Part:BBa_K1172501:Experience

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Applications of BBa_K1172501

Combination with pBad to Reduce Replication Stress (Newcastle iGEM 2016)

As observed by the [http://2013.igem.org/Team:Bielefeld-Germany/Project/Porins Bielefeld team] in 2013, expression oprF leads to lower growth due to the higher replication stress. This effect is amplified by the stronger promoters such as a T7 promoter. By changing the promoter to a pBAD promoter which is regulated by L-arabinose we were able to reduce replication stress during the exponential phase of bacteria growth. This is important for fuel cell use as it allows a large population to be generated quickly. Porin expression can then later be triggered by the addition of L_arabinose. Cells containing our device (both the novel porin and the equivalent to Bielefeld’s device) grow normally allowing induction when the culture is established.

We used a variation on the [http://2016.igem.org/Team:Newcastle/Protocols#Microbial-fuel-cell University of Reading's NCBE fuel cell protocol] for our experiments. The variation we made was dissolving 1g of arabinose as well as 9g of glucose into 50 ml of potassium phosphate buffer solution. 4 ml of the L-arabinose orpF expression constructs [BBa_K1895005] cell cultures were added to the fuel cell.

In our first experiment we verified that the expression of orpF increased the output voltage of the fuel cell. We did this by leaving the fuel cells to run for an hour whilst voltage taken every 3 minutes.

Figure 1: Reading microbial fuel cell output (mean±SE, mV). BBa_K1895005 caused overexpression of large porins, BBa_K1895004 caused overexpression of small porins. Separately, BBa_K1895004 with glucose only (no porin expression induction by arabinose) and LB broth only were used as negative controls. For both negative controls error bars are negligible and therefore difficult to distinguish on the graph. Voltages were measured every 3 minutes via digital voltmeter and the experiment stopped after 60 minutes.

As can be seen from figure 1 BBa_K1895005, the OprF expression device results in a larger voltage as experience by the Bielfeld team. The subsequent step was to show that without arabinose in the growth media these bacteria experience less replication stress.

Growth curves of the different battery constructs at 37°C were created. This was done by growing cells with the devices in a 96 well-plate and measured and OD600 readings were taken every five minutes. The cultures were shaken between each measurements. The cells grown in the prescence of arabinose were found to have a lower growth rate due to the overexpression of the porins. However, the cells grown in LB broth without arabinose have a much higher growth rate, see figure 2.

Figure 2: Growth curves of the different battery constructs at 37°C for 20 hours. These were grown in a 96 well-plate and measured using a BIOTEK Powerwave HT. OD600 readings were taken every five minutes and the cultures were shaken inbetween measurements. The cells grown in the prescence of arabinose were found to have a lower growth rate due to the overexpression of the porins. However, the cells grown in LB broth without arabinose have a much higher growth rate.

As evidenced by the growth curve in figure 2, we can confirm that by placing this construct under the pBAD arabinose-induced promoter, we can increase the overall growth of the culture.

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