Team:Westminster UK/Results
Labwork
Promoter Assay
Optical density was measured for quantification of promoter efficiency at 588nm and 600 nm. Untransformed E. Coli Top10 and Shewanella oneidensis MR-1 were used as a control and reference. Optical density measurements were taken in triplicates to obtain mean values using the Nanodrop 200 Thermo scientific every hour for 5 consecutive hours.
Figure 1: Optical density values for BBa_J23100 transformed E.coli top10, control E.coli and controll S.oneidensis Mr-1 for every hour across a 5 hour period at 588nm (OOD588) and 600nm (OD600)
Results show that although significantly lower growth was observed in Shewanella oneidensis MR-1, data collected is meaningful, providing characterisation of widely used BBa_J04450 part in a different chassis.
TodE
TodE 3-methylcatechol 2,3-dioxygenase enzyme was ligated to BBa_J23100 constitutive promoter, BBa_B0034 strong Ribosome Binding Site (RBS) in order to improve expression and fulfill the requirement of improving a biobrick. E. Coli TOP10 and 2mM 3-methylcatechol and AV at 384nm were used as controls for comparison. Expression was monitored by means of absorbance values where measurements were taken in triplicates to obtain a mean value using the Nanodrop 200 Thermo scientific every half hour for 3 hours.
Figure 2: Mean absorbance values of TodE with BBa_J23100 promoter and BBa_B0034 RBS, E.coli TOP10 and AV control at half hour intervalss acrossss a 3 hour period
It can be observed that the addition of a different RBS and Promoter to the TodE coding sequence resulted in a significant improvement in expression when compared to the AV E-coli control by more than 14 fold at 3 hours. However, this improvement was not as significant as results obtained from the previous years where expression was more than 20 fold higher in comparison to AV control at 3 hours.
Co-culturing S.oneidensis with C.reinhardtiCo-culture assay
The co-culture assay was performed in collaboration with the Kaiserslautern iGEM team, who were working with transforming PETase and MHETase genes into Chlamidomonas reinhardti. With a bacterial sample they kindly provided us with we attempted to fulfil our sustainability objective by exploring the possibility of both S. oneidensis and C.reinhardti living in an engineered symbiotic relationship in order to increase final electrical output. Four different combinations of S.oneidensis, C.reinhardti along with TAP and MSM media were tested and, voltages were recorded over a period of 5 days.
|
MFC Number |
Shewanella oneidensis MR-1 culture % |
Chalamidonoas reinhardti culture % |
MSM media % |
TAP media % |
|
MFC 1 |
100 |
0 |
100 |
0 |
|
MFC 2 |
50 |
50 |
100 |
0 |
|
MFC 3 |
100 |
0 |
75 |
25 |
|
MFC 4 |
50 |
50 |
75 |
25 |
Table 1: Combinations of Shewanella oneidensis and Chalamidonoas reinhardti in 4 MFC's
Figure 3: Electrical output of S.oneidensis and C.reinhardti MFC's at different combinationss; 100% S.oneidensis, 0% C.reinhardti, MSM 100% and 0% TAP (S_MSM). 50% S.oneidensis, 50% C.reinhardti, MSM 100% and 0% TAP (SC_MSM), 100% S.oneidensis, 0% C.reinhardti, MSM 100% and 0% TAP (S_MSM), 100% S.oneidensis, 0% C.reinhardti, MSM 75% and 25% TAP (S_MSMTAMP) and 50% S.oneidensis, 50% C.reinhardti, MSM 75% and 25% TAP(SC_MSMTAP)
Results display that the pure MSM media MFC setup (S_MSM) produced the highest voltage, between 19 and 13mV, indicating the importance of media content on electrical output of MFCs. Similarly, the use of pure MSM media with both bacteria produced the second highest voltage MFC, reinforcing this idea and suggesting the possibility of co-culturing. The use of TAP in both the co-cultured and only S.oneidensis bacteria MFC displayed similar trends to each other with the later only producing a slightly higher electrical output than the former. These results also show that all 4 MFCs which constituted of S.oneidensis and MSM media ran successfully to produce an electrical output thus once again reinforcing the notion that media content, especially MSM media, is an important constituent of an MFC and, S.oneidensis is acceptable to use in an MFC system.
Cell count ratios
Ratios of cell counts were performed on SC_TAPMSM and SC_MSM MFCs in order to confirm the survival of both bacterial species in a co-culture.
Figure 4: Cell count ratios for MFCs SC_TAPMSM and SC_MSM
Results for cell count ratios suggest that MSM media is more suitable for co-culture in comparison to MSM media with TAP as the original ratio of the species were maintained. The results also indicate the survival of both organisms during co-culturing suggesting a symbiotic relationship existing between the two.
MFC’s
MFC’s permease channel and 4 MFC’s test constructs supplemented with 100ng/ul of Chlorampheniucol and 100ng/ul Amphicillin for selection. MFC output data for CRP, EtrA, RhlA and RpoE genes were collected over 4 days.
Figure 5 : MFC output data for the 6 MFCs: CRP, EtrA, RhLA and RpoE, no antibiotic control (Shewanella WT) and TPHP-PPSB1C3 control (TPHP control) over a running period of 4 days
It can be observed by these results that the over expression of oxygen-responsive transcriptional regulator Fnr EtrA showed the most significant increase from 0mV to over 100mV in comparison to both controls and other gene constructs. This therefore indicates that we succeeded in testing and producing an MFC using the Shewanella oneidensis strain MR-1 However, over expression of the RpoE and RhlA genes in their constructs were observed to be unsuccessful at increasing MFC output when compared to both controls by achieving around 10mV electrical output. Yet, the CRP construct was observed to generally produce a higher electrical output than the no antibiotic control. Interestingly, the TPHP-PSB1C3 containing control showed significantly higher electrical output compared to the no antibiotic control and RhlA, RpoE and CRP gene constructs.
