were measured periodically with the automatic microplate reader. For green fluorescence, excitation and emission wavelengths were 395 and 509 nm.

Fig2.

The LacI repressor is able to inhibit transcription to some extent in E. coli. This inhibition can be  relieved by adding  isopropyl-beta-D-thiogalactopyranoside (IPTG).
To optimize the condition of repression, strain was cultured in the test tubes with liquid minimal medium. And the concentration gradients of arabinose were set in these tubes(2×10-5M,4×10-5M,8×10-5M,1.6×10-4M,3.2×10-4M）.

Fig3.

When the concentration of arabinose is 2×10-5M,4×10-5M and 8×10-5M,the repression effect of LacI repressor is relatively close.Compared with these concentration,repression effect of LacI repressor increases after 30h under the concentration of arabinose is 16×10-5M.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 2640
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  Illegal BamHI site found at 3159
  Illegal XhoI site found at 3060
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961