Part:BBa_K2253001:Experience

Experience and quantification by iGEM BOKU Vienna 2021

We used the promotor for our Lactobacillus plantarum expression cassette. Team Costa Rica 2019 already quantified its strength in E.Coli, but one in Lactobacillus is still missing in the registry. Thus, we set up an experiment for it. We compared it with 7 other promoters.

Experimental set up

5ml cultures of the eight different transfected Lactobacillus plantarum NC8, as well as an untransfected control, were incubated at 30°C overnight under anaerobic conditions and constant shaking. On the following day, conditions were changed to aerobic for 3-4 hours. Afterwards, the cell suspensions were diluted to different densities (OD 600 of 2, 1 and 0.1). The measurement was carried out using a Tecan reader. Samples where excited at 587 nm and emission measured at 640 nm. Sterile media was used as a reference. Experiments were carried out in triplets.

Construct	Promotor
1	BBa_K3855000
2	BBA_K3855001
3	BBa_K3855002
4	BBA_K3855003
5	BBA_K3855004
6	BBA_K3855005
7	BBa_K2253000
8	BBa_K2253001

Results

We found that the promotor expression is of medium strength. P1,P2 and P3 were significantly stronger. While P8, P5 and P4 were significantly weaker. The two promotors by Team Austin Texas 2017 were of comparable strength.

Figure 1. Excitation at OD 2,1 and 0.1 of different mCherry transfected L.plantarum suspensions. Wildtype emission was subtracted. Raw values can be found in our Benchling write up (Cloning, Friday 8.10.2021).

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