Basic parts

-- Part:BBa_K2856001

Modification of bi-functional glutamate--cysteine ligase/glutathione synthase (gshF)

The BBa_K2856001 harbors a coding sequence of bi-functional glutamate--cysteine ligase/glutathione synthase (gshF) derived from S.agalactiae. Codon-optimization has been made for Lactococcus Lactis. gshFp catalyzes the conversion of Cys, Glu and Gly to GSH.

Figure. 1 Schematic diagram of GSH module.

Usage and Biology

Bifunctional glutamate--cysteine ligase/glutathione synthase (gshF) is an enzyme involved and responded to synthetic reaction of GSH. In this reaction, one Cysteine and one Glutamate are converted to one γ-GC, then one γ-GC and one Glycine are converted to one GSH (Figure 2). The Lactococcus Lactis NZ9000 has inability to synthesis GSH. In our project, we construct a plasmid harboring gshF in order to produce GSH in Lactococcus Lactis NZ9000.

Figure. 2 Enzymatic reaction catalyzed by gshF.

Construction and validation of plasmid pNZ-gshF

Gene gshF was amplified from genomic DNA of S. agalactiae and cut with restriction enzyme Hind III and NcoI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol.

Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 3, all the picked colonies had gene gshF, illustrating that the plasmid pNZ-gshF was successfully constructed.

Figure. 3 Validation of plasmid pNZ-gshF. M represented marker. 1, 2 and 3 represented three randomly picked colonies.

Protein Analysis

After transferring the plasmid pNZ-gshF to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of gshF gene. The cells were washed twice with 0.1 M PBS after centrifugation. Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the supernatant of the samples were used to analysis the protein expression. As shown in Figure. 4, expected bands of the GshF protein were observed on the gel in the lane of recombinant L. lactis containing pNZ-gshF induced with different nisin concentration while no GshF protein existed in L. lactis NZ9000.

Figure. 4 SDS PAGE validation of gene gshF expression in L. lactis. M represented marker. WT represented L. lactis NZ9000. 1-3 represented L. lactis/pNZ-gshF induced with 100, 50 and 20 ng/ml nisin.

Validation of glutathione (GSH) by HPLC analysis

To confirm the synthetic glutathione in L. lactis/pNZ-gshF, HPLC was performed to analyze the extracts from the strain. Glutathione was identified on the basis of retention times related to standard sample. According to the retention time of standard glutathione sample, it can be confirmed that glutathione was synthesized in L. lactis/pNZ-gshF.

Figure. 5 Validation of glutathione (GSH) by HPLC.

-- Part:BBa_K2856002

Modification of S-adenosylmethionine synthetase (MetK)

The BBa_K2856001 harbors a coding sequence of S-adenosyl-methionine synthetase (MetK) derived from Lactococcus lactis NZ9000 genome. The MetK protein catalyzes methionine and ATP to form S-adenosyl-methionine.

Figure. 1 Schematic diagram of SAM module.

Usage and Biology

S-adenosyl-methionine synthetase encoded by gene metK is an enzyme involved and responded to the synthetic reaction of S-adenosyl-methionine (SAM). In this reaction, one molecule methionine and one molecule ATP are converted to one molecule SAM (Figure 2). The L. lactis NZ9000 has the ability to synthesize SAM, but just enough for itself. In our project, we constructed a plasmid harboring metK in order to produce more SAM in L. lactis.

Figure. 2 Enzymatic reaction catalyzed by metK.

Construction and validation of plasmid pNZ-metK

Gene metK was amplified from genomic DNA of Lactococcus lactis NZ9000 and cut with restriction enzyme Hind III and KpnI, and ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol.

Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 3, all the picked colonies had gene metK, illustrating that the plasmid pNZ-metK was successfully constructed.

Figure. 3 Validation of plasmid pNZ-metK. M represented marker. 1-5 represented three randomly picked colonies.

Protein Analysis

After transferring the plasmid pNZ-metK to L. lactis NZ9000, SDS-PAGE was performed to detect the protein expression level of metK gene. The cells were washed twice with 0.1 M PBS after centrifugation. Crude protein was extracted through cell breaking using ultrasonication and centrifugation. Then the supernatant of the samples were used to analysis the protein expression. As shown in Figure. 4, expected bands of the MetK protein were observed on the gel. Recombinant L. lactis containing pNZ-metK induced with different nisin concentration showed higher expression of MetK protein.

Figure. 4 SDS PAGE validation of gene metK expression in L. lactis. M represented marker. WT represented L. lactis NZ9000. 1-3 represented L. lactis/pNZ-metK induced with 100 50 and 20 ng/ml nisin.

Validation of S-adenosyl-methionine (SAM) by HPLC analysis

To confirm the synthetic S-adenosyl-methionine in L. lactis/pNZ-metK, HPLC was performed to analyze the extracts from the strain. S-adenosyl-methionine was identified on the basis of retention times related to standard sample. According to the retention time of standard S-adenosyl-methionine sample, it can be confirmed that S-adenosyl-methionine was synthesized more in L. lactis/pNZ-metK than original L. lactis NZ9000.

Figure. 5 Validation of S-adenosyl-methionine (SAM) by HPLC.

-- Part:BBa_K2856003

Modification of cell wall anchor domain-containing protein (CwaA)

BBa_K2856003 harbors an adhesion-associated protein called cwaA. The cwaA encodes a protein containing multiple domains, including five cell wall surface anchor repeat domains and an LPxTG-like cell wall anchor motif.

Figure. 1 Schematic diagram of adhesion factor module.

Usage and Biology

The cwaA gene encodes 923 amino acids with a predicted molecular weight of 93.7 kD. The C terminus of CwaA contains an LPQTDE (LPxTG-like cell wall anchoring) motif belonging to the gram-positive LPxTG anchor superfamily. CwaA possesses five cell wall surface anchor repeat domains. The LPxTG-like motif and three of the five cell wall surface anchor repeat domains. Therefore, CwaA is a cell wall-anchored protein. The specific hit domains of CwaA also included epiglycanin (tandem-repeating region of mucin, pfam05647), OmpC (outer membrane protein, COG3203), PT (the tetrapeptide XPTX repeat, pfam04886) and BF2867_like_N (N-terminal domain found in Bacteroides fragilis Nctc 9343 BF2867 and related proteins, cd13120), probably with a role in cell adhesion

Figure. 2 Enzymatic reaction catalyzed by metK.

Construction and validation of plasmid pNZ-cwaA

Gene cwaA is from genomic DNA of Lactobacillus plantarum NL42 .In order to remove some illegal restriction enzyme sites and add a HIS tag, the gene cwaA was synthesized by Shanghai Generay Biotech (Shanghai, China). The gene cut with restriction enzyme Hind III and NcoI was ligased with plasmid pNZ8148 cut with the same enzyme. Then the ligation product was transferred to E.coli and spread on plates containing 10 mg/L chloramphenicol. Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. As shown in Figure. 3, all the picked colonies had the brand of cwaA, illustrating that the plasmid pNZ-cwaA was successfully constructed.

Figure. 3 Validation of plasmid pNZ-cwaA. M represented marker. 1-4 represented three randomly picked colonies.

Validation of self-aggregation of the recombinant L. lactis/pNZ-cwaA

To evaluate whether CwaA was effective for adhesion, we performed self-aggregation assays. The cells were collected and washed twice with o.1M PBS (pH=7.0).Then the cells were resuspended in 4ml PBS buffer with an initial OD600=0.5(A0) and the final OD600(At) of the upper most layer was measured after 4 hour cultivation in 30℃ without shaking.

Self-aggregation (%)=100-(At/A0)X100

Figure. 4 Self-aggregation value of L. lactis NZ9000 and L. lactis/pNZ-cwaA.

Composite parts

Plasmid pNZ-GM

Plasmid pNZ-GM harbors gene gshF and gene metK. Combing the modules in one plasmid to validate the function of simultaneously expression of protein GshF and MetK.

Construction of plasmid pNZ-GM

Gene gshF and metK were expressed in tandem and controlled by the same inducible promoter PnisA. Gibson assembly method was used to join gshF and metK to form pNZ-GM and then transferred to E.coli DH5α. RBS site was added in front of gene metK.

Validation of plasmid pNZ-cwaA

Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl culture were added to the PCR system as template. The length of the fragment obtained by PCR using the primers for verification was 3494 bp theoretically if plasmid pNZ-GM was constructed successfully. As shown in Figure. 2, all the picked colonies had gene gshF and metK, illustrating that the plasmid pNZ-GM was successfully constructed.

Figure. 1 Validation of plasmid pNZ-GM. M represented marker. 1 to 6 represented randomly picked colonies.

Functional characterization of plasmid pNZ-GM

After we introduced the plasmid pNZ-GM to L. lactis NZ9000, we tested the GSH and SAM production. Compared with wild type L. lactis NZ9000, the engineered strain L.lactis/pNZ-GM produced more GSH and SAM.

Figure. 2 GSH and SAM production of strain L. lactis NZ9000 and L. lactis/pNZ-GM. Asterisk represented none production.

Plasmid pNZ-GMcA

Plasmid pNZ-GMcA harbors gene gshF, gene metK and cwaA. Combing three modules in one plasmid in order to improve the colonization ability of Lactococcus lactis while producing GSH and SAM.

Construction of plasmid pNZ-GMcA

Plasmid pNZ-GM and adhesion module amplified from plasmid pNZ-cwaA were cut by Pst I and Sal I, then were linked with T4 DNA ligase and transferred to E.coli DH5α.

Validation of plasmid pNZ-GMcA

Colonies on the plates were randomly picked and inoculated in 1ml LB medium for 3 hours at 37℃, 200 rpm. 1 μl cultures were added to the PCR system as template. The length of the fragment 1 obtained by PCR for verification was 3760 bp theoretically and fragment 2 was 3079 bp if plasmid pNZ-GM was constructed successfully. As shown in Figure. 1, all the picked colonies contains gene gshF, metK and cwaA, illustrating that the plasmid pNZ-GMcA was successfully constructed.

Figure. 1 Validation of plasmid pNZ-GMcA. M represented marker. gm-1 to gm-5 and cA-1 to cA-5 all represented the same five randomly picked colonies.

Functional characterization of plasmid pNZ-GMcA

After we introduced the plasmid pNZ-GMcA to L. lactis NZ9000, we tested the GSH and SAM production and self-aggregation value to evaluate adhesivity of the strain. As depicted in Figure. 2, compared with wild type L. lactis NZ9000, the engineered strain L.lactis/pNZ-GMcA produced more GSH and SAM and showed better adhesivity.

Figure. 2 GSH and SAM production and self-aggregation value of strain L. lactis NZ9000 and L. lactis/pNZ-GMcA. Asterisk represented none production.