Composite parts description
1. araBAD_SaSSy-FPPS:(BBa_K5181014)
About:
This part codes for the enzyme Santalene Synthase(SaSSy) and farnesyl pyrophosphate synthase(FPPS) derived from Santalum album. Both enzymes are a part of Santalol synthesis pathway.
Components(Basic parts):
1. araBAD_RBS(BBa_K5181006): This part is a self-sufficient arabinose promoter along with its Supporting gene AraC and RBS(Ribosome Binding site) (1)
2. SaSSy_opt(BBa_K5181004):SaSSy gene codes for the enzyme Santalene Synthase converts FPP into several isomers namely: alpha santalene, beta-santalene, epi-beta santalene, and alpha-exo-bergamotene which are the last precursors for santalol synthesis.(ref to project design pg)
3. IRES(BBa_K5181013):This part is an Inter Ribosomal Entry Site which contains a sequence of ribosome binding site, and NdeI cut site, including ATG as start codon. The difference between ATG and RBS is 8 base pairs.
4. FPPS_opt(BBa_K5181005):This gene encodes for FPPS which synthesizes FPP from precursors IPP/DMAP (MEP pathway_ref to design page)and sequence is codon optimised for P. putida.
Order of basic parts:
araBAD_RBS:BBa_K5181006,SaSSy_opt:BBa_K5181004,IRES:BBa_K5181013,FPPS_opt:BBa_K5181005
Map:
Design strategy:
This part is designed as a bicistronic insert with appropriate components and can be used with pSEVA backbones. Gene SaSSy is placed after the araBAD promoter and RBS, followed by the FPPS gene. is a bicistronic part where araBAD promoter(2) was chosen, which is suitable for P. putida to induce our genes SasSY and FPPS stably in P. putida. The spacer sequence of the Inter Ribosomal Entry Site is chosen such that both genes are transcribed under the same promoter. The part is designed such that gene SaSSy is positioned before FPPS to get optimal expression, given that the P. putida genome codes for FPPS natively.This is a bicistronic part. After the assembly of 3 fragments, Two cut sites NcoI and NdeI gets formed.The NcoI cut site (CCATGG) gets assembled in a way that makes the ATGG a part of the reading frame, starting at 6 bp after the first RBS. The NdeI cut site(CATATG) is a part of IRES, which gets formed after the assembly, and the 'ATG' marks the start of the reading frame 8 bp after the second RBS.
Backbones:
pSEVA backbones with cut sites EcoRI and HindIII can be used. This part is Gibson compatible with the pSEVA backbones such that it is inserted within the MCS region with the above-mentioned cut site. Experiments performed by our team with this composite part and detailed results can be viewed here(link to the Results page)
Application:
Any gene starting with 'ATGG' can be placed under the arabinose promoter using the NcoI cut site in an experimentally verified ORF. For a dual expression system, one can use the IRES mentioned part: BBa_K5181013 and put their second gene in an experimentally verified ORF under the same promoter promoter using the NdeI cut site. The components chosen in this part are suitable for expression in P. putida and are codon-optimized. Please refer to the corresponding registry page for Alphafold2 predicted structures of FPPS, SaSSy proteins and their substrate interaction sites.
2. tet_RBS_P450-CPR_fusion protein (BC_linker):(BBa_K5181015)
About:
This part is a bicistronic composite part designed by team IISER-TVM. It consists of genes Cytochrome P450 monooxygenase and Cytochrome P450 reductase from Santalum album.
It codes for a fusion protein CPR-P450 Which is involved in the last step of Santalol synthesis (design page ref)
Components:
1. Self-sufficient tet_RBS: BBa_K5181009:This part is a self-sufficient tetracyclin promoter with its supporting gene tetR and Ribosome Binding Site.
2. P450-CPR_fusion protein (BC_linker): BBa_K5181008: This part is an artificial fusion protein. It is a bifunctional self-sufficient p450 that contains the cytochrome p450 monooxygenase domain for CYP736A167 from Santalum album and the reductase domain of CPR1 of the same species connected by linker from the naturally existing bifunctional p450 of Bacillus cereus.
Plasmid map:
Design strategy:
This part has been designed specifically to achieve expression of eukaryotic Sandalwood P450/CPR, our prokaryotic system. By understanding the structure and function of native P450 CAM in P. putida we devised a design to truncate the transmembrane domain of both CPR and P450. Further, the architecture of a soluble, naturally existing bifunctional P450 present in Bacillus cereus was followed to design a fusion protein.
Application:
Cytochrome P450 is a diverse superfamily of heme-thiolate proteins found in various organisms. They play crucial roles in biosynthetic pathways for natural products, degradation of xenobiotics, steroid biosynthesis, and drug metabolism. One of such CYP736A167 from the Santalum album is the key player in our engineered Santalol synthesis pathway. It requires a redox partner i.e. CPR1 from the same species for its function and catalysing conversion of Santalene to Santalol. From literature and structural studies of naturally occurring P450 CAM from P. putida and bifunctional P450 from Bacillus cereus, we adopted the strategy of P450-CPR fusion protein mimicking the structural architecture of these two bacterial P450 This part design caters to the expectation of a idel modular part where the promoter, RBS is a replaceable insert which will help to check expression of our GOI under different inducible promoters and suitable RBS. Also we can experiment with linker sequences in the fusion protein (replace them with BM linker,GST linker etc)
3. araBAD_RBS_P450-CPR_fusion protein (BM_linker):(BBa_K5181020)
About:
This part is a variant of a bicistronic composite part BBa_K5181015. It codes for a fusion protein of Cytochrome P450 mono-oxygenase and cytochrome P450 reductase with a linker sequence obtained from Bacillus megaterium, and the part is controlled by araBAD promoter. The enzymes P450 and CPR are involved in the last step of Santalol synthesis and catalyze the oxidation of santalene to santalol.
Components:
1. P450-CPR_fusion protein (BM_linker): BBa_K5181016- This part consists of truncated CPR-fusion protein genes with BM linker.
2. araBAD_RBS_2: (BBa_K5181007): This is a part consisting of araBAD promoter and RBS which as a whole replaceable part due to NsiI and HindIII cut sites. The linker is abotained from Bacillus megaterium.
Plasmid map:
Design strategy:
This part has been designed specifically to achieve expression of eukaryotic Sandalwood P450/CPR, our prokaryotic system. It is a modified version of BBa_K5181015 .The promoter-RBS is replaceable because of HindIII and NsiI cut sites, making the part modular. Similarly, the fusion protein has a BM linker obtained from Bacillus megaterium. The linker sequence is flanked by ScaI sites such that linker can also be replaced with GST or BC linker to experiment with expression of variants of fusion protein.
Application:
This part design caters to the expectation of an ideal modular part where the promoter, RBS is a replaceable insert that will help check the expression of our GOI under different inducible promoters and suitable RBS. Also, we can experiment with linker sequences in the fusion protein (replace them with BM linker, GST linker, etc)
4. rhaBAD_P450-CPR_fusion protein (BM_linker):(BBa_K5181019)
About:
This part is a variant of a bicistronic composite part BBa_K5181020. It codes for a fusion protein of Cytochrome P450 mono-oxygenase and cytochrome P450 reductase with a linker sequence obtained from Bacillus megaterium, and the part is controlled by araBAD promoter. The enzymes P450 and CPR are involved in the last step of Santalol synthesis and catalyze the oxidation of santalene to santalol.
Components:
1. P450-CPR_fusion protein (BM_linker): BBa_K5181016- This part consists of truncated CPR-fusion protein genes with BM linker.
2. rhaBAD_RBS: BBa_K5181010- This is a part consisting of rhaBAD promoter and RBS which as a whole replaceable part due to NsiI and HindIII cut sites. The linker is obtained from Bacillus megaterium.
Plasmid map:
Design strategy:
This part has been designed specifically to achieve expression of eukaryotic Sandalwood P450/CPR, our prokaryotic system.It is a modified version of BBa_K518102 and has rhaBAD promoter-RBS. The linker sequence is flanked by ScaI sites such that linker can also be replaced with GST or BC linker to experiment with expression of variants of the fusion protein. All the gene sequences are optimised for P. putida.
Application:
This part design caters to the expectation of an ideal modular part where the promoter, RBS is a replaceable insert that will help check the expression of our GOI under different inducible promoters and suitable RBS. Also, we can experiment with linker sequences in the fusion protein (replace them with BM linker, GST linker, etc).
5. pRGPDuo3_lac_P50_CPR_truncated(BM_linker):(BBa_K5181017)
About:
This plasmid is a dual expression vector having two genes, Cytochrome P450 monooxygenase(CYP450) and its redox partner Cytochrome P450 reductase (CPR1), obtained from the genome of Santalum album.
Components:
Ori- pBBR1-suitable for gene expression in Pseudomonas putida
Antibiotic resistance- Gentamicin
Tac promoters
LacIq
P450_truncated
CPR_truncated
Plasmid map:
Design strategy:
Modifications have been made to the original backbone sequence of pRGPDuo3 as mentioned below:
1. Both genes CYP450 and CPR are required to be expressed in the same proportion for their composite function. Hence, we replaced the araBAD promoter and its supporting protein with the tac promoter, resulting in a design where both genes are under the control of the same type of promoter( tac). Both tac promoters are regulated by single LacI repressor
2. Also, we noticed that the complete tac promoter (Herman et.al 1983) contains the tac promoter, lac operator, and an RBS, and the complete set of 80 bp was present in pRGPDuo3. The araBAD promoter, with its supporting gene accounting for 1190 bp, was replaced with the 80 bp tac promoter, with a 10 bp spacer sequence between the complete tac promoter and the CPR1 gene
3. We flanked our CPR1 gene with NotI and NheI sites to make our gene in the plasmid replaceable, which added to its modularity.
4. The p450 gene was placed under the natively present tac site flanked by restriction sites BamHI and SacI. Hence, we reduced the size of the original pRGPDuo3 by 1100 bp and named it pRGPDuo_lac.
5. All the genes were codon optimised for our chassis P. putida
Application:
We designed this plasmid in the form of 5 Gibson assembly-compatible fragments assembled to form the complete functional plasmid. This part can be used to express these genes required for the conversion of Santalene to santalol.
6. pRGPDuo4_SaSSy_FPPS:(BM_linker):(BBa_K5181018)
About:
This plasmid is a dual expression vector having two genes, Farnasyl pyrophosphate synthase (FPPS) and Santalene synthase(SaSSy) obtained from the genome of Santalum album. These enzymes synthesise FPP and convert FPP to Santalene respectively.
Components:
Ori: pRO1600 - suitable for gene expression in P. putida.
Antibiotic resistance- Kanamicin
tac promoter, araBAD promoter(along with supporting genes)
LacIq
FPPS(BBa_K5181005)
SaSSy(BBa_K5181004)
Plasmid map:
Design strategy:
pRGPDuo4 is a dual expression vector with two different promoters. Its backbone contained two inducable promoters, tac, and araBAD, and their supporting genes. We placed SaSSy under tac promoter and FPPS under araBAD promoter
Two vectors pET 28a(+) and pBbE8k-fcs from addgene, were used to modify the existing pRGPDuo4 to reduce its length to ease bacterial transformation and cost of plasmid synthesis. We swapped bases from 3858th position to 1259th position(1259 bp ) pertaining to araBAD promoter , ara C supporting gene and 69 bp of nucleotides following the araBAD promoter with position 7 to 1242th position(1242 bp) of pBbE8k-fcs as it contains a strong RBS(link in wp) (AGGAGA) 7 bp from the start codon of the gene, The start codon is determined by the NdeI cut site for entering the gene, and any gene with start codon as ATG can be expressed under this promoter.
Kannamycin and Ori modification:
We needed our plasmid to express two genes under different inducible vectors Thus after 20 bp from the Amp promoter 702 bp(2331-3032) were deleted which included (M13 reverse primer site, a lac operator, a lacpromoter and a cap binding site).The ColE1 ori was taken from from pET28a(+) along with its kanamycin resistance gene till the start of F1 ori of pET28a(+) (3285-4902) was taken codon optimized for P. putida and replaced with the Ori and Kannamycin resistance gene of pRGPDuo4(3033-5426) by theses modifications we reduced our backbone size by 1617 bp
Application:
We designed this plasmid in the form of 5 Gibson assembly-compatible fragments assembled to form the complete functional plasmid. This part can be used to express these genes required for conversion of Santalene to santalol
Primers
This section details the primers we designed and used throughout our project. We used SnapGene to design primers and further refined the sequence based on results such as hairpin loops and melting temperatures from the IDT oligo analyzer online tool.
The main purpose of the following primers was to amplify our Gibson assembled insert to yield higher concentration for downstream experiments.
Sr. no. | Primer information | Primer sequence | 1st round Tm(°C) | Tm(°C) |
---|---|---|---|---|
1 | Forward primer for the amplification of araBAD-SaSSy-FPPS | 5’ CGC CTA GGC CGC GGC CGC GCG AAT TCT TAG TGG 3’ | 71.3 | 77.7 |
2 | Reverse primers for amplification of araBAD-SaSSy-FPPS | 5’ TTT TCC CAG TCA CGA CGC GGC CGC AAG CTT TTA TGA CAA CTT GAC GGC TAC ATC ATT CAC 3’ | 74.9 | 76.8 |
3 | Forward primer for amplification of CPR-P450 | 5’ CGC CTA GGC CGC GGC CGC GCG AAT TCT TAG TGA TG 3’ | 69.2 | 76.8 |
4 | Reverse primer for the amplification of CPR-P450 | 5’ TTT TCC CAG TCA CGA CGC GGC CGC AAG CTT TTA AGA CCC 3’ | 70.8 | 75.7 |
5 | Forward primer to replace tet promoter with araBAD promoter | 5’ ATATGCATATATCTCCTTCTTAAAAGATC 3’ | 49.5 | 56.9 |
6 | Reverse primer to replace tet promoter with araBAD promoter | 5’ CGCAAGCTTTTATGACAAC 3’ | 54.9 | same |
Supplementary information:Tools
1. Pseudomonas putida codon usage table (5)
We used this table to codon optimise the genes along with GenScript’s codon optimization tool.
2. Plasmid compatibility groups:
These groups refer to classifications of plasmids based on their ability to coexist within the same bacterial cell without interfering with each other's replication and function. Understanding these compatibility groups is crucial for genetic engineering in cases where we are cotransforming the bacteria with more than one plasmid. To achieve independent unhindered function of multiple plasmids in a single cell, the compatibility groups(Ori) of each of the plasmids should be different.
- Plasmid: pBbE8k-fcs: https://www.addgene.org/83068/sequences/
- Sathesh-Prabu, C., Tiwari, R., Kim, D. et al. Inducible and tunable gene expression systems for Pseudomonas putida KT2440. Sci Rep 11, 18079 (2021).https://doi.org/10.1038/s41598-021-97550-7
- Broad-Host-Range ProUSER Vectors Enable Fast Characterization of Inducible Promoters and Optimization of p-Coumaric Acid Production in Pseudomonas putida KT2440
Patricia Calero, Sheila I. Jensen, and Alex T. Nielsen
ACS Synthetic Biology 2016 5 (7), 741-753
DIO: 10.1021/acssynbio.6b00081 - Gauttam R, Mukhopadhyay A, Simmons BA, Singer SW. Development of dual-inducible duet-expression vectors for tunable gene expression control and CRISPR interference-based gene repression in P. putida KT2440. Microb Biotechnol. 2021 Nov;14(6):2659-2678.
DIO10.1111/1751-7915.13832. - Codon usage table: https://www.kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=160488&aa=1&style=N
- .Plasmid compatibility groups: https://tse3.mm.bing.net/th?id=OIP.5eiaoJOgmNm-CY4pKhC83QHaEj&pid=Api&P=0&h=180