chAMBER Toolbox

The chAMBER toolbox is the first toolbox in the iGEM history to be entirely dedicated to protein compartmentalisation in bacteria. 

Plasmids for compartment formation

pENC (pMIC_Enca)

Plasmid for the expression of EncA in E. coli, a protein from Myxococcus xanthus, which forms a rigid shell compartment called encapsulin [1]. In this plasmid, the encA gene is under the control of the PBAD promoter (arabinose-inducible). BioBrick: BBa_K4229057

pWiffull (pHT1spysnpT2T3)

Plasmid for the expression of the H, T1, T2 and T3 proteins in E. coli from the myxobacterium Haliangium ochraceum, which form a rigid-shell compartment, called wiffleball [2]. The genes are arranged in an operon and are under the IPTG-inducable PLac 0-1 promoter. The T1 protein is fused to the SpyTag REF and SnoopTag REF for the recruitment of proteins of interest (POIs) in the compartment. This plasmid was a kind gift of Cheryl Kerfeld.BioBrick: BBa_K4229049

pWiffmin (pHT1spysnp)

Plasmid for the expression of the H and T1 proteins in E. coli from the myxobacterium Haliangium ochraceum, which form a rigid-shell compartment, called minimal wiffleball [2]. The genes are arranged in an operon and are under under the IPTG-inducable PLac 0-1 promoter. The T1 protein is fused to the SpyTag and SnoopTag for the recruitment of proteins of interest (POIs) in the compartment. This plasmid was a kind gift of Cheryl Kerfeld. BioBrick: BBa_K4229047

pLiquidro (pMic_spySPD5_snoop)

Plasmid for the expression of the spindle-deficient protein 5 (SPD-5) protein in E. coli from Caenorhabditis elegans, forming liquid droplets in vitro [3] and in vivo [4]. The spd-5 gene is under the control of the PBAD promoter (arabinose-inducible). The SPD-5 protein is fused to the SpyTag and SnoopTag for the recruitment of POIs in the liquid droplets. BioBrick: BBa_K4229078

Plasmids for recruitment of proteins into the compartments

pVenusCatch (pBbA2CmVenus2)

Plasmid for the expression of mVenus fused to the SpyCatcher in E. coli REF. The gene is under the TetR-responsive promoter (doxyxycline-inducible). This plasmid was a kind gift of Cheryl Kerfeld. BioBrick: BBa_K4229066

pTurquoiseCatch (pBbA2CmTurqiouse)

Plasmid for the expression of mTurqious fused to the SpyCatcher in E. coli REF. The gene is under the TetR-responsive promoter (doxyxycline-inducible). This plasmid was a kind gift of Cheryl Kerfeld.BioBrick: BBa_K4229067

pGFPenc (pVEN_sfGFP_TP)

Plasmid for the expression of sfGFP fused to the targetpeptide in E. coli for recruitment into encapsulins REF. BioBrick: BBa_K4229061

Usage of the toolbox

Researchers interested in assessing the effect of compartmentalisation on their POI shall simply select any of the plasmids for recruitment and either swap the gene with their gene of interest (GOI) or make a fusion between their GOI and the fluorescent protein-encoding gene already present in the plasmid. We suggest to test both N- and C-terminal tagging with the SpyCatcher or SnoopCatcher since proteins can differentially be affected by fusions at their termini.

After co-transformation of the appropriate compartment-forming plasmid and the POI-expression plasmid (be aware of the incompatibility groups of the plasmids [5] and/or of the resistance cassette present on the plasmid of choice) in E. coli, recruitment can be assessed via fluorescence microscopy or functionality tests can be conducted directly. 

Compatibility of the compartments

While we did not explicitly test this, we believe that at least two different compartments can be co-expressed in the same E. coli cell. Specifically, the encapsulins use a peptide for recruitment of POIs which is independent of the SpyCatcher/SpyTag – SnoopCatcher/SnoopTag system used for the wiffleball (full and minimal) and the SPD-5-mediated liquid droplets. Therefore, we envisage that researchers could perform multiplexing and compartmentalise several enzymes using encapsulins and either the wiffleball or the liquid droplets. Of course, plasmid incompatibility should be considered. Moreover, since it is not possible to co-transform four plasmids in E. coli, one plasmid would have to encode for all the GOIs and respective recruitment tags, or, alternatively, the cargo should be cloned into the same plasmid as encA itself.