Results

1. New Advance

BBa_K562000. The E. coli tat Promoter – A constitutive promoter (neither inducible or repressible) that will express genes at functional levels.

2. New Advance

BBa_K562009. The complete synthetic bacterial microcompartment (sphereactor) – microcompartment genes pduABTUNJK from Salmonella were assembled into a synthetic operon. Gene products of pduB, pduU, pduN and pduK all have hexa-Histidine tags for easy isolation or identification. Western immunoblots show proteins produced both individually and as a complex when expressed in the E. coli host. As well as biobricking the whole microcompartment (BBa_K562009), we also biobricked parts producing PduAB (BBa_562001), PduTU (BBa_K562006), PduN (BBa_K562005) and PduJK (BBa_K562004) separately, in case synthetic biologists wish to assemble their own microcompartments. Parts producing PduABTU (BBa_K562007) and PduABTUN (BBa_K562008) are also available.

3. New Advance and Proof of Principle

A) BBa_562001. This is a microcompartment targeting tag (PduD20) driven by the tat promoter from Bba_K562000). The Pdu20 tag is the first 20 amino acid residues of the PduD protein, which is normally inside the microcompartment. Part BBa_K562012 is a further modified version of BBa_562001 that produces a fusion between the Pdu20 tag and GFP. We hypothesized that the Pdu20 sequence would be sufficient to target GFP to the microcompartment and that this could be shown by confocal fluorescence microscopy. GFP was found to localize in the cytoplasm and within the microcompartment. The GFP also carries a haemagluttinin (HA) epitope tag in case future synthetic biologists need to follow its production.

B) BBa_562002. This is a microcompartment targeting tag (PduD40) driven by the tat promoter from Bba_K562000). The Pdu40 tag is the first 40 amino acid residues of the PduD protein, which is normally inside the microcompartment. Part BBa_K562013 is a further modified version of BBa_562002 that produces a fusion between the Pdu40 tag and GFP. We hypothesized that the Pdu40 sequence would target GFP to the microcompartment and that this could be shown by confocal fluorescence microscopy. GFP was found to localize in the cytoplasm and within the microcompartment. The GFP also carries an HA tag in case future synthetic biologists need to follow its production.

C) BBa_K562019. This is a further modified version of BBa_562002 that produces a fusion between the Pdu40 tag and GFP carrying a C-terminal ssrA tag. This targets the protein for destruction by ClpXP. We hypothesized that the Pdu40 sequence would target GFPssrA to the microcompartment and that this would protect GFP from degradation. Some GFP was found to by protected by-coexpression with the microcompartment.

4. Proof of Principle

A) BBa_K562010. This is a further modified version of BBa_562001 that produces a fusion between the Pdu20 tag and mCherry carrying a C-terminal HA tag. We hypothesized that the Pdu20 sequence would target mCherry to the microcompartment. Some mCherry was found in the microcompartment upon purification.