Difference between revisions of "Team:UESTC-China/Demonstrate"

Pathway construction for degrading 1,2,3-TCP

1.1 Degrading Pathway construction based on the expression of DhaA31, HheC-W249P & EchA in tobacco

In order to express the three enzymes (DhaA31 isolated from Rhodococcus rhodochrous, HheC-W249P and EchA isolated from Agrobacterium radiobacter AD1) in tobacco, we firstly did the codon-optimized of the three enzymes in dicotyledonous plants. And then the genes of DhaA31, HheC-W249P and EchA were commercially synthesized. Finally, with the application of 2A peptide and Golden Gate strategy, we successfully introduced DhaA31, HheC-W249P and EchA into the plant expression vector piGEM2017-backbone which contained the resistance of kanamycin (NptII) (Fig 1). The expression of these three enzymes were driven by the common constitutive promoter CaMV35s in plants.

Figure 1. The introduction of piGEM2016-001 to piGEM2016-005

Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful (Fig 2).

Figure 2. The image of agarose gel electrophoresis by double enzyme digestion.

(a)piGEM2017-001 (Line1,enzyme digested by PstI+BamHI; Line2,enzyme digested by ScaI)

(b)piGEM2017-002 (Line1,enzyme digested by PstI+BamHI; Line2, enzyme digested by ScaI)

(c) piGEM2017-003 (Line1,enzyme digested by EcoNI+BamHI; Line2,enzyme digested by ScaI)

(d) piGEM2017-004 (Line 1, enzyme digested by EcoRI+BamHI; Line 2, enzyme digested by PstI+BamHI)

(e) piGEM2017-005 (Line 1, enzyme digested by EcoRI+BamHI; Line 2, enzyme digested by ScaI)

M: DNA marker

1.2 Degrading Pathway construction based on the root expression of DhaA31, HheC-W249P & EchA in tobacco

Considering that TCP mainly existed in the soil and groundwater, we chose the plant root specific expression promoter pYK10 from Arabidopsis thaliana to replace the constitutive promoter pCaMV35s in order to obtain better degradation efficiency of TCP by enriching these three enzymes at the root of the plants.

At the same time, in order to strengthen the root growth of tobacco, a gene that coded the expression of the cytokinin, CKX3, was also introduced into piGEM2017-backbone after the codon optimization of dicotyledonous plants (Fig 3).

Figure 3. The introduction of piGEM2017-008, piGEM2017-021 and piGEM2017-024

Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful (Fig 4).

Figure 4. The image of agarose gel electrophoresis by double enzyme digestion.

(a) piGEM2017-008 (b) piGEM2017-021 (c) piGEM2017-024

(M: DNA marker; Line 1, enzyme digested by PstI; Line 2, enzyme digested by EcoRI +BamHI)

1.3 Degrading Pathway construction based on the extracellular expression of DhaA31, HheC-W249P & EchA in tobacco

In order to transport the three enzymes out of tobacco to play their functions, we chose AO-S, a plant cell wall localization peptide, to help achieve this goal. Besides, AO-S can also stabilize the expression of the proteins. Based on these, the vectors which can fulfil the fusion expression of AO-S together with the three enzymes were constructed.

Finally, in order to intuitively confirm whether the multi-gene plant expression vectors we designed can play their roles, GUS reporter gene was also introduced into the piGEM2017-backbone (Fig 5).

Figure 5. The introduction of piGEM2017-022 023 025 026 and 027

Before DNA sequencing, those vectors were verified by restriction enzyme digestion. After electrophoresis analysis, the samples which contained all desired bands were selected and sent for sequencing. The sequencing results showed that all the above constructed vectors were successful (Fig 6).

Figure 6. The image of agarose gel electrophoresis by double enzyme digestion.

(a) piGEM2017-022 (b) piGEM2017-023 (c) piGEM2017-025 (d) piGEM2017-026 (e) piGEM2017-027

(M: DNA marker; Line 1, enzyme digested by PstI; Line 2, enzyme digested by EcoRI +BamHI)

Tobacco Transformation

In order to introduce foreign genes into tobacco and achieve the stable expression of the exogenous proteins in tobacco, Agrobacterium mediated transformation which was a common transgenic method for dicotyledonous plants was used.

We firstly transferred our plasmids into Agrobacterium EHA105 and then the T-DNA fragment which contained the target genes of the Agrobacterium EHA105 was integrated into the chromosome of tobacco through the Agrobacterium mediated transformation.

For obtaining the transgenic tobacco, the whole steps can be divided into the following eight parts. Seed germination, pre-culture, infection, co-culture, resistance screening, calli induction, root culture, transplanting. The cycle of obtaining a transgenic tobacco was about ten weeks (Fig 7).

Figure 7. The steps for obtaining transgenic tobacco

Positive validation of transgenic plants

After Agrobacterium mediated transformation, we successfully obtained the callis which have the resistance of kanamycin from the leaves of tobacco. The callis began to sprout in about 3 weeks. Another 3 weeks later, we obtained several transgenic tobacco. In order to verify whether the transgenic tobacco was false positive or not, we extracted the genomic DNA of T0 generation tobacco and designed pairs of primers to do PCR amplification of DhaA31, HheC-W249P& EchA. Compared with wild type, we successfully amplified the target bands in T0 transgenic tobacco, which indicated that our multi-gene expression system worked successfully and we obtained the positive transgenic tobacco (Fig 8).

Figure 8. The image of agarose gel electrophoresis by PCR detection

a.piGEM2017-001 b.piGEM2017-002

c.piGEM2017-003 d.piGEM2017-004

e. piGEM2017-004 f.piGEM2017-005

( PC, positive control (plasmid)，WT, negative control (wild-type tobacco))

In order to confirm whether DhaA31, HheC-W249P, EchA and CKX3 can be normally transcribed in tobacco, and determine the transcription level of these genes in tobacco, we extracted the RNA from the leaves and roots of the transgenic tobacco. And then RT- PCR was used to detect. Tubulin, a common used endogenous gene of the tobacco was selected as internal reference. Compared with the wild type, the cDNA of transgenic tobacco amplified the corresponding band of target genes, indicating that the above four exogenous genes were successfully transcribed in tobacco. At the same time, the cDNA of genes expressed by pYK10 promoter amplified a clearly band in root tissue than in leaves or other tissues, indicating that pYK10 promoter could successfully drive DhaA31, HheC-W249P, EchA and CKX3 these four exogenous genes expressed in the root of tobacco (Fig 9).

Figure 9. The image of agarose gel electrophoresis by RT-PCR detection

a.piGEM2017-001 b.piGEM2017-002 c.piGEM2017-003

d.piGEM2017-004 e.piGEM2017-005 f.piGEM2017-008

(PC, positive control (plasmid)，WT, negative control (wild-type tobacco), WT-L(leaves of

wild-type tobacco),WT-R(roots of wild-type tobacco),Tubulin was an internal control in tobacco)

Construct validation of multi-gene expression system

β-D - glucuronidase (encoding GUS gene) was an acidic hydrolase isolated from the E. coli K-12 strain that catalyzed the hydrolysis of many beta-glucoside esters. In the histochemical staining with X-gluc, the plants containing the GUS gene will appear blue. Based on this, transgenic tobacco (piGEM2017-026) which had GUS gene and wild type tobacco were identified after the histochemical staining with X-gluc for 24h at the same time. Compared with wild type tocacco, the transgenic tobacco was dyed blue, proved that the multi-gene expression system in tobacco worked as we expected again. (Fig 10).

Enzyme activity assay for the validation of transgenic tobacco

In order to detect that the transgenic tobacco can work properly, the concentration of the reaction products was measured by gas chromatography (GC) as described in our protocol. Gas chromatograph Network GC System (Fuli 9750, China) equipped with a capillary column ZB-FFAP 30 m x 0.25 mm x 0.25 µm (Phenomenex, USA) was used to the quantitative analyses of all metabolites of the TCP degradation pathway except for GLY. The concentration of GLY was determined by GC (Fuli 9790) equipped with a DB-5 column (L60 m×i.d. 0.25 mm and df 0.25μm thin coating film) (Supelco, Bellefonate, PA, USA).

5.1 Measure the standard curves of reaction products

For quantitative assay, standard curves of 1,2,3-TCP, 2,3-DCP, ECH, CPD, GDL, GLY ranging from 0-5 mM in 200 mM of Tris-SO4 buffer(pH=8.5) were measured (Fig 11).

Figure 11. Standard curves of reaction products

5.2.1 Activity determination of DhaA31 (piGEM2017-001) in transgenic tobacco

Figure 12. The Concentration of 2,3-DCP produced within 7 hours. The activity of DhaA31 was

determined in 6.5 mL supernatant of tobacco carrying piGEM2017-001 (2 g leaves (FW) were

rinsed thoroughly with 6 mL 200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and

6.5mL supernatant was stored) and 5 mM 1,2,3-TCP. Each data represented the mean value ± standard deviation from three independent experiments.

5.2.2 Activity determination of HheC-W249P (piGEM2017-002) in transgenic tobacco

Haloalcohol dehalogenase (HheC) could degrade 2,3-DCP and CPD to ECH and GDL, respectively. So we detected its activity with 2,3-DCP and CPD as substrates in piGEM2017-002, respectively. While no ECH or GDL were detected at the selected time in wild-type or piGEM2017-002 (data not shown), indicating that no active HheC-W249P was expressed in transgenic tobacco carrying piGEM2017-002. This could be either due to the fact that there was no HheC-W249P expression or the expressed enzyme was less stable in plants. Among these three enzymes, HheC-W249P was the only enzyme that was active as a tetramer, while the other two enzymes were monomer. This could also explain why the activity of HheC-W249P was not detected in transgenic tobacco carrying piGEM2017-002. Due to the limitation of time, we could’t further optimize the expression of HheC-W249P.

Instead, we tested the activity of HheC-W249P by mimicking the environment of tobacco in vitro. For this, HheC produced by recombinant E.coli was added into the supernatant of wild-type tobacco, rinsed in Tris-SO4 buffer. As shown in Figure 13, the concentration of ECH in the supernatant of tobacco and Tris-SO4 buffer was similar, indicating that components existed in tobacco did not show a significant effect on the activity of HheC-W249P. In the following experiments, HheC produced by E.coli was added in the TCP degradation pathway.

Figure 13. Concentration of ECH within 7 hours. 2 g leaves (FW) were rinsed thoroughly with 6 mL

200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and 6.5mL supernatant was

stored for the determination of HheC enzymatic activity with 5 mM ECH . 6.5mL 200mM Tris-SO4

buffer (pH=8.5) with same amount HheC was used as control. Each data represents the mean value ± standard deviation from three independent experiments.

5.2.3 Activity determination of EchA (piGEM2017-003) in transgenic tobacco

Figure 14. Concentration of CPD within 7 hours. 2 g leaves (FW) were rinsed thoroughly with 6 mL

200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and 6.5mL supernatant was

stored for the determination of EchA enzymatic activity with 5 mM ECH. Each data represents the

mean value ± standard deviation from three independent experiments.

5.2.4 Work validation of multi-enzyme system (piGEM2017-004, piGEM2017-005) in transgenic tobacco

DhaA31, HheC-W249P and EchA catalyzed five-step reactions converting toxic TCP to harmless glycerol. To test whether this multi-enzyme conversion system could work in tobacco successfully or not, we detected the time consumption curve of 6 mM TCP by using the grinded leaves. The concentrations of TCP, GLY and other intermediates were monitored. In this case, HheC produced by E.coli was added in the reaction mixture. The conversion of TCP to GLY within 30 h was determined as shown in Figure 15. Glycerol produced gradually with the consumption of 1,2,3-TCP. After 30 h, less than 1 mM of TCP was left, indicating that our system worked well in tobacco.

Figure 15. Time courses of conversions of 1,2,3-TCP with enzymes extracted from leaves of

transgenic tobacco carrying piGEM2017-004 and HheC produced by E.coli . 4 g leaves(FW) were

rinsed thoroughly with 8 mL 200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and

10 mL supernatant was stored for the determination of multi-enzyme activity with 6 mM 1,2,3-TCP.

As described above, tobacco contained endogenous epoxide hydrolase so as shown in Figure 16, the efficiency of converting TCP to GLY by piGEM2017-005 was detected and showed a similar degradation efficiency as the one shown in Figure 15.

Figure 16. Time courses of multi-enzyme conversions of 1,2,3-TCP with enzymes extracted from

leaves of piGEM2017-005-7 and added HheC. 4 g leaves(FW) of piGEM2017-005-7 were rinsed

thoroughly with 8 mL 200mM Tris-SO4 buffer (pH=8.5), the extraction was centrifuged and10 mL

supernatant was stored for the determination of multi-enzyme activity with 6 mM 1,2,3-TCP.

5.2.5 Glycerol measurement using gas chromatography–mass spectrometry (GC-MS)

The glycerol production was monitored by periodically taking samples from the supernatant of piGEM2017-004 and piGEM2017-005. The final extract was used for the detection of glycerol by GC-MS (Fig 17). As shown in Figure 17, the peak of glycerol after the pre-column derivatization was observed from samples with 0h and 20h incubation. Most importantly, the peak of glycerol after the pre-column derivatization increased within 20 hours while glycerol after the pre-column derivatization was not found in sample of wild-type tobacco.

Figure 17. Chromatogram of glycerol and internal 1,2,3-butanetriol derivatives of supernatant of

transgenic tobacco carrying piGEM2017-004 and wild-type tobacco after the pre-column derivatization. Samples were taken from reaction mixture at 0h, 20h.

Figure 18. Mass spectrums of tris (trimethylsilyl) glycerol ether and 1,2,3-tris (trimethylsilyl) butanetriol ether

Activity determination of multi-enzyme system in hydroponic solutions

To investigate whether our “super tobacco” could work or not when placed in the environment, hydroponics method was used to demonstrate. Plants were incubated in liquid nutrient solutions containing a certain concentration of substrate. Samples were taken from medium at selected time. Gas chromatography was used to detect the degradation products in periodic sampling. Figure 19 showed that DhaA31 and EchA could work successfully in the culture, which indicated that our transgenic tobacco could work properly in the hydroponic environment.

Figure 19. The concentration of 2,3-DCP within 3 days.

Figure 20. The concentration of CPD within 3 days.

Figure 21. The hydroponics devices designed by us

Work going on