Part:BBa_K3457054

J23109-Olac-RBS-sfGFP

    This biological part is the expression sequence of Super-folder Green Fluorescent Protein, also called sfGFP. We use it in the experiment to test J23109, J23107 and J23100 promoters.

Group: QHFZ-China iGEM 2020

Design

Figure 1. The Schematic cartoon of the DNA construct of BBa_K3457054.

    This part was modified from BBa_K3457037 T7-Olac-RBS-CAHS 106094 by Golden Gate technique.

Documentation:

Protocol:

    We modified a frequently and widely used vector, pet28a+ and put this part into it (Fig. 2). Then we transformed the plasmid into E. coli BL21 strain.

Figure 2. The Schematic cartoon of the vector.

    We used the following protocol to detect fluorescence:
(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 2mM iPTG 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) The bacteria solution was centrifuged and the LB medium was removed. Then the bacteria was resuspended by PBS. 100 μL such 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 PBS was deducted from the result above. GFP / OD₆₀₀ was calculated.

Results:

Figure 3. Via a reporter, sfGFP, the expression function of the vector was verified.

    sfGFP was normally expressed. J23109 is a weak promoter.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 11
  Illegal NheI site found at 34
  Illegal NheI site found at 978
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]