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_K562003), 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_K562001. 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_K562001 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_K562002. 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_K562002 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_K562002 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_K562001 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.

B) BBa_K562011. This is a further modified version of BBa_K562002 that produces a fusion between the Pdu40 tag and mCherry carrying a C-terminal HA tag. Some mCherry was found in the microcompartment upon purification.

5. Application - magnetic bacteria

BBa_K562014. This is a further modified version of BBa_K562002 that produces a fusion between the Pdu40 tag and bacterioferritin (Bfr) from Escherichia coli. The Pdu40-Bfr fusion also carries a C-terminal HA tag. We hypothesized that the Pdu40 sequence would target Bfr to the microcompartment. Bfr forms a large storage protein for iron and iron oxides It was hoped that targeting Bfr to the sphereactor would concentrate iron in large particles in the cytoplasm.

We also designed a new part, but didn’t complete it yet, based on BBa_K562002 that produces a fusion between the Pdu40 tag and the HybA 16-Fe ferredoxin from Escherichia coli. The Pdu40-HybA fusion was designed so that the native Tat signal peptide and C-terminal transmembrane helix from HybA were removed . The construct also carries a C-terminal HA tag and is currently in the pT7.5 vector awaiting transfer into pSB1C3. It is hypothesised that the co-expression of PduD40-BfrHA and PduD40-HybAHA would fill the sphereactor with ferric oxide- and iron sulfide-containing proteins. Finally, in order to make an inorganic metal particle within the sphereactor it was planned to target a temperature-sensitive protease into the microcompartment with a view to destroying the protein backbone holding the iron compounds. The protease chosen was DegP from Escherichia coli, which is a chaperone at 30 C and a protease at 37 C. We designed a new part, but again did not complete it in time, based on BBa_K562002 that produces a fusion between the Pdu40 tag and the DegP protease lacking its N-terminal Sec signal peptide and carrying a C-terminal HA tag. The PduD40-DegPHA construct has been assembled in pT7.5 but has not been moved into pSB1C3 yet.

What could a magnetic bacterium be used for? We imagine that engineering magnetic particles in bacteria could be coupled to other bioremediation processes. For example, iGEM teams working on environmental or medical problems may want to recover the engineered organisms at the end of the treatment. Being able to recover all engineered bacteria by use of a magnet may be one solution.

Biobricks we improved upon

1. Application - arsenic bioremediation

BBa_K562018. This is a further modified version of BBa_K562002 that produces a fusion between the Pdu40 tag and the fMT gene from BBa_K190019 (Groningen, 2009) - EXPERIENCE. This part encodes metallothionein from Fucus vesiculosus (a type of seaweed), which is a protein that is good at binding arsenic. We hypothesised that targeting fMT into a microcompartment would enable bacteria to sequester even more toxic arsenic than ever before. BBa_K562018 thus produces a fusion between Pdu40 and fMT, which also carries a C-terminal HA epitope tag, and should be targeted into the sphereactor.

2. Application - bacterial lemonade

A) BBa_K562015. This is a further modified version of BBa_K562002 that produces a fusion between the Pdu40 tag and the limonene synthase from BBa_I742111 (Edinburgh, 2007) - EXPERIENCE. We improved the part by adding a PduD40 microcompartment targeting tag and a C-terminal HA epitope tag for easier characterisation of the protein product.

B) BBa_K562016. This is a modified version of BBa_K562002 that produces a fusion between the Pdu40 tag and the D-xylose isomerase from Escherichia coli K-12. We added a PduD40 microcompartment targeting tag and a C-terminal HA epitope tag for easier characterisation of the protein product.

C) BBa_K562017. This is a modified version of BBa_K562002 that produces a fusion between the Pdu40 tag and the aspartate ammonialyase from BBa_C0083 (Polkadorks, IAP, 2004) - EXPERIENCE. We improved the part by adding a PduD40 microcompartment targeting tag and a C-terminal HA epitope tag for easier characterisation of the protein product.

This overall aim of this project was to increase reaction effiencies and concentrate lemonade ingredients into the microcompartments to allow purification of tiny balls of flavour using the Histags on the sphereactor.

Our Favourite Parts

1. The synthetic bacterial microcompartment - The sphereactor: BBa_K562009

Capsid protein like structure which in bacteria such as Salmonella functions as a metabolic organelle. Bacterial microcompartment consist of a few thousand shell proteins which contain two or more enzymes which enhances or sequesters of specific metabolic pathway, normally involving toxic or volatile intermediates. The seven genes encoding the BMC Pdu AB, TU, N and JK were ligated into firstly pT7.5 and then into the biobrick plasmid pSB1C3. BMC protein expression was measured using western blotting, staining and also by radioactive labeling. BMC’s were also visualized using fluorescence microscopy and also using electron microscopy. We call our synthetic BMC the ‘sphereactor’.

2. The PduD20 targeting tag: BBa_K562001

A 20 amino sequence from the PduD protein which we hypothesized would act as a signaling sequence to target our proteins of interest to the interior of the BMC. It was previously thought that 40 amino acids was essential for targeting however our experiments where our PduD20 was fused to GFP have shown that it is possible to use a 20 amino acid sequence as a signaling sequence.

3. GFP with an ssrA destruction tag: BBa_K562019

GFP with an ssrA tag which would target GFP for degradation by the protease ClpXP. This allowed us to test our signal sequence (PduD40) since any GFP which left in the bacterial cytoplasm would be broken down, while the GFP targeted to the BMC would be safe from ClpXP provided that it reaches the BMC before it is broken down. Our blots of GFPssrA expressed without the microcompartment show no detectable levels of GFP, however when the microcompartment is present GFP was detected suggesting that GFP was being sequestered inside the sphereactor and so protected.