Part:BBa_K5181014
araBAD_RBS_SaSSY_FPPS
This is a bicistronic composite part designed by team PETal iGEM IISER-TVM. It consists of basic parts, namely an araBAD promoter with RBS(BBa_K5181006), SaSSy(BBa_K5181004), IRES (BBa_K5181013), FPPS(BBa_K5181005) genes forming a complete insert as a composite part. It codes for FPP synthase(FPPS), which converts GPP into Farnasyl pyrophosphate(FPP). The subsequent step converts FPP to santalene by santalene synthase(SaSSy).
The AlphaFold2 predicted structures of FPPS and SaSSy are shown below.
Interaction Studies
The Interaction of farnesyl pyrophosphate synthase (FPPS) with geranyl pyrophosphate (GPP) is depicted below:FPPS interaction sites
The Polar interaction sites are: Lys-47, Asn-49, Arg-50, Glu-83
The Van der waals sites are: Gly-46, Leu-48, Ser-53, Gln-86, Phe-196
The Interaction of Santalene synthase (SaSSy) with farnesyl pyrophosphate (FPP) is depicted below:
SaSSy interaction sites
The Polar interaction sites are: Arg-460, Asn-463, Glu-471, Arg-474, Lys-479
The Van der waals sites are: Arg-284, Trp-293, Thr-318, Tyr-396, Tyr 539
The linear map of the insert is shown below.
Linear map
| Annotation | Range in basepairs | length (bp) | Misc. info |
|---|---|---|---|
| misc_DNA | has 100% sequence similarity with pSEVA631 MCS region and contain HindIII,NotI and EagI sites | ||
| araC_gene | araC: Represses araBAD promoter in absence of L-arabinose (complement) | ||
| misc_DNA | Gap between araBAD promoter and araC gene | ||
| araBAD promoter | arabinose induced promoter | ||
| misc_DNA | Gap between araBAD promoter and RBS | ||
| RBS | Ribosome Binding site AAGGAGA | ||
| misc_DNA | Region between RBS and SaSSy | ||
| SaSSy | gene coding for santalene synthase, has a 6X his tag in the C-terminal end | ||
| IRES | IRES included an RBS AGGAG 8 bp away from the transcription start site (also contains a partial NdeI cut site, which is completed with the ATG of FPPS) | ||
| FPPS | gene coding for Farnesyl pyrophosphate synthase, contains a 6X his tag in C-termial end |
*The Map contains an EcoRI site which is a part of our plasmid, This insert is compatible with pSEVA Default MCS
This insert can be used with any pSEVA (default MCS) backbone with EcoRI and HindIII as cut sites. The part is codon optimized for our strain Pseudomonas putida, and all the experiments that we successfully performed, including Gibson assembly, PCR, Transformation, ligation and expression of enzymes to synthesise santalene, are explained thoroughly in the next section.
PCR amplification of the insert araBAD_SaSSy_FPPS_
Figure 1: Bridge PCR to confirm Gibson assembly and amplification
We performed Gibson assembly to obtain this part from three fragments synthesized by GenScript.
The following Agarose gel electrophoresis map shows the amplified araBAD_SaSSY_FPPS insert after bridge PCR to confirm Gibson assembly. The polymerase chain reaction was tested at four temperatures (66 °C, 68 °C, 70 °C,72°C) marked in Lanes 1,2,3,4. Band of size 4082 bp corresponds to amplified insert. Refer to the design section for the forward and reverse primer sequence. The PCR amplified samples of SaSSy-FPPS were cleaned using the Macherey-Nagel™ NucleoSpin™ PCR Clean-up XS kit.
Ligation of the insert with pSEVA631 backbone:
The insert araBAD_SaSSy-FPPS from the PCR clean-up and the backbone pSEVA631 were ligated using NEB T4 DNA Ligase.
Single and double digestion of the ligated plasmid (pSEVA631-araBAD_SaSSy-FPPS) was performed to verify the ligation, followed by gel electrophoresis. The 1% Agarose gel electrophoresis results confirmed the successful ligation of the insert into the backbone, ensuring the proper assembly of the construct.
pSEVA 631 (backbone) size = 3519 bp araBAD_SaSSy-FPPS ( insert) size = 4082 bp
In the case of single digestion, a thick band was found at 7Kb, corresponding to the size of our ligated plasmid (7005bp), confirming the ligation process.
Similarly, double digestion with HindIII and EcoRI of the ligated plasmid gave two thick bands of 4082 bp and 3519 bp.
Transformation
The ligated plasmids were subsequently transformed into MACH-1 E.coli electrocompetent cells to amplify the plasmids. These amplified plasmids were isolated using Macherey-Nagel™ NucleoSpin Plasmid QuickPure™ Kit. Finally, these plasmids were electroporated into Pseudomonas Putida TA7-EG electrocompetent cells for transformation.
Figure: Successful transformation of the ligated mixture into MACH-1
Upon isolating the plasmids, they were further electroporated using Gene Pulser®/Micropulser™ electroporation cuvettes into the Pseudomonas Putida TA7-EG competent cells.
Figure: SaSSy-FPPS P. putida TA7-EG transformed plate image
Expression of SaSSy_FPPS in P. putida TA7-EG
To assess the expression of SaSSy and FPPS, we transformed the ligated pSEVA631-SaSSy-FPPS plasmid into P. putida TA7-EG in TPA+EG. The culture was then induced with and was allowed to grow for 16 hours at 20°C. The bacteria was later pelleted and lysed using sonication. Further, the Ni-NTA column was performed to purify SaSSy protein tagged with His protein. SDS-PAGE was conducted, followed by Coomassie staining. We obtained two prominent bands at 65kDa and 45kDa corresponding to protein SaSSY and FPPS, respectively, confirming their successful expression in P. putida TA7-EG.
Figure: Expression of SaSSy and FPPS proteins
Quantitative analysis of santelene through GCMS
To assess the expression of SaSSy and FPPS, we transformed the ligated pSEVA631-araBAD_SaSSy-FPPS plasmid into P. putida TA7-EG in TPA+EG media. We induced our transformed Pseudomonas putida strains with multiple concentrations of arabinose(0.2%,0.7%,1.5%,2%). After reaching the desired OD, the cultures were incubated at 20°C for approximately 16 hours. The bacterial cells were then harvested by centrifugation. The transformed pellets with an untransformed pellet as control were sent for GC-MS analysis at the Instrumental Facility in the Chemistry Department of IISER TVM. It showed maximum expression at 0.7% arabinose induction. The GC chromatogram displayed intensities of the detected peaks at their respective retention times, while the mass spectra showed relative intensities versus m/z. The internal santalene standard produced peaks at 204 m/z in the chromatogram. A review of the MS spectrum revealed a distinct pattern, confirming santalene, which was quantified at 10.25% abundance. In contrast, the GC-MS output for the wild-type P. putida strain did not exhibit any santalene peak, indicating no production in the wild-type. The santalene peaks from modified strains matched the m/z ratio of sesquiterpene, which confirms the production of santalene, given that santalene synthase is expressed.
Figure: GCMS sample- pSEVA631_araBAD_SaSSY_FPPS induced with 0.7% arabinose in P.putida TA7-EG transformed cells.
Figure: GCMS sample- Wild-type P.putida TA7-EG cells
Hence, the Wild-type Pseudomonas putida TA7-EG cells and the transformed P. putida showed different peaks at the 19-minute retention time. Hence, the 204 peak corresponds to the santalene m/z ratio, expressed by the pSEVA631_SaSSY_FPPS plasmid incorporated into our chassis.
Consistent with our hypothesis, the modified P. putida strains expressing the SaSSy-FPPS proteins with the pSEVA631 backbone exhibited successful santalene synthesis, supporting our approach of achieving the first-ever production of santalene in Pseudomonas putida growing on PET monomers.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NotI site found at 17
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1244
Illegal BglII site found at 2615
Illegal BglII site found at 4039 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1009
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2727
Illegal SapI site found at 991
Illegal SapI.rc site found at 1440
Illegal SapI.rc site found at 3837
| None |