Part:BBa_K3457056

Arac-Pbad-GFP

    This biological part, given by iGEM Team NEFU-China 2020, is the expression sequence of Green Fluorescent Protein, also called GFP. We use it in the experiment to test arabinose sensor. It also used as the control group in some of our freeze-drying experiments.

Author: Yixian Yang

     TDPs (Tardigrade intrinsically Disordered Proteins) show protective effect to bacteria. However, ralated studies are all use T7 promoter and E. coli BL21 (DE3) strain. This year, we used the Inducible pBad/araC promoter to express a TDP, CAHS 106094, in another starin, when this part was used as the control and reporter. For all the experiments below, we use E. coli DH5α strain.

Design

Figure 1. The Schematic cartoon of the DNA construct.

Documentation:

Protocol:

    The protocol is as below:
(1) Pick clones which are in good condition and put them into 500 μL LB medium containing antibiotics. Shake them to grow at 37℃ for 5~7 hours until the bacteria solution becomes turbid.
(2) Add inducer into 3 mL LB medium containing antibiotics. Add 3 μL of the bacteria solution mentioned in step 1 to dilute the bacteria by the ratio of 1:1000. Shake the solution to grow the bacteria at 37℃ overnight.
(3) 100 μL bacteria solution solution was put into a well of a 96-well palte. The GFP fluorescence and OD₆₀₀ were detected by a microplate readers (Bio-Teck). The parameters are: exciting light: 488 nm, light reception: 520 nm, gain: 50.
(4) The value of LB medium was deducted from the result above. GFP / OD₆₀₀ was calculated.
Note: For step (3), The inducer usually was 0.2% L-arabinose. However, it can be changed if we want to study the concentration of the inducer and the effect of D-arabinose. To study whether glucose and trehalose affect the promoter, we even added D-glucose or D-trehalose of different concentrations with 0.2% L-arabinose into the medium.

Result

Figure 2. GFP can be induced by 0.2% L-arabinose, but not D-arabinose. Glucose and trehalose afected the promoter.

     The result is quite clear. First, L-arabinose efficiently induced the expression of GFP. 0.2% arabinose seemed enough. However, D-arabinose did not have any effect. Secondly, glucose and trehalose supressed the promoter, even though 0.2% L-arabinose was added. At the concentration of 0.3%, the effect of trehalose was even wore than that of glucose. The effect of glucose is well-known. However, that of trehalose is not. We give a hypothesis here. One trehalose can be hydrolyzed into two glucose. So 0.3% trehalose equals to 0.6% glucose. Thus, at a low concentration, trehalose is more unsufferable to the promoter than glucose.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 1704
  Illegal EcoRI site found at 2016
  Illegal SpeI site found at 2007
  Illegal PstI site found at 2034
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 1704
  Illegal EcoRI site found at 2016
  Illegal SpeI site found at 2007
  Illegal PstI site found at 2034
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 1704
  Illegal EcoRI site found at 2016
  Illegal BglII site found at 1998
  Illegal BamHI site found at 1144
  Illegal BamHI site found at 1913
  Illegal XhoI site found at 1281
  Illegal XhoI site found at 1989
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 1704
  Illegal EcoRI site found at 2016
  Illegal SpeI site found at 2007
  Illegal PstI site found at 2034
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 1704
  Illegal EcoRI site found at 2016
  Illegal SpeI site found at 2007
  Illegal PstI site found at 2034
  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961