Part:BBa_K562010:Experience

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••••• iGEM Dundee 2011

This part was seen to work in practice. The PduD20 tag fused to mCherry resulted in fluorescent cells (Figure 1). The crude extract after cell breakage was bright pink (Figure 2) and, when co-expressed in the presence of the microcompartment encoded by BBa_K562009 was found to co-purify with the microcompartment (Figure 3).

Results

E. coli was transformed with BBa_K562010 and grown overnight in LB medium under aerobic conditions. A sample of whole cells was taken, washed in phosphate-buffered saline, and applied to a microscope slide using CellTak adhesive. The cells were then observed by confocal microscopy and shown to be fluorescent (Figure 1).

Figure 1: mCherry is produced and fluorescent when fused to the PduD20 signal sequence.

Next, E. coli was transformed with BBa_K562009 and BBa_K562010 and grown anaerobically for 16 hours in LB medium containing 0.4% (w/v) glucose. Cells were harvested, washed and broken in 50 mls B-PER solution, which resulted in a bright pink extract (Figure 2), before being loaded onto an IMAC column.

Figure 2: A crude extract of broken E. coli cells expressing BBa_K562010.

Next, proteins bound to the IMAC column were eluted with an imidazole gradient and analysed by SDS-PAGE. Samples (10 microlitres from each 1 ml fraction) were taken and mixed with Laemmli disaggregation buffer before being analysed by SDS-PAGE and Western immunoblotting. The PduD20-mCherry fusion had been further engineered to encode an HA epitope tag at its C-terminus - thus allowing easy recognition of full-length fusion proteins.

mCherry clearly co-purifies with the microcompartment (Figure 3) giving a strong indication that it has been targeted to - or into- the microcompartment.

Figure 3: Pdu20-mCherry-HA co-purifies with the His-tagged microcompartment. (A) SDS-PAGE analysis, followed by Coomassie staining, shows the elution of microcompartment proteins from the IMAC column. (B) Western immunoblotting of exactly the same fractions run in (A) above using a mouse monoclonal antibody raised against the HA epitope tag. This clearly identified PduD20-mCherry-HA in the eluted fractions.

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