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Parts & Composite Parts

This section contains the parts and composite parts used and developed by our group. The composite parts list includes the plasmids for each of the four enzymes we inserted into the minimal cell, namely PFOR, OAH, ACS, and PYC. The parts list contains the genomic sequences of the enzymes, as well as the promoters and primers used for our work.

Composite Parts

This composite part combines a mycoplasma promoter and Pyruvate carboxylase (PYC) from the baceria Bacillus subtilis (BsPYC). The mycoplasma promoter allows for the expression of the gene of interest inside of mycoplasma and mycoplasma derived cells (JCVI-Syn3B).

This composite part combines a complementary fragment of DNA to pCC1BAC EcoRV fixed HindIII Exp Vector backbone, an intermediate expression level mycoplasma promoter, and pyruvate:ferredoxin oxidoreductase from the baceria Desulfocurvibacter africanus (DaPFOR). This composite part allows for the integration of this gene of interest into the pCC1BAC EcoRV fixed HindIII Exp Vector backbone using Gibson Assembly or E. Coli Plasmid Assembly through the overlapping complementary region. The mycoplasma promoter allows for the expression of the gene of interest once the assembled plasmid is used to transform mycoplasma and mycoplasma derived cells (JCVI-Syn3B).

This composite part combines a complementary fragment of DNA to pCC1BAC EcoRV fixed HindIII Exp Vector backbone, an intermediate expression level mycoplasma promoter, and Acetyl-CoA synthetase (ACS) from the baceria Pseudomonas putida (PpACS). This composite part allows for the integration of this gene of interest into the pCC1BAC EcoRV fixed HindIII Exp Vector backbone using Gibson Assembly or E. Coli Plasmid Assembly through the overlapping complementary region. The mycoplasma promoter allows for the expression of the gene of interest once the assembled plasmid is used to transform mycoplasma and mycoplasma derived cells (JCVI-Syn3B).

This composite part combines a complementary fragment of DNA to pCC1BAC EcoRV fixed HindIII Exp Vector backbone, an intermediate expression level mycoplasma promoter, and oxalacetate acetylhydrolase from the fungus Aspergillus niger (AnOAH). This composite part allows for the integration of this gene of interest into the pCC1BAC EcoRV fixed HindIII Exp Vector backbone using Gibson Assembly or E. Coli Plasmid Assembly through the overlapping complementary region. The mycoplasma promoter allows for the expression of the gene of interest once the assembled plasmid is used to transform mycoplasma and mycoplasma derived cells (JCVI-Syn3B).

Parts

Aspergillus niger (An) Oxalacetate acetylhydrolase (OAH) is an enzyme that facilitates the conversion of oxaloacetate into acetate and oxalate. In this reaction, one molecule of oxaloacetate and one molecule of water is converted into one molecule of acetate and one molecule of oxalate.

Desulfocurvibacter africanus (Da) Pyruvate:ferredoxin oxidoreductase (PFOR) is an enzyme that facilitates a reversible breakdown of pyruvate into acetyl-CoA. Because this reaction is reversible, this means that PFOR can either facilitate the breakdown of pyruvate or synthesize pyruvate based on conditions.

Pseudomonas putida (Pp) Acetyl-CoA synthetase (ACS) is an enzyme that facilitates the conversion of acetate into acetyl-CoA. More specifically, ACS converts one acetate, one ATP, and one CoA to yield one acetyl-CoA, one AMP, and one diphosphate.

Bacillus subtilis (Bs) pyruvate carboxylase (PYC) is an enzyme that facilitates the conversion of pyruvate into oxaloacetate. More specifically, PYC converts one pyruvate, one hydrogen carbonate, and one ATP into one oxaloacetate, one phosphate, one ADP, and one H+.

F_0001 is a primer that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). This primer anneals from nucleotide 2362 to nucleotide 2382 in the vector (counting from the first nucleotide of the registered sequence).

R_0001 is a primer that complements to the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). When used in combination with other primers, R_0001 can facilitate the PCR of a specific region of the plasmid.

F_0013 is the first of the forward primers that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). This primer anneals from nucleotide 2351 to nucleotide 2370 in the vector (counting from the first nucleotide of the registered sequence).

F_1213 is the second of the forward primers that complements a region of the Desulfocurvibacter africanus PFOR (BBa_K4921011). This primer constitutes 27 nucleotides and can be used to facilitate PCR of a region of the PFOR gene in combination with other primers.

F_2425 is the third of the forward primers that complements a region of the Desulfocurvibacter africanus PFOR (BBa_K4921011). This primer constitutes 27 nucleotides and can be used to facilitate PCR of a region of the PFOR gene in combination with other primers.

F_3635 is fourth of the forward primers that complements a region of the Desulfocurvibacter africanus PFOR (BBa_K4921011). This primer constitutes 26 nucleotides and can be used to facilitate PCR of a region of the PFOR gene in combination with other primers.

R_2425 is the first of the reverse primers that complements a region of the Desulfocurvibacter africanus PFOR (BBa_K4921011). This primer constitutes 26 nucleotides and can be used to facilitate PCR of a region of the PFOR gene in combination with other primers.

R_3635 is the second of the reverse primers that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). This primer anneals from nucleotide 2241 to nucleotide 2268 in the vector (counting from the first nucleotide of the registered sequence).

F_0001 is the first of the forward primers that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). This primer anneals from nucleotide 2362 to nucleotide 2382 in the vector (counting from the first nucleotide of the registered sequence).

R_0001 is the first of the reverse primers that complements to the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). When used in combination with other primers, R_0001 can facilitate the PCR of a specific region of the plasmid.

R_1207 is the second of the reverse primers that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector (BBa_K4921000). This primer anneals from nucleotide 2241 to nucleotide 2268 in the vector (counting from the first nucleotide of the registered sequence).

This is a segment of DNA that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector backbone. This is used for the insertion of a gene of interest into this backbone via Gibson Assembly or E. Coli Plasmid Assembly. This complementary region, when attached to a gene of interest, will allow the fragment to anneal to the backbone via this overlapping/complementary region.

This is a segment of DNA that complements a region of the pCC1BAC EcoRV fixed HindIII Exp Vector backbone. This is used for the insertion of a gene of interest into this backbone via Gibson Assembly or E. Coli Plasmid Assembly. This complementary region, when attached to a gene of interest, will allow the fragment to anneal to the backbone via this overlapping/complementary region.

A promoter used in the bacteria Mycoplasma. Its strength was characterized in association with the expression levels of mycoplasma genes that use this promoter. From this characterization, this promoter is classified to have intermediate expression level.

A promoter used in the bacteria Mycoplasma. Its strength was characterized in association with the expression levels of mycoplasma genes that use this promoter. From this characterization, this promoter is classified to have intermediate expression level.

A promoter used in the bacteria Mycoplasma. Its strength was characterized in association with the expression levels of mycoplasma genes that use this promoter. From this characterization, this promoter is classified to have intermediate expression level.

A mycoplasma promoter used for the minimal cell (JCVI-Syn3B) which is derived from Mycoplasma mycoides.