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Citrobacter freundii Characterisation:
Replicon compatibility
To even consider a new chassis for synthetic biology (and especially iGEM), it should first of all be able to replicate the various types of plasmids that are used to insert genes/BioBricks into it. To test this, our C. freundii was transformed with several plasmids containing the most commonly used replicons. The transformations were done according to standard protocol written for E. coli and the transformants were plated out onto media as indicated in Table 1 and shown in Figure 1.
Table 1 - Replicon compatibility media and results
Figure 1 - Plates containing E. coli or Citrobacter freundii transformed with plasmids that have different replicons. Note that the top row shows Citrobacter freundii cells and the bottom row shows E. coli cells, except for the first plates in each row, which are switched around, for colour comparison.
These results show that all but one of the plasmids have successfully been transformed into both E. coli and C. freundii, therefore these replicons are compatible with C. freundii.
C. freundii cells with the multi-host plasmid (pTG262) did not grow at all. The most probable reason is that the cmlR gene (which confers chloramphenicol resistance) in pTG262 is a Gram positive one (from Lactobacillus) which works much less well even in E. coli than the standard iGEM cmlR gene, so if C. freundii is a little more chloramphenicol-sensitive or expresses it a little worse, it would explain why no growth was seen on this plate.
pTG262 characterisation
One reason why the C. freundii cell transformed with pTG262 did not grow may be that the C. freundii cells are less resistant to chloramphenicol than E. coli, so they were plated onto plates with varying concentrations of chloramphenicol (Table 2) to see whether they grow at all. pSB2K3 was used as a negative control, as it does not have chloramphenicol resistance.
Table 2 - Table indicating the amount of chloramphenicol added to each (20ml) plate.
Method
The plates were incubated for two days and growth was observed on the 5 and 10 μl chloramphenicol plates. In order to assess whether growth on these plates was due to the activity of the resistance gene on the plasmid or due to some innate resistance to chloramphenicol, 20 μl of C. freundii containing the plasmid or 20 μl untransformed C. freundii were plated out onto LB agar containing 5, 6, 7, 8, 9 or 10 μl chloramphenicol and incubated at 37°C overnight.
Close the method.
The C. freundii + pTG262 plasmid grew on all plates to some extent whereas the no plasmid control grew only below 8 μl chloramphenicol and there were fewer colonies on the plates compared to the C. freundii + pTG262. This suggests that the plasmid works, but very weakly.
Conclusions
- We characterised the compatibility of Citrobacter freundii with the major replicon types used by iGEM
- We extensively characterized the pTG262 plasmid to figure out why it was not working well in Citrobacter freundii
- We have concluded that the other replicons are compatible with this organism
