Part:BBa_K3457037

T7-Olac-RBS-sfGFP

    This biological part is the expression sequence of Super-folder Green Fluorescent Protein, also called sfGFP. It was mainly used as the control group of our project this year. We also use it to test J23109, J23107 and J23100 promoters.

Group: QHFZ-China iGEM 2020

Design

Figure 1. The Schematic cartoon of the DNA construct of BBa_K3457037.

    This part should be composed of a T7 promoter, RBS B0034, CDS of sfGFP BBa_K3457015, and a T7 terminator. However, we added another terminator B0015 before the T7 terminator. The reason is that if the T7 promoter is changed into other promoters, such as J23100, the corresponding RNA polymerase changed from T7 RNAP to endogenous RNAP of E. coli, which can be stopped by terminator B0015. In other words, the added terminator made the part easier to be modified. In the cartoon (Fig. 1), there are two points around the T7 promoter. They are two BsaI cutting site, which can be used to change the T7 promoter by Golden Gate technique.

Documentation:

Introduction:

    This year, we tried to introduce a new biopreservation method. We used freeze-drying to make the engineered into dry powder. Then the powder can be stored at room temperature for a long time. This method can make the storage of bacteria get rid of ultra-low temperature freezer, so that it will promote the practical application of engineered bacteria out of laboratory. However, the stresses during freeze-drying and subsequent dry storage, including freeze, dry and vacuum, are lethal to bacteria. We use TDPs, to help bacteria survive the situation. In these tests, sfGFP was the control group, as well as a reporter.

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) 100 μL bacteria 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.

Results:

Figure 3. sfGFP was expressed with T7 promoter.

    In the modified pet28a vector, sfGFP was normally expressed.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 972
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 27
  Illegal BsaI.rc site found at 2