"
Citrobacter freundii Characterisation:
Lac promoter characterisation
As the lac promoter is often used in synthetic biology, we wanted to test the its activity out in C. freundii by measuring the fluorescence of the RFP gene tied to this promoter. The reason for doing this is because it is not yet known whether our strain of C. freundii has a lacI gene. If the lacI gene is present on the host’s chromosome, we expect the fluorescence to be much lower when the cells are grown in media that contain no IPTG than in the ones that contain IPTG as the promoter will not be on. If there is no lacI gene, or if the C. freundii lacI cannot inhibit the E. coli Lac promoter, we expect the fluorescence in all 3 bottles will fall into a similar range.
Method
Three different sets of E. coli and C. freundii containing the pSB1C3 + Plac-RFP plasmid were grown overnight in LB+chloramphenicol, their OD was measured in order to normalize the number of cells and the normalized dilutions were used to inoculate 2.5ml M9 media that contained chloramphenicol and either no IPTG or 1, 2, 3, 4 or 5 μl IPTG. These bottles were then incubated at 37°C for 24 hours. The overnight LB cultures were inoculated into M9 in order to minimize background fluorescence to get clearer results.
The fluorescence of the cultures was measured just after inoculation (using a green filter with the fluorimeter) and it was fairly even within the two species, averaging 1669.88 FSU for E. coli and 1235.91 FSU for C. freundii. Their fluorescence and OD was again measured after 24 hours in order to quantify RFP expression. These readings were normalized by dividing the fluorescence with the OD and the averages of the three sets were calculated.
Results
Figure 1 - Graphs showing RFP fluorescence when cells were grown in M9 with or without IPTG
In Figure 1 above, the peaks that can be seen in C. freundii at 2 and 4 μl IPTG are due very high fluorescence readings in one of the sets, which have skewed these averages somewhat. These results do, however, show that there is a significant difference in RFP expression in E. coli with and without IPTG, while no significant difference in the levels of RFP expression is observable in C. freundii. This suggests that E. coli has got a native LacI gene that represses the Lac promoter on the plasmid, while C. freundii lacks a native LacI gene, or that C. freundii can’t regulate the E. coli Lac promoter, which results in the RFP gene being constitutively expressed in C. freundii.
Genome sequencing
The genomes of two C. freundii strains (the type strain, ATCC 8090 and another strain our lab had, called SBS 197) were sequenced in Newcastle by Dr Wendy Smith and Prof Anil Wipat with IonTorrent Sequencing. We hoped that these sequences would help elucidate the mystery of the constitutive lac promoter, but unfortunately the sequence reads were very poor in the region of the lacI gene in both sequences, so for the time being, this still remains a mystery.
Is an unregulated Plac promoter a bad thing?
Not necessarily, as regulation can still be obtained if the E. coli lacI gene is supplied in addition to the Plac construct. By controlling lacI expression levels, expression can be controlled without IPTG, as in the repressilator, toggle switch and other devices, without worrying about endogenous lacI.
