FIG.1 p15Aori-pGPP 2019XJTU-Igem parts BBa-K3052010
FIG.2 Replicon optimized plasmid 2 (421ori-pGPP)
2022-XJTU-China parts BBa_K4182012
FIG 3 MVA pathway and constructed gene cluster
(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)
The MVA pathway mainly exists in eukaryotes. It starts from acetyl-CoA to form IPP and DMAPP via mevalonate, with the help of several enzymes including AtoB, HMGS, HMGR, MK, PMK, MPD and Idi. Then IPP and DMAPP are further condensed to form geranyl pyrophosphate (GPP) catalyzed by geranyl pyrophosphate synthase (trGPPS). GPP is a very common precursor of fragrance molecules, terpenes and our herbicide aspterric acid (AA).
FIG 4 Structure of RK2 ori
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 under its native BHR promoter. This origin of replication is among the least restrained and it keeps its copy number per cell very low (1-3 copies).
Our plasmid 2 derivate from RK2 ori, and its low copy number is regulated by iterons mechanism. TrfA protein is a replication regulator belonging to RepA protein family, which can regulate plasmid replication by binding to the repeat sequence (iterons) near the replication start site. TrfA will initiate plasmid DNA replication once binding to the origin. However, with the increase of plasmid copy, the concentration of the iterons and TrfA protein increases, leading to the TrfA-TrfA protein interaction and lock the two plasmid DNA like a handcuff, then replication terminates and the copy-number of the plasmid is regulated.
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 (See the results of team 2019-XJTU-China ). So we tried to replace the replicon p15Aori (5-10 copies) with the lower copy RK2 ori (1-3 copies), to reduce the pressure of cell growth and metabolism as well as protein expression by reducing the copy number of plasmid.
Because of the large size of the plasmid and the difficulty of gene manipulation, the Gibson Assembly method was used in our study: oriV-trfA fragment was obtained by PCR, while the linear pGPP fragment (~12 kb) was obtained by enzyme digestion of Sac I and Bln I. For the recovery of linear large pGPP fragment, a variety of strategies were employed and a new direct recovery method by the silica-gel particle was found to be efficient, mainly because the silica-gel particles were uniformly dispersed in buffer, which can avoid the DNA fragment break caused by excessive shear force. So centrifugation was conducted at 8000 rpm instead of 12000 rpm, and be gentle during the purification. Finally we obtained pure pGPP fragment with a concentration of 4.1 μg/μL.
 FIG.5 Fragment used for construction of plasmid 2 (1: M, DL10000, 2: oriV+TrfA 3: pGPP linear fragment) |
FIG.6 Colony PCR verification of plasmid 2. M, DL5000 |
After Gibson assembly, we obtained only 6 colonies on LB medium with low cloning efficiency, and the colony PCR of clone 1-4 showed positive results. Then plasmids were extracted and sent to the company for sequencing. Sequencing results showed No.4 was correct.
The strain harboring our plasmid 2 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 replicon, which proved the effectiveness of the strategy. When the metabolic pressure was reduced, cell growth and metabolism were enhanced, leading to the increased protein expression. It will also facilitate the synthesis of GPP and AA, our final product.
Our EPS synthesis circuit was constructed based on the previous work by team 2020-XJTU-China (https://2020.igem.org/Team:XJTU-China/Engineering) . It contains the key enzymes GalU and PmgA under the regulation of LacI-Ptrc promoter.
FIG 8 EPS synthetic pathway and EPS synthesis circuit
We hope to produce extracellular polysaccharide EPS in engineered bacteria by EPS synthesis circuit (plasmid 4) to achieve water conservation and sand fixation of soil. Figure 9 shows the EPS biosynthetic pathway: Glucose can be converted to glucose-1-phosphate via EMP pathway or by enzyme phosphoglucose mutase (PgmA), and then UDP is synthesized by UDP glucose pyrophosphorylase (GalU) and served as precursor of EPS. Fredrik at al reported that the overexpression of pgmA and galU genes in Streptococcus thermophiles enhanced the EPS production from 0.17 g/mol to 0.31 g/mol. The same strategy was also employed in our study and the team 2020-XJTU-China. (https://2020.igem.org/Team:XJTU-China/Engineering)
FIG 9 The biosynthetic pathway of EPS
Based on the work of the XJTU-2020 team (https://2020.igem.org/Team:XJTU-China/Engineering), we selected the pgmA from E.coli (GenBank: CP041425.1) and galU gene from E.coli (GenBank: CP104721.1) which proved highest EPS production in previous study. LacI-Plac promoter fragment, pgmA and galU amplified from previous EE plasmid, were ligated into the backbone pSB1K3 by Golden Gate Assembly (Figure 10). Several clones were obtained after assembly and transformation, yet it is difficult to extract recombinant plasmid after several attempts. So another high copy number vector backbone pSEVA341 was employed, and the recombinant plasmid successfully constructed and verified by colony PCR as shown in Figure 11, and further confirmed by sequencing. The iGEM ID for plasmid 4 is BBa_K4182009.
FIG10：PCR fragments used for construction of plasmid 4
FIG11 Colony PCR verification of plasmid 4
As shown in the FIG12, compared to the EE plasmid constructed by team 2020-XJTU-China (https://2020.igem.org/Team:XJTU-China/Engineering) , our EPS synthesis plasmid were constructed with replicon optimization from medium-copy-number pMB1ori (15-20 copies) of EE plasmid to high-copy-number pRO1600 ori (100-150 copies) of pSEVA341 vector, and promoter optimization from low efficient P43 promoter to inducible high efficient PlacUV5. The optimization leads to the increased transcriptional levels for both galU and pgmA gene as shown in Figure 13. After IPTG induction, the expression levels of galU and pgmA gene were 3.4 and 2.8 fold compared to that of EE plasmid, demonstrating the improvement after promoter and replicon optimization.
FIG12: Optimization of plasmid 4 compared to EE plasmid
(BBa_K3331012 Vs BBa_K4182009)
FIG 13 RT-qPCR result of plasmid 4 compared to previous EE plasmid
(IPTG was induced at a concentration of 1mM for 6h at 37℃.)
Anthrone-sulfuric acid colorimetry is a fast and convenient sugar fixation method. Under strong acidic conditions, anthrone can be associated with free or polysaccharides present in hexose, pentose and hexoclonic acid to generate blue-green glycal derivatives, and sugar content can be determined by the different color of glycal product, whose maximum absorption peak at 620 nm. In this experiment we can quantitatively compare the EPS content of engineered bacteria by this method.
First, we used glucose at concentrations of 0, 0.2, 0.4, 0.6, and 0.8 (units of mg/ml) as standard solutions to determine the standard curve at 620 nm. (FIG 14)
FIG 14: Standard curve using glucose as a standard solution and their different colors.
In order to avoid the influence of the sugar in LB medium, we purified the the intracellular EPS by alcohol precipitation and re-dissolved in water. The EPS content was detected by anthrone-sulfuric acid method and the results are shown in FIG8.
FIG15: 620 nm detection S of three strains harboring EE plasmid or plasmid 4. (calculated as the ratio of absorbance to liquid concentration), *indicating that there are significant differences in this sample compared to EE (based on T-TEST).
Compared with the previous EE plasmid, the EPS yield of the engineering bacteria with plasmid 4 has been significantly improved by about 3 fold (P<0.05), demonstrating the construction of plasmid 4 is successful and effective, and also confirming our improvement.