Part:BBa_K4182012:Design
RK2 Ori
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 399
Illegal NgoMIV site found at 523 - 1000COMPATIBLE WITH RFC[1000]
Profile
Base Pairs
532
Source
E.coli
Usage&Biology
Optimize and improve the replicon ori of 2019XJTU-iGEM parts BBa-K3052010 to reduce the metabolic pressure of cells and improve the protein expression level
1. Plasmid map before and after replicon optimization
FIG.1 p15Aori-pGPP 2019XJTU-Igem parts BBa-K3052010
FIG.2 Optimized plasmid 2 (i.e. 421ori-pGPP)
2. Principle and process of plasmid 2 construction
2.1 mevalonate (MVA) pathway
The MVA pathway mainly exists in eukaryotes. It uses acetyl-CoA as raw material to form IPP and DMAPP through the precursor mevalonate. Then, under the action of polyisoprenyl diphosphate synthase, IPP and DMAPP are further condensed to form geranyl pyrophosphate (GPP). The reaction is also called the cytoplasmic pathway because it occurs in the cytoplasm.
According to works of literature, carbon sources like glucose are first metabolized to acetyl-CoA which is then converted into IPP and DMAPP, the two universal precursors of all terpenes. Then IPP and DMAPP will be further converted to geranyl pyrophosphate (GPP) by a geranyl pyrophosphate synthase (GPPS) which is a common precursor of fragrance molecules.
acetyl-CoA is catalyzed by acetyl-CoA acetyltransferase to produce acetyl-CoA, which is catalyzed by 3-hydroxy-3-methylglutaryl synthase hydroxymethylglutaryl-CoA (HMG-CoA) is formed under the catalysis of coenzyme A synthase. HMG-CoA is then reduced by hydroxymethylglutaryl-CoA reductase (HMGR) to form mevalonate (MVA). mevalonate kinase (MK), phosphomevalonate kinase (MVA), MVA, MVA, HMGR, MVA, MVA, MVA, MVA, MVA, MVA. PMK, mevalonate diphosphate decarboxylase, MPD, and isopentenyl diphosphate isomerase (Idi) catalyze the formation of IPP and DMAPP. Finally, they are further condensed to form geranyl pyrophosphate (GPP).
atoB（acetyl-CoA acetyltransferase）；
HMGS（3-hydroxy-3-methylglutaryl coenzyme A synthase）；
HMGR（3-hydroxy-3-methylglutaryl-coenzyme A reductase）；
MK（mevalonate kinase）:
PMK（phosphomevalonate kinase）；
MPD（mevalonate diphosphate decarboxylase）；
2.2 Promoter optimization oriV+trfA
RK2 is a 2.22-kb replication origin of the broad-host-range IncPa plasmid formed by the vegetative origin (oriV) and the replication protein trfA. The standardized ori segment is formed by oriV followed by the gene that encodes the replication protein TrfA, which is expressed from its native BHR promoter. The immediate source of this RK2 origin was pJB785TT and the relevant sequence of this origin was used for formatting the corresponding module. This origin of replication is among the least restrained and it keeps its copy number per cell very low.
The 421-oripGPP vector is pSEVA421, and the replication start site is from RK2, which belongs to the repeat plasmid. trfA on the vector sequence belongs to the repA gene, and its expressed protein RepA protein can regulate plasmid replication by binding to the repeat sequence near the replication start site. According to the steric hindrance model, the regulation of plasmid copy by RepA depends on the concentration of duplicates. The function of RepA is essentially DNA polymerase, and the first effect of RePA binding to repeats is to initiate plasmid DNA replication. With the replication of the plasmid, the concentration of the repeats increases. The RepA bound to the duplicator interacts with the RepA bound to the other duplicator, coupling two segments of plasmid DNA through the RePA-RePA interaction, forming steric hindrance between the two duplicators and terminating replication initiation.
Since the MVA pathway involves many genes (the P15AORI-PGPP plasmid is 15kb in length) and the previous data showed that the expression level of each enzyme in the cell was not high, we tried to replace the replicating P15Aori (5-10 copies) with the lower copy oriV+trfA (1-3 copies). To reduce the pressure of cell growth and metabolism by reducing the copy number, to improve the protein expression.
3. Construction process and verification of plasmid 2
Because of the large size of the plasmid and the difficulty of gene manipulation, the Gibson ligation method was used: oriV-trfA PCR was used to obtain the linear pGPP fragment, while the linear PGPP fragment was obtained by enzyme digestion. Restriction enzymes Sac I and Bln I were used to digesting the linearized 12-kb GPP fragment. For the recovery of linear large fragments, we optimized a variety of protocols and tried a new direct purification method using silica gel particles. Compared with the silica gel column, the silica gel particles are uniformly dispersed in the sol buffer system, which can avoid the breakage of DNA fragments caused by excessive shear force on the DNA adsorbed to the silica gel particles. Compared with the conventional silica gel column, the centrifugal speed involved in the operation is also reduced from 12000 rpm to 8000 rpm. Be careful and gentle during operation to avoid damaging the integrity of DNA. GPP fragments were eventually recovered to a lower concentration (4.1 μg/μl) and visualized on agarose gels (Figs. 3-9, GPP).
FIG.3: M, DL10000, 2: oriV+TrfA 3: pGPP linear fragment The high molecular weight of plasmids was associated with low ligation efficiency and only 6 colonies on two solid media. Monoclonal colonies were selected for colony PCR (Figure 3-11) and 1-4 showed positive results. Four positive Wells were selected and transferred to liquid culture, bacteria were stored, and plasmids were extracted and sent to the company for sequencing. Sequencing results showed that fragments 1 and 4 had been correctly ligated, and strain 4 was stored.
FIG.4 pGPP plasmid colony PCR. M GL Marker for 5000
The positive strain was cultured to OD600= 0.6-0.8, and IPTG with a final concentration of 1mM was added for 6 hours. RT-qPCR was performed after induction. The results showed that the transcription level of key enzyme GPPS was significantly increased after the replacement of lower copy number replicons, which proved the effectiveness of the strategy. When the metabolic pressure of cells was reduced, cell growth and metabolism were enhanced, and protein expression was also increased. This also facilitates the synthesis of GPP and AA, our final product.
FIG.5 RT-qPCR results of GPPS in three strains
References
[1]Rafael Silva-Rocha, Esteban Martínez-García, Belén Calles, Max Chavarría, Alejandro Arce-Rodríguez, Aitor de las Heras, A. David Páez-Espino, Gonzalo Durante-Rodríguez, Juhyun Kim, Pablo I. Nikel, Raúl Platero, Víctor de Lorenzo, The Standard European Vector Architecture (SEVA): a coherent platform for the analysis and deployment of complex prokaryotic phenotypes, Nucleic Acids Research, Volume 41, Issue D1, 1 January 2013, Pages D666–D675, https://doi.org/10.1093/nar/gks1119
[2]Jorge Alonso-Gutierrez, Rossana Chan, Tanveer S. Batth, Paul D. Adams, Jay D. Keasling, Christopher J. Petzold, Taek Soon Lee, Metabolic engineering of Escherichia coli for limonene and perillyl alcohol production, Metabolic Engineering, Volume 19,2013, Pages 33-41, ISSN 1096-7176,https://doi.org/10.1016/j.ymben.2013.05.004.
[3]Figurski, D.H. and Helinski, D.R. (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl Acad. Sci. USA, 76, 1648–1652.
[4]Santos,P.M., Di Bartolo,I., Blatny,J.M., Zennaro,E. and Valla,S.(2001) New broad-host-range promoter probe vectors based on the plasmid RK2 replicon. FEMS Microbiol. Lett., 195, 91–96.