Targeting GFP brick

Description: This targeting sequence consist in the large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme, RuBisCO, (BBa_K1113701) attached with a flexible linker (BBa_K105012) to the superfolder GFP (BBa_I746916) under the regulation of the pBad/araC promoter (BBa_I0500) and includes a RBS (BBa_B0034). The targeting sequence is constructed to deliver the fluorescence to the Carboxysome under the induction with L-arabinose. Since this is a targeting sequence, in order to work it needs to be expressed in the presence of the Carboxysome, (BBa_K1113100).

This part was constructed with a mutation in the Prefix (from “cggccgc” to “atacctg”), which erases the NotI restriction site. This modification allows the correct design of primers for Gibson Assembly. This part it’s also constructed on the pSB4K5 standard vector.
The large subunit of RuBisCO was obtained from the pHnCBS1D plasmid (plasmid provided by David Savage, Departments of Molecular & Cell Biology and Chemistry, University of California, Berkeley).

Carboxysome brick

Description: This Carboxysome operon includes 10 genes necessaries for the expression of the microcompartment: the large and small ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme (RuBisCO) subunits, 7 shell-associated proteins and the Carbonic Anhydrase. The whole operon is under the regulation of an ITPG-inducible promoter. It includes a RBS and a terminator. The operon it’s originally from Halothiobacillus neapolitanus but adapted to E. coli by P. Silver and D. Savage (“Modularity of a carbon-fixing protein organelle” Walter Bonacci, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver and David F. Savage) in which they took the original operon pHnCB and added a tenth gene, csoS1D, located just outside the core operon and show to be necessary for the correct assembly of the carboxysome.

The pHnCBS1D plasmid was provided by David Savage (Departments of Molecular & Cell Biology and Chemistry, University of California, Berkeley).

Backbone brick

Description: This composite part corresponds to the low-copy backbone vector pSB4K5 with the pBad/araC promoter (BBa_I0500) and a RBS (BBa_B0034). This will allow an easier assembly and expression for testing coding sequence as its expression is under an arabinose inducible promoter.

This part was constructed with a mutation in the Prefix (from “cggccgc” to “atacctg”) which erases the NotI restriction site. This modification allows the correct design of primers for Gibson Assembly.

Carbo + Biotinylation tag

Description: The carboxysome operon includes 10 genes necessaries for the expression of the microcompartment: The large and small RuBisCO subunits, 7 shell-associated proteins and the Carbonic Anhydrase. The whole operon is under the regulation of an ITPG-inducible promoter and includes a RBS and a terminator. The operon it’s originally from Halothiobacillus neapolitanus but adapted to E. coli by P. Silver and D. Savage (“Modularity of a carbon-fixing protein organelle” Walter Bonacci, Poh K. Teng, Bruno Afonso, Henrike Niederholtmeyer, Patricia Grob, Pamela A. Silver and David F. Savage) in which they took the original operon pHnCB and added a tenth gene, csoS1D, located just outside the core operon and show to be necessary for the correct assembly of the carboxysome. It has also been incorporated a red fluorescent protein (RFP) (BBa_E1010) fused with an ALA-ALA-ALA linker to one of the shell proteins (csoS1A) and a tag sequence that expressed the biotin ligase recognition peptide (BLRP) (BBa_), which can be biotinylated in the presence of the biotin ligase BirA enzyme native from Escherichia coli. This process allows later on a strong interaction between the biotin and streptavidin that can be used for purification purposes, specifically as an extraction method for recombinant or protein complexes.

The operon was obtained from the pHnCBS1D plasmid (plasmid provided by David Savage, Departments of Molecular & Cell Biology and Chemistry, University of California, Berkeley). The BLRP sequence was an adaptation from the plasmid provided by the INTACT method (“The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana”. Roger B Deal1 & Steven Henikoff)

Rub L - Bgal brick

Description: Includes the lacZ gene (BBa_I732005), which encodes for the β-galactosidase attached with a linker (BBa_K105012) to the large subunit of the RuBisCO (BBa_K1113701), under the regulation of the pBad/araC promoter (BBa_I0500), and includes a RBS (BBa_B0034). The construct was made to prove the delivery of the β-galactosidase to the inside of the BMC and to test reactions in vitro within the carboxysome.

The large subunit of the RuBisCO was obtained from the pHnCBS1D plasmid (plasmid provided by David Savage, Departments of Molecular & Cell Biology and Chemistry, University of California, Berkeley).

This part was constructed with a mutation on the prefix (from “cggccgc” to “atacctg”), which erases the NotI restriction site. This modification allows the correct design of primers for Gibson Assembly. This part was constructed on the pSB1C3 standard vector.

Rub L - Bgal brick

Description: Includes the lacZ gene (BBa_I732005), which encodes for the β-galactosidase attached with a linker (BBa_K105012) to the large subunit of the RuBisCO (BBa_K1113701) under the regulation of the pBad/araC promoter (BBa_I0500), and includes a RBS (BBa_B0034). The construct was made to prove the delivery of the β-galactosidase to the inside of the BMC and to test reactions in vitro within the carboxysome.

The large subunit of the RuBisCO was obtained from the pHnCBS1D plasmid (plasmid provided by David Savage, Departments of Molecular & Cell Biology and Chemistry, University of California, Berkeley).

This part was constructed with a mutation on the prefix (from “cggccgc” to “atacctg”), which erases the NotI restriction site. This modification allows the correct design of primers for Gibson Assembly. This part was constructed on the pSB4K5 standard vector.

RubL-GFP-ssrA

Description: This part consist on the superfolder GFP (BBa_I746916) attached to two targeting sequences: The large subunit of the ribulose-1,5-bisphosphate carboxylase/oxygenase enzyme, RuBisCO, (BBa_K1113701), which targets into the inside of Carboxysome, and the ssrA degradation tag (BBa_M0052), which is a targeting sequence for degradation by the protease ClpXP. This construct was used as a reporter for the correct assembly of the Carboxysome. It works with the competition of the two targeting sequence., In case of a correct assembly of the Carboxysome, the microcompartment will keep the fluorescent protein protected inside it, in any other case this will be degraded by the protease.

The large subunit of RuBisCO was obtained from the pHnCBS1D plasmid (plasmid provided by David Savage, Departments of Molecular & Cell Biology and Chemistry, University of California, Berkeley). All the others are bricks available on the Registry.

This part was constructed with a mutation on the prefix (from “cggccgc” to “atacctg”), which erases the NotI restriction site. This modification allows the correct design of primers for Gibson Assembly. This part is also constructed on the pSB4K5 standard vector.

Biotinylation Tag brick

Description: The sequence codifies for the biotin ligase recognition peptide (BLRP) ( 5’ GLNDIFEAQKIEWHE 3’) which can be biotinylated in the presence of the biotin ligase BirA enzyme native from Escherichia coli. This Biotinylation process allows a strong interaction between biotin and streptavidin that can be used for protein purification purposes, specifically as an extraction method for the Carboxysome. The sequence is preceded by a methionine start codon along with a second codon consistent with efficient translation in E. coli and a terminator of transcription.

This part is an adaptation from the INTACT method (“The INTACT method for cell type–specific gene expression and chromatin profiling in Arabidopsis thaliana”. Roger B Deal1 & Steven Henikoff)