Team:Hamburg/Parts

Parts

Parts Overview

 Terpenoids are an important group of natural products used as biofuels, drugs or fragrances. Naturally occuring in plants it has been shown that microbial terpenoid production in microorganisms like yeast, E. coli or cyanobacteria is possible. Nevertheless iGEM projects seem to rarely focus on this interesting class of natural products which is correlated with a lack of useful parts inside the iGEM registry. Fortunately we were able to change that and designed a novel golden gate based toolbox which allows.
  1. production of terpenoid precursors and simple terpenoids
  2. creation of CYP P450-reductase fusion enzymes to optimise processing of terpenoid precursors and production of bioactive target products
  3. modularity of the system to enable exchange of linker sequences/promoters/etc. (MoClo-compatible toolbox)
We achieved that by first introducing a multitude of different enzymes used in the biosynthesis of our target products and precursors to the registry (Link zur Seite, wo wir das genau beschreiben/Engineering/Description). The codon optimised sequences (E. coli or Synechocystis sp. PCC 6803) of these novel enzymes can be found on the registry pages BBa_K3846000 to BBa_K3846024. We then optimised the metabolic pathway and designed fusion proteins aided by bioinformatic models (see Model). Unfortunately we weren’t able to characterise most of the parts in the lab due to time and corona restriction constraints.

Part Design

Part design Generation 0.0:

 Initially we designed our parts to be assembled by golden gate assembly for each construct (e.g. fusion protein expression cassette) individually, which made it difficult to exchange parts of the construct like promoters. Therefore we aimed to improve the modularity of our construct design.

Linker design V 1.0:

 In a first step to allow more variability and easier combination of CYP P450 enzymes and reductases, different linkers restriction enzymes sites for MfeI (CAATG) and BamHI (GGATCC) were introduced at the N-terminus and C-Terminus of the CYP 450 enzymes and reductases respectively. Linkers were then created by annealing DNA oligos with AATG and GATC overhangs. Flexible linkers mainly containing Glycine (BBa_K3846500, BBa_K3846502) and rigid linkers mainly containing proline (BBa_K3846501, BBa_K3846503) of different length were created. Due to the introduced restriction sites, the linker sequence could not be chosen freely. Two amino acids at the N-Terminus (Glutamine + Leucine) and two amino acids at the C-terminus (Glycine + Serine) were fixed. CYP1A1-CPR fusion proteins with different linkers using this design can be found in the registry BBa_K3846510-BBa_K3846513. Fig. 1: Linker design V 1.0. Expression cassette, MfeI and BamHi restriction sites Conjugation Shuttle Vector BBa_K3846050 An important feature of useful golden-gate assembly (MoClo) compatible backbones is the possibility to easily recognise successful integration of fragments inside the vector to avoid the necessity to test multiple clones. iGEM provides the universal acceptor plasmid (BBa_P10500/link) for creating new phytoparts containing lacZ-α for blue-white screening. Substrates for blue-white screening are not cheap and a better way to also reduce costs would be using fluorescent proteins as visible markers as was shown to be possible by the iGEM Marburg Team 2018 with their improvement backbone BBa_K2560002 (link). In our project, we are using cyanobacteria and wanted to have a universal acceptor plasmid compatible with both species. Therefore, we modified an RFS1010-based broad-host range vector by introducing BsaI and BsmbI restriction sites flanking an eYFP expression cassette to obtain a cyanobacteria compatible fluorescence dropout acceptor plasmid. Successful integration of golden gate assembly fragments leads to the removal of the eYFP coding sequence from the plasmid and it is then possible to distinguish between wrong (yellow/green) and correct (white) colonies. An additional feature of this vector is that it can be used for conjugation of cyanobacteria by triparental mating. The characterisation of this part is unfortunately still pending. Fig. 2: MoClo-based Part Design 2.0 To improve the usefulness of our parts, we then aimed to make our parts compatible with the MoClo standard of golden? gate based IIS restriction enzyme assembly. Thereby we expanded the Common Genetic Syntax for fusion sites to allow the creation of a) fusion proteins connected by linker sequences and b) multiple CDS expressed in an operon. (see Best Composite Part, Part Collection) alt_text Fig. 3: (v) Backbone:
Bba_K3846050 modified pAM5411 (phytobrick acceptor backbone)
Connectors: To enable the assembly of several translational units (TU) short DNA sequences connecting the fusion sites are needed. We designed several such short connecting sequences to enable the creation of multi operon constructs and substitute parts of our constructs.
BBa_K3846110 TU connector (CGCT-GGAG)
BBa_K3846128 TU connector (cgct-agct)
BBa_K3846129 TU connector (agct-ggag)
BBa_K3846130 TU connector (cgct-aaca)
BBa_K3846131 TU connector (aaca-ggag)
BBa_K3846136 TU Connector (ATCG-GCTT)
BBa_K3846139 TU connector (AATG-ATCG)
BBa_K3846140 TU stop codon connector (TTCG-GCTT)
Promoter (regulatory):
BBa_K3846100 T7 promoter (phytobrick)
BBa_K3846111 J23119 (phytobrick)
BBa_K3846112 J23111 (phytobrick)
BBa_K3846113 rhaA (phytobrick)
BBa_K3846114 rhaA mutated (phytobrick)
BBa_K3846115 rhaS (phytobrick)
BBa_K3846126 lacUV5 promoter (phytobrick)
RBS
BBa_K3846102 Strong RBS (phytobrick)
CDS CYPs
BBa_K3846101 CYP1a1 (phytobrick) - with N-terimanl 6xHis tag
BBa_K3846106 Terminal olefin-forming fatty acid decarboxylase (OleT) - phytobrick with N-terminal 6xHis tag
BBa_K3846107 CYP71AV1 (phytobrick) - with N-Terminal 6xHis tag
BBa_K3846132 Costunolide synthase (CYP71BL2) (phytobrick)
BBa_K3846135 Parthenolide synthase (CYP71CA1) - phytobrick
Reductases
BBa_K3846104 NADPH-cytochrome P450 reductase (CPR) - phytobrick
BBa_K3846105 NADPH-P450 reductase (BM3R) - phytobrick
BBa_K3846108 Ferredoxin-1 (phytobrick)
BBa_K3846109 NADPH-cytochrome P450 reductase 2 (atr2) - phytobrick
MevT/MBIS Pathway
BBa_K3846118 Acetyl-CoA acetyltransferase (atoB) - MevT pathway (phytobrick)
BBa_K3846119 Hydroxymethylglutaryl-CoA synthase (HMGCS1) - MevT Pathway (phytobrick)
BBa_K3846120 Hydroxymethylglutaryl-CoA synthase (HMGCS1) - MevT Pathway (phytobrick)
BBa_K3846121 Mevalonate kinase (MVK) - MBIS pathway (phytobrick)
BBa_K3846122 Phosphomevalonate kinase (PMVK) - MBIS pathway (phytobrick)
BBa_K3846123 Diphosphomevalonate decarboxylase (MVD) - MBIS pathway (phytobrick)
BBa_K3846124 Isopentenyl-diphosphate Delta-isomerase (idi) - MBIS pathway (phytobrick)
BBa_K3846125 Farnesyl diphosphate synthase (ispA) - MBIS pathway (phytobrick)
Terpenoid metabolism
BBa_K3846116 Alpha-bisabolene synthase (phytobrick) - with N-terminal 6xHis tag
BBa_K3846127 Amorpha-4,11-diene synthase (AMS1) - phytobrick
BBa_K3846133 Germacrene A hydroxylase - phytobrick
BBa_K3846134 Germacrene A synthase (GAS) - phytobrick
BBa_K3846137 Alpha-humulene synthase (ZSS1) - phytobrick
Energy metabolism
BBa_K3846138 Glucose 1-dehydrogenase 2 (gdh2) - phytobrick
linker sequences
BBa_K3846150 10 aa flexible linker
BBa_K3846151 20 aa flexible linker
BBa_K3846152 10 aa rigide linker
BBa_K3846153 20 aa rigide linker
BBa_K3846154 linker sequence (Bacillus megaterium CYP102A1)
BBa_K3846155 linker sequence (Beauveria bassiana CYP505)
BBa_K3846156 linker sequence (Fusarium oxysporum CYP505)
BBa_K3846157 linker sequence (Aspergillus terreus NIH262, CYP505E3)
BBa_K3846158 linker sequence (Bacillus licheniformis ATCC 14580, CYP102A7)
BBa_K3846159 linker sequence (CYP505D6, Phanerochaete chrysosporium)
BBa_K3846175 CYP1A1 shortened sequence for linker test (1)
BBa_K3846176 CYP1A1 shortened sequence for linker test (2)
BBa_K3846177 RAT CPR shortened sequence for linker tests (1)
BBa_K3846178 RAT CPR shortened sequence for linker tests (2)
Teminator
BBa_K3846103 double terminator (BBa_B0014 - phytobrick)
BBa_K3846117 rrnB T1 and T2 terminator (phytobrick)
fusion proteins Our project focuses on enzymatic reactions catalysed by cytochrome P450 enzymes. This group of enzymes always rely on a reductase to be functional. To increase the activity of the P450 enzyme we decided to create fusion proteins, consisting of an N-terminal cytochrome P450 enzyme and a C-terminal NADPH-cytochrome P450 reductase connected by a linker sequence. It is likely that the composition of the linker sequence influences the activity of the fusion protein since electrons have to be transferred from the reductase to the P450 enzyme and the structural arrangement of the domains should have an effect on this process. Optimising the linker sequence is therefore crucial for reaching optimal enzyme activity. To allow modular combinations of different P450 enzymes, reductases and linkers we designed these parts interchangeably using the following MoClo compatible fusion sites. fusion sites: * CYP P450 enzyme (AATG -TTCG) * linker sequence (TTCG - ATCG) * reductase (ATCG - GCTT)
Best Basic Part: Amorpha-4,11-diene 12-monooxygenase (CYP71AV1) - phytobrick (BBa_K3846107) Amorpha-4,11-diene 12-monooxygenase (CYP71AV1) is a P450 enzyme from Artemisia annua which catalyses the reaction of amorpha-4,11-diene to artemisinic acid, which then can be oxidised to artemisinin. Artemisinin is an important anti-malaria drug and since 2002 recommended first-line treatment for uncomplicated malaria [Approaches and Recent Developments for the Commercial Production of Semi-synthetic Artemisinin ]. As Artemisinin is mainly extracted from Artemisia annua its availability and price fluctuates greatly. Therefore it was necessary to exploit other non-plant derived sources to stabilise price and availability. These efforts led to the establishment of a semi-synthetic artemisinin production pathway in Saccharomyces cerevisiae. CYP71AV1 catalyses the last enzymatic reaction leading to the production of artemisinic acid which is then oxidised chemically. CYP71AV1 is the key enzyme of this semi-synthetic artemisinin production pathway we are establishing in Escherichia coli and we created the phytobrick BBa_K3846107 by codon-optimising the coding sequence of CYP71AV1 and adding fusion sites to enable modular cloning. Previously, only a few kinetic studies were performed for CYP71AV1 [sources needed] and 3d crystal structure data is also not available. Our plan was to use this part to characterize the functionality of CYP71AV1 further and get more kinetic data. Unfortunately we weren’t able to achieve this goal. Fig.1: A Prediction of 3D Model of CYP71AV1. (Source: Comparative functional analysis of CYP71AV1 natural variants reveals an important residue for the successive oxidation of amorpha-4,11-diene) Best Composite Part Artemisinic acid production cassette (BBa_K3846354) Following up to our best basic part we present here an artemisinic acid production cassette, an IPTG inducible multi-operon protein expression composite part enabling the complete synthesis of artemisinic acid in Escherichia coli. We combine the expression of the MevT pathway operon (acetoacetyl-CoA synthase (ackA), HMG-CoA synthase (HMG), and an N-terminally truncated version of HMG-CoA reductase (tHMGR)) and the MBIS pathway operon (mevalonate kinase (MK), phosphomevalonate kinase (PMK), phosphomevalonate decarboxylase (PMD), isopentenyl diphosphate isomerase (idi), and farnesyl diphosphate synthase (ispA)) to obtain the terpenoid basic module farnesyl diphosphate (FPP) with the expression of amorpha-4,11-diene synthase and CYP71AV1-ATR2 fusion protein to synthesize amorphadiene and artemisinic acid. Enzymes of the MevT and MBIS pathway are arranged in two separate operons under control of the lacUV5 promoter. Amorpha-4,11-diene synthase and CYP71AV1-ATR2 fusion protein are each expressed behind a T7 promoter. Similar engineering studies with the same enzymes in Saccharomyces cerevisiae resulted in product titers of up to 100 mg l<sup>-1</sup>. [Production of the antimalarial drug precursor artemisinic acid in engineered yeast] The different parts of the cassette can be assembled on the same or different plasmid or alternatively integrated into the genome. The parts making up the MevT and MBIS pathway are mainly based on previous work conducted by Keasling et al. [Keasling 2187, JBEI-3716, Optimization of the mevalonate-based isoprenoid biosynthetic pathway in Escherichia coli for production of the anti-malarial drug precursor amorpha-4,11-diene]. Our contribution is making the parts compatible with Modular Cloning and adding the CYP71AV1-ATR2 to the pathway. Unfortunately we were unable to produce significant amounts of artemisinic acid with this construct. But we are sure that our groundbreaking work in the field of terpenoid fermentation will make it easier for future iGEM teams to start their project in this field. Yield optimization through cultivation and part modification (or integration in the genome) as well as industrial scale up have the potential to increase availability and accessibility of artemisinin as an antimalarial therapeutic.
Best Part Collection THE L.E.T.Z. F.E.T.Z PROTEIN COLLECTION Fusion Electron Transfer enZyme Collection We proudly present the LETZ FETZ Collection, a MoClo golden-gate based toolbox for the expression of fusion proteins involved in the biosynthesis of terpenoids. It contains 39 phytobricks, 10 linker sequences, 21 fusion proteins, a phytobrick acceptor backbone and 34 protein expression cassettes. It expands existing toolboxes like the Marburg Collection by enabling the creation of multi domain proteins connected by linker sequences and expression of enzymes organised in operons. Additionally it includes all the necessary enzymes to allow metabolic engineering of E. coli and Synechocystis PCC6803 for the production of terpenoids from scratch. This collection also features an eYFP dropout shuttle vector for cyanobacteria (BBa_K3846050) based on the RSF1010 broad-host range design compatible with conjugating cyanobacteria. To our knowledge there is no standardised syntax for the creation of multidomain fusion proteins compatible with modular cloning, so we provide you with syntax rules for the creation of two-domain proteins connected by a linker sequence. We also developed a bioinformatic tool (Link) to aid the rational design of these proteins. In prokaryotes enzymes involved in the same metabolic pathway are often organised in operons. We therefore expanded the MoClo syntax to accommodate the expression of up to five enzymes from the same promoter. In combination with existing MoClo Toolboxes (like the Marburg Collection) our L.E.T.Z. F.E.T.Z. Collection allows de novo plasmid assembly with striking flexibility and simple workflow. We hope that our collection will revolutionise the way future iGEM teams create fusion proteins and the number of teams focussing their projects on terpenoids will increase with this contribution to the registry. Phytobricks
BBa_K3846100 T7 promoter (phytobrick)
BBa_K3846101 CYP1a1 (phytobrick) - with N-terimanl 6xHis tag
BBa_K3846102 (http://parts.igem.org/Part:BBa_K3846102) Strong RBS (phytobrick)
BBa_K3846103 …. double terminator (BBa_B0014 - phytobrick)
BBa_K3846104 NADPH-cytochrome P450 reductase (CPR) - phytobrick
BBa_K3846105 NADPH-P450 reductase (BM3R) - phytobrick
BBa_K3846106 Terminal olefin-forming fatty acid decarboxylase (OleT) - phytobrick with N-terminal 6xHis tag
BBa_K3846107 CYP71AV1 (phytobrick) - with N-Terminal 6xHis tag
BBa_K3846108 Ferredoxin-1 (phytobrick)
BBa_K3846109 NADPH-cytochrome P450 reductase 2 (atr2) - phytobrick
BBa_K3846110 TU connector (CGCT-GGAG)
BBa_K3846111 J23119 (phytobrick)
BBa_K3846112 J23111 (phytobrick)
BBa_K3846113 rhaA (phytobrick)
BBa_K3846114 rhaA mutated (phytobrick)
BBa_K3846115 rhaS (phytobrick)
BBa_K3846116 Alpha-bisabolene synthase (phytobrick) - with N-terminal 6xHis tag
BBa_K3846117 rrnB T1 and T2 terminator (phytobrick)
BBa_K3846118 Acetyl-CoA acetyltransferase (atoB) - MevT pathway (phytobrick)
BBa_K3846119 Hydroxymethylglutaryl-CoA synthase (HMGCS1) - MevT Pathway (phytobrick)
BBa_K3846120 Hydroxymethylglutaryl-CoA synthase (HMGCS1) - MevT Pathway (phytobrick)
BBa_K3846121 Mevalonate kinase (MVK) - MBIS pathway (phytobrick)
BBa_K3846122 Phosphomevalonate kinase (PMVK) - MBIS pathway (phytobrick)
BBa_K3846123 Diphosphomevalonate decarboxylase (MVD) - MBIS pathway (phytobrick)
BBa_K3846124 Isopentenyl-diphosphate Delta-isomerase (idi) - MBIS pathway (phytobrick)
BBa_K3846125 Farnesyl diphosphate synthase (ispA) - MBIS pathway (phytobrick)
BBa_K3846126 lacUV5 promoter (phytobrick)
BBa_K3846127 Amorpha-4,11-diene synthase (AMS1) - phytobrick
BBa_K3846128 TU connector (cgct-agct)
BBa_K3846129 TU connector (agct-ggag)
BBa_K3846130 TU conncector (cgct-aaca)
BBa_K3846131 TU connector (aaca-ggag)
BBa_K3846132 Costunolide synthase (CYP71BL2) (phytobrick)
BBa_K3846133 Germacrene A hydroxylase - phytobrick
BBa_K3846134 Germacrene A synthase (GAS) - phytobrick
BBa_K3846135 Parthenolide synthase (CYP71CA1) - phytobrick
BBa_K3846136 TU Connector (ATCG-GCTT)
BBa_K3846137 Alpha-humulene synthase (ZSS1) - phytobrick
BBa_K3846138 Glucose 1-dehydrogenase 2 (gdh2) - phytobrick
BBa_K3846139 TU connector (AATG-ATCG)
BBa_K3846140 TU stop codon connector (TTCG-GCTT)
BBa_K3846175 CYP1A1 shortened sequence for linker test (1)
BBa_K3846176 CYP1A1 shortened sequence for linker test (2)
BBa_K3846177 RAT CPR shortened sequence for linker tests (1)
BBa_K3846178 RAT CPR shortened sequence for linker tests (2)
Linker
BBa_K3846150 10 aa flexible linker
BBa_K3846151 20 aa flexible linker
BBa_K3846152 10 aa rigide linker
BBa_K3846153 20 aa rigide linker
BBa_K3846154 linker sequence (Bacillus megaterium CYP102A1)
BBa_K3846155 linker sequence (Beauveria bassiana CYP505)
BBa_K3846156 linker sequence (Fusarium oxysporum CYP505)
BBa_K3846157 linker sequence (Aspergillus terreus NIH262, CYP505E3)
BBa_K3846158 linker sequence (Bacillus licheniformis ATCC 14580, CYP102A7)
BBa_K3846159 linker sequence (CYP505D6, Phanerochaete chrysosporium)
Fusion proteins
BBa_K3846200 CYP1A1-CPR fusion protein (10 aa flexible linker)
BBa_K3846201 CYP1A1-CPR fusion protein (20 aa flexible linker)
BBa_K3846202 CYP1A1-CPR fusion protein (10 aa rigid linker)
BBa_K3846203 CYP1A1-CPR fusion protein (20 aa rigid linker)
BBa_K3846204 CYP1A1-CPR fusion protein (Bacillus megaterium CYP102A1 linker)
BBa_K3846205 CYP1A1-CPR fusion protein (Beauveria bassiana CYP505 linker)
BBa_K3846206 CYP1A1-CPR fusion protein (Fusarium oxysporum CYP505 linker)
BBa_K3846207 CYP1A1-CPR fusion protein (Aspergillus terreus NIH262, CYP505E3 linker)
BBa_K3846208 CYP1A1-CPR fusion protein (Bacillus licheniformis ATCC 14580, CYP102A7 linker)
BBa_K3846209 CYP1A1-CPR fusion protein (CYP505D6, Phanerochaete chrysosporium linker)
BBa_K3846210 CYP1A1-Ferredoxin fusion protein
BBa_K3846211 CYP1A1-ATR2 fusion protein
BBa_K3846212 CYP1A1-BM3R fusion protein
BBa_K3846213 OleT-CPR fusion protein
BBa_K3846214 OleT-BM3R fusion protein
BBa_K3846215 OleT-ATR2 fusion protein
BBa_K3846216 OleT-Fd fusion protein
BBa_K3846217 CYP71AV1-CPR fusion protein
BBa_K3846218 CYP71AV1-BM3R fusion protein
BBa_K3846219 CYP71AV1-ATR2 fusion protein
BBa_K3846220 CYP71AV1-Fd fusion protein
Backbone
BBa_K3846050 modified pAM5411 (phytobrick acceptor backbone)
Expression cassettes
BBa_K3846300 Alpha-bisabolene synthase expression cassette (rhamnose inducible)
BBa_K3846301 OleT-BM3R expression cassette (rhamnose inducible) - cyanobacteria
BBa_K3846302 OleT-ATR2 expression cassette (rhamnose inducible) - cyanobacteria
BBa_K3846303 OleT-CPR expression cassette (rhamnose inducible) - cyanobacteria
BBa_K3846304 OleT-Fd expression cassette (rhamnose inducible) - cyanobacteria
BBa_K3846305 CYP1A1-CPR fusion protein (10 aa flexible linker) expression (T7 promoter)
BBa_K3846306 CYP1A1-CPR fusion protein (20 aa flexible linker) expression (T7 promoter)
BBa_K3846307 CYP1A1-CPR fusion protein (10 aa rigid linker) expression (T7 promoter)
BBa_K3846308 CYP1A1-CPR fusion protein (20 aa rigid linker) expression (T7 promoter)
BBa_K3846309 CYP1A1-CPR fusion protein (Bacillus megaterium CYP102A1 linker) expression (T7 promtoer)
BBa_K3846310 CYP1A1-CPR fusion protein (Beauveria bassiana CYP505 linker) expression (T7 promtoer)
BBa_K3846311 CYP1A1-CPR fusion protein (Fusarium oxysporum CYP505 linker) expression (T7 promoter)
BBa_K3846312 CYP1A1-CPR fusion protein (Aspergillus terreus NIH262, CYP505E3 linker) expression (T7 promoter)
BBa_K3846313 CYP1A1-CPR fusion protein (Bacillus licheniformis ATCC 14580, CYP102A7 linker) expression (T7 promoter)
BBa_K3846314 CYP1A1-CPR fusion protein (CYP505D6, Phanerochaete chrysosporium linker) expression (T7 promoter)
BBa_K3846315 CYP1A1-Ferredoxin fusion protein expression (T7 promoter)
BBa_K3846316 CYP1A1-ATR2 fusion protein expression (T7 promoter)
BBa_K3846317 CYP1A1-BM3R fusion protein expression (T7 promtoer)
BBa_K3846318 CYP71AV1-CPR fusion protein expression (T7 promoter)
BBa_K3846319 CYP71AV1-BM3R fusion protein expression (T7 promoter)
BBa_K3846320 CYP71AV1-ATR2 fusion protein expression (T7 promoter)
BBa_K3846321 CYP71AV1-Fd fusion protein expression (T7 promoter)
BBa_K3846350 MevT pathway
BBa_K3846351 MBIS pathway
BBa_K3846352 Amorpha-4,11-diene synthase expression cassette (T7 promoter)
BBa_K3846353 Amorpha-4,11-diene production cassette
BBa_K3846354 Artemisinic acid production casette
BBa_K3846355 Costunolide operon
BBa_K3846356 Parthenolid operon
BBa_K3846357 Costunolide production casette
BBa_K3846358 Parthenolide production cassette
BBa_K3846359 Humulen synthase expression casette (T7 promoter)
BBa_K3846360 Humulen production casette
BBa_K3846361 ATR2 expression casette (T7 promoter)
BBa_K3846362 RAT CPR expression cassette (T7 promoter)
BBa_K3846363 BM3R expression cassette (T7 promoter)
BBa_K3846364 Ferredoxin expression cassette (T7 promoter)
BBa_K3846365 CYP1A1 expression cassette (T7 promoter)
BBa_K3846366 OleT expression cassette (T7 promoter)
BBa_K3846367 CYP71AV1 expression cassette (T7 promoter)
BBa_K3846368 ATR2 + CYP1A1 expression control

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