Part:BBa_K116001:Experience

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UNIPV-Pavia

Characterization experiment on BBa_K116001 - UNIPV-Pavia Team (test performed by M. Meroso, G. Zambianchi) We received the BioBrick measurement system (which has a GFP protein generator downstream) of BBa_K116001 from iGEM HQ in September (it is called BBa_K116002). The bacterial strain that contained the plasmid was NEB 10-beta, so we decided to transform it into E. coli TOP10. We wanted to perform some experiments to better understand how it works and if can be successfully used. We performed several experiments with different LB medium and we got almost the same results. We used: LBK (NaCl 0M) (pH 5.5 - 6.6 - 7.5 - 8.5) LB NaCl 70mM (pH 5.5 - 6.6 - 7.5 - 8.5) LB NaCl 171mM (pH 5.5 - 6.6 - 7.5 - 8.5) LB NaCl 250mM (pH 5.5 - 6.6 - 7.5 - 8.5) LB NaCl 600mM (pH 10 - 11.2) Here we show just one experiment to explain our work. A complete report can be downloaded from this link. Experiment Na+ 250mM Motivation We’ll try again to make E. coli producing GFP at the variation of pH in presence of Na+. Methods Compatibility: E. coli TOP10 in pSB1A2 We prepared falcons of LB NaCl 250mM and adjusted pH using KOH and HCl to values 5.5, 6.6, 7.5 and 8.5. We inoculated 8ul of Invitrogen TOP10 containing BBa_K116002 into 4ml of LB + Amp and incubated overnight at 37°C, 220 rpm. We did the same for TOP10 with BBa_K173001 and BBa_B0033 inside. Next morning we put 50ul from each of the three falcon into 5ml of LB NaCl 250 mM pH 6.6 and incubated again for five hours and at 37°C, 220 rpm. We measured the final O.D. with TECAN F200 and diluted each genetic circuit into four falcons with LB NaCl 250mM at different pH (5.5 - 6.6 - 7.5 - 8.5) in order to obtain a same O.D. equal to 0,02 (12 falcons overall). Then we performed an experiment of 21 hours duration with measures of absorbance and fluorescence every 5 minutes with TECAN F200. Each value is the mean of three measurements and cultures were shaked for 15 seconds every five minutes. Results BBa_K116002 Absorbance BBa_K116002 Fluorescence pH 5,5: Fluorescence pH 6,6: Fluorescence pH 7,5: Fluorescence pH 8,5: Fluorescence Comments BBa_K116002 didn't produce any GFP as well as negative control (BBa_B0033): after the transient due to noise their GFP production rate goes to zero while positive control (BBa_K173001) has a significantly higher one. After looking better for a motivation in some articles ([Rachel Karpel et al.]) we think this could be because of the E. coli strain: we use TOP10 while a special strain (delta-nhaA) without some membrane proteins that regulate E. coli homeostasis is used in other experiments. Final considerations In our opinion this sensor (primarily sodium sensor and secondarily pH sensor) needs very particular conditions to work (first of all a specific bacterial strain) we couldn’t reproduce, so we consider it almost unusable. Sequence analysis from iGEM freezer The UNIPV-Pavia iGEM 2009 team requested this part from iGEM Headquarters, but sequence was not correct: actually, the part contained in the plasmid was BBa_K116002 and it also has an additional undocumented "c" nucleotide at the end of nhaA promoter (found after sequencing). Because BBa_K116002 is the measurement system of this BioBrick, we used it to characterize the activity of nhaA promoter (see Application section above).

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