Team:AISSU Union/Engineering

Engineering success

Abstract

In the process of achieving production of naringenin and catechin, the most critical issue is to accomplish the assembly of gene that perform two metabolism pathways. To achieve this goal, we started from constructing basic parts (Level 0 ), followed by assembling each individual parts (Level 1) into a composite part plasmid (Level 2) and finally construct a complete metabolism pathway. The assembly of naringenin and catechin, however, involves numerous fragments with relatively great total length, which increased difficulty of construction in molecular level.Various methods been tested during the stage, including Gibson Assembly®, Golden gate assembly and yeast homologous recombination. Although many different assembly methods had been tested, only the yeast homologous recombination achieved the assembly of metabolism pathway finally.

1. Obtaining basic parts required for first combination

In the first stage of the experiments, we need to obtain all basic parts, including promoters, terminators, selective markers, and origin sequences involved in the yeast toolkit we used. Meanwhile, we constructed the vector for Level 1plasmids assembly, which is type8-pSB1K3-GFP and type8-pSB1C3-GFP respectively.

1.1 Obtaining Toolkit plasmids

For the basic parts involved in the toolkit, we entrust BGI to synthesize most of sequences, excepting promoters pPDC1 and terminator tCYC1, of which relevant sequences were obtained using yeast genome as template. The sequences were inserted into the plasmids using Gibson assembly. The picture demonstrates the results of enzyme-digested verification to constructed toolkit plasmids using restriction enzyme BsaI.

Fig.1 Results of yeast toolkit plasmids enzyme-digested verification M: RB- MK8000 Ladder, 1:Type-1-ConL1(194bp), 2: Type-1-ConL2(194bp),3: Type-1-ConL3(194bp),4: Type-1-ConL4(194bp),5: Type-1-ConL5(194bp),6: Type-1-ConLS(194bp), 7:Type-1-ConLS’(194bp),8:Type-2-pAOX1(943bp), 9:Type-2-pGAL1(542bp), 10:Type-2-pPOP6(709bp), 11:Type-2-pRET2(709bp),12: Type-2-pPOP6(709bp) 13:Type-2-pTDH3(689bp), 14:Type-2-pTEF1(709bp), 15:Type-2-pTEF2(709bp),16:Type-4-tADH1(238bp), 17:Type-4-tTDH1(237bp), 18:Type-4-tENO1(238bp), 19:Type-4-tPGK1(238bp), 20:Type-4-tSSA1(238bp), 21:Type-4-tENO2(238bp),22:Type-5-ConR1(173bp), 23:Type-5-ConR2(173bp), 24:Type-5-ConR3(173bp), 25:Type-5-ConR4(173bp), 26:Type-5-ConR5(173bp), 27:Type-5-ConRE(173bp), 28:Type-5-ConRE’(173bp), 28:Type-6-URA3(1063bp), 29:Type-7-2micron(1005bp),

According to Fig.1, we obtain two clear bands after BsaI digestion, the length of the vector(1622bp) and the inserted fragments matched with previous expectations by comparing with Marker MK8000 ladder, confirming the successful assembly of Toolkit plasmids and availability in subsequent construction.

1.2 Assembly of construction vectors Type8K, Type 8C

We obtain three purified vectors fragments used in Level 1 construction through the PCR. We assemble these fragments through Gibson Assembly®. The construction strategy and results is demonstrated by Fig.2.

Fig.2 A: Results of type8-pSB1K3-GFP and type8-pSB1C3-GFP plasmids maps; B: Bsal digestion verification results; C: results of sequencing analysis

Using BsaI digestion to verify the constructed plasmids, shown in Fig.2 B, two clear bands occur. By comparing with MK8 Ladder, the length of vector plasmid(1622bp) and length of inserted fragment (1351bp), matched with the previous expectations. The sequence analysis results, in Fig. 2C showed no significant mutation or deletion.(The details of vector plasmid type 8K is generally same as type8C, so here is not demonstrated).

Fig.3 Characterization of the type8-pSB1K3-GFP and type8-pSB1C3-GFP

These vector plasmids were inserted with gene producing green fluorescent protein. Therefore, to test the results of construction, we transfer constructed transformants into selection medium in plates and liquid (Fig.3A and Fig.3B). Under the exposure of the U.V , the green fluorescents from the colonies shows successful performance of type8-pSB1K3-GFP and type8-pSB1C3-GFP in DH5α.

2 Assembly of Level 1 plasmids

After obtaining all individual parts in yeast toolkit, the next step involving construction of the seven different parts with type8-pSB1K3-GFP, adding coding sequences with corresponding promoters and terminators, which is one difficulty we encounter during the process of assembly.

2.1 Naringenin pathway relevant sequences Level 1 plasmids construction

The construction results of coding sequence RtTAL are shown by Fig.4:

Fig.4 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence RtTAL

The construction schematic of RtTAL sequence is demonstrated in Fig.4. The initiation of the RtTAL sequence is done by promoter pRET2, with termination done by tSSA1. The sequence ConLS and ConR1 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR are used to enact selection through colonies PCR, results are shown in Fig.4 B. The band length 3432bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence Pc4CL are shown by Fig.5:

Fig.5 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence Pc4CL

The construction schematic of Pc4CL sequence is demonstrated in Fig.5. The initiation of the Pc4CL sequence is done by promoter pPGK1, with termination done by tCYC1. The sequence ConL1 and ConR2 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.5 B The band length 3004 bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence GbCHS are shown by the Fig.6:

Fig.6 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GbCHS

The construction schematic of GbCHS sequence is demonstrated in Fig.6. The initiation of the GbCHS sequence is done by promoter pTEF1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.6 B The band length 2529bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence MsCHI are shown by Fig.7:

Fig.7 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence MsCHI

The construction schematic of MsCHI sequence is demonstrated in Fig.7. The initiation of the MsCHI sequence is done by promoter pAOX1, with termination done by tTDH1. The sequence ConL3 and ConRE are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.7 B The band length 2021bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

2.2 Catechin pathway relevant sequences Level 1 plasmids construction

The construction results of coding sequence CrCPR are shown by Fig.8:

Fig.8 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence CrCPR

The construction schematic of CrCPR sequence is demonstrated in Fig.8. The initiation of the CrCPR sequence is done by promoter pGAL1, with termination done by tTDH1. The sequence ConLS and ConR1 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.8 B The band length 3327bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence CsF3H are shown by Fig.9:

Fig.9 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence CsF3H

The construction schematic of CsF3H sequence is demonstrated in Fig.9. The initiation of the CsF3H sequence is done by promoter pGAL7, with termination done by tENO2. The sequence ConL1 and ConR2 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.9 B The band length 2755bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence GhF3’H are shown by Fig.10:

Fig.10 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence GhF3’H

The construction schematic of GhF3’H sequence is demonstrated in Fig.10. The initiation of the GhF3’H sequence is done by promoter pPGK1, with termination done by tADH1. The sequence ConL2 and ConR3 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.10 B The band length 2892bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence AaDFR are shown by Fig.11:

Fig.11 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence AaDFR

The construction schematic of AaDFR sequence is demonstrated in Fig.11. The initiation of the AaDFR sequence is done by promoter pTDH3, with termination done by tENO1. The sequence ConL3 and ConR4 are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.11 B The band length 2377bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

The construction results of coding sequence DuLAR are shown by Fig.12:

Fig.12 A: the construction strategy; B: results of colony PCR; C: results of sequencing analysis of coding sequence DuLAR

The construction schematic of DuLAR sequence is demonstrated in Fig.12. The initiation of the DuLAR sequence is done by promoter pPOP6, with termination done by tSSA1. The sequence ConL4 and ConRE are connector sequences within the Level 1 plasmid assembly. Furthermore, type9-KVF and type9-VR were used to enact selection through colonies PCR, results are shown in Fig.12 B The band length 2502bp matched with expectations. The sequence analysis results show no significant mutations or deletions, representing the success of the assembly.

3 Assembly of Level 2 plasmids

Initially, we choose to use Golden Gate assembly to achieve construction of naringenin and catechin pathway, involving four and five composite parts are shown above. However, after the screening of transformants through colony PCR, we did not find individual that matched with the design expectations, which was the largest bottleneck we encounter during the assembly. Thus, we refer to research done by Ando et.al. and choose yeast homologous recombination to accomplish assembly of final metabolism pathways.

3.1 Yeast transformation, transferring and selection

To achieve yeast homologous recombination, we take each composite part in Level 1 as PCR template, designing primers involved homologous arms with length 50bp to ensure high construction efficiency. According to yeast recombination strategy developed by our teams, we manage to achieve the transformation of yeast monoclonals. In addition, corresponding monoclonals were dissolved in ddH2O in order to facilitate subsequent yeast colony PCR.

Fig.13 Schematic of yeast homologous recombination, transferring and culturing

4 Yeast colony PCR verification

To test validity of the junctions between each sequence, we design corresponding primers to verify the inserted sequences. The results of junctions PCR are shown in Fig.14.

Fig.14 A: The results of colony PCR of upstream transformant; B: The results of colony PCR of downstream transformant; C: Construction of catechin metabolic pathway; D：Construction of naringenin metabolic pathway

The results in Fig.14 show clear bands with length that matched with expectations. The subsequent sequence analysis shows no significant mutations or deletions, confirming the success of construction of two metabolism pathways.