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
Author: Yixian Yang
This part was modified from BBa_K3457037 T7-Olac-RBS-CAHS 106094 by Golden Gate technique.
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.
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.
sfGFP was normally expressed. J23107 is a moderate promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12Illegal NheI site found at 11
Illegal NheI site found at 34
Illegal NheI site found at 978
- 21COMPATIBLE WITH RFC
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC