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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 confirms that E. coli has got a native LacI protein that represses the Lac promoter on the plasmid. C. freundii may lack a native LacI protein or 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.
One reason the Lac promoter coupled to our BioBricks is not regulated is because its LacI binding sequence might be different from that of the native Citrobacter freundii operator sequence. To test this, we have done a sequence alignment of the region where we think the Citrobacter freundii operator region might be with the consensus operator sequence in E. coli (5'-T GGAATTGTGAGCGGATAACAATT-3'). The sequence alignment can be seen in Figure 2 below.
Figure 2 – Sequence alignment between our Citrobacter freundii sequence and the consensus E. coli operator region sequence
As it can be seen from this sequence alignment, the Citrobacter freundii sequence, while showing some similarities, is not completely identical to the E. coli consensus sequence, which might be the reason.
We then looked at the sequence of the LacR repressor protein in Citrobacter freundii, as it is known that the N-terminal sequence of this protein is what binds to DNA. We wanted to see whether there are any differences between this protein’s N-terminal sequence and that of E. coli MG1655, the strain that is most often used in iGEM and in labs in general. The protein BLAST results can be seen in Figure 3.
Figure 3 – protein BLAST results showing homology between the Citrobacter freundii (Query) and E. coli
K-12 MG1655 (Subject) Lac repressor protein sequence.
This result shows that the N-terminus of this protein is well conserved between these two organisms, but there are a few differences in amino acids that may account for this protein not being able to regulate a foreign Lac promoter.
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 activity.
