iGEM 2024 — Team pinghe-mca

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Notebook

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| 2023 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| December |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Background research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Experiment research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| 2024 | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| January |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Background research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Experiment research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Febuary |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 |  |  |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Background research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Experiment research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| March |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Experiment research |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| April |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |  |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| May |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| June |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |  |
| Online theoretical learning |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| July |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Preparing culture medium |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Construction of plasmid - PCR |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Promotion video producing |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| August |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 | 31 |
| Construction of plasmid - PCR |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Acquiring carrier - shake culture |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Plasmid extraction |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Enzyme digestion of pPICZαA & Electrophoresis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Enzyme digestion of Target gene & Electrophoresis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Copy and reconnection of plasmid |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Monoclonal colony verification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Shake culture |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Plasmid extraction from E. Coli |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Enzyme digestion & Electrophoresis |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Gel extraction & Transformation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Monoclonal colony verification |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Shake culture |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Enlargement |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Protein extraction & Purification & SDS-PAGE |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| PTVA screening high copy transformants |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Promotion video producing |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| September |  | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | |
| Date | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 | 21 | 22 | 23 | 24 | 25 | 26 | 27 | 28 | 29 | 30 |  |
| Yield test |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Enzyme activity test |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Promotion video producing |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Defence preparation |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |
| Wiki building |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |

###### 2023

###### December

* Online theoretical learning
* Background research
* Experiment research

![](https://static.igem.wiki/teams/5528/notebook1.jpg)

###### 2024

###### January

* Online theoretical learning
* Background research
* Experiment research

###### Febuary

* Online theoretical learning
* Background research
* Experiment research

###### March

* Online theoretical learning
* Experiment research

###### April

* Online theoretical learning

###### May

* Online theoretical learning

###### June

* Online theoretical learning

![](https://static.igem.wiki/teams/5528/notebook2.jpg)

###### July

* Preparing culture medium
* Construction of plasmid - PCR
* Promotion video producing

![](https://static.igem.wiki/teams/5528/notebook3.jpg)

###### August

* Construction of plasmid - PCR
![](https://static.igem.wiki/teams/5528/notebook4.jpg)* Acquiring carrier - shake culture
![](https://static.igem.wiki/teams/5528/notebook5.jpg)* Plasmid extraction
* Enzyme digestion of pPICZαA & Electrophoresis
![](https://static.igem.wiki/teams/5528/notebook6.jpg)* Enzyme digestion of Target gene & Electrophoresis
* Copy and reconnection of plasmid
* Monoclonal colony verification
![](https://static.igem.wiki/teams/5528/notebook7.jpg)* Shake culture
![](https://static.igem.wiki/teams/5528/notebook8.jpg)* Plasmid extraction from E. Coli
* Enzyme digestion & Electrophoresis
![](https://static.igem.wiki/teams/5528/notebook9.jpg)* Gel extraction & Transformation
![](https://static.igem.wiki/teams/5528/notebook10.jpg)* Monoclonal colony verification
* Shake culture
* Enlargement
* Protein extraction & Purification & SDS-PAGE
* PTVA screening high copy transformants
* Promotion video producing September
* Yield test
![](https://static.igem.wiki/teams/5528/notebook11.jpg)* Enzyme activity test
* Promotion video producing
* Defence preparation
* Wiki building

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![](https://static.igem.wiki/teams/5528/candys.png)
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Engineering

![](https://static.igem.wiki/teams/5528/logo.png)

* [Overview](#content-1)
* [Reference](#content-2)

Overview

D-psicose 3-epimerase (DAE) plays an important role in the biotransformation of D-psicose, but the thermal
stability of D-psicose 3-epimerase is poor and the expression level is low, which cannot meet the
requirements of industrial production. In this study, we synthesized the coding genes of
D-psicose-3-epimerase from different sources, ligated the coding genes into the expression vectors, and
transformed them into Pichia pastoris GS115 strain. The target protein was induced and purified, and the
yield and enzymatic properties of D-psicose and D-psicose 3-epimerase with good thermal stability were
detected. On this basis, the liquid PTVA method was used to obtain high-copy strains by Zeocin antibiotic
screening to further improve the expression level of D-psicose 3-epimerase and the yield of D-allulose. This
study will provide a new strategy for the industrial production of D-psicose (Figure
1).

![](https://static.igem.wiki/teams/5528/engineering1.png)

Figure 1.The engineering schematic diagram of the project design

###### Part 1 [BBa\_K5528005](https://parts.igem.org/Part%3ABBa_K5528005)(pPICαA-TcDAE)

###### Design

We was conducted to select target DNA(TcDAE) in NCBI database. The genes encoding DAE fused with 6×His-tag
at its C-terminus were optimized and synthesized.The plasmid map was constructed by snapgene software(
2).
And TcDAE inserted into the expression vector pPICZαA between restriction endonucleases EcoRI
and SalI
sites, the recombinant plasmids pPICZαA-TcDAE were further transformed into E.Coli DH5α to construct
plasmids.

![](https://static.igem.wiki/teams/5528/engineering2.png)

Figure 2. The plasmid map of pPICZαA-TcDAE

###### Build

We transferred the plasmid pPICZαA-TcDAE into E.coli-DH5α and did single clone verification. Figure 3-A
shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of
the target gene TcDAE. Figure 3-B, showed the E. coli plates we cultured and
the location of sample for
single clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure
3-C showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing
to conﬁrm
its sequence ﬁdelity, and the positive recombinant plasmids were named as pPICZαA-TcDAE (Figure 3).

![](https://static.igem.wiki/teams/5528/engineering33.png)

Figure 3. The single clone verification and sequencing comparison of
plasmid pPICZαA-TcDAE ( E.coli DH5α)

###### Part 2 [BBa\_K5528006](https://parts.igem.org/Part%3ABBa_K5528006)(pPICαA-TtDAE)

###### Design

Selection of novel DAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its
C-terminus were optimized , and chemically synthesized.The plasmid map was constructed by snapgene
software(Figure 4). And it inserted into the expression vector pPICZαA between
restriction endonucleases
EcoRI and SalI sites, the recombinant plasmids pPICZαA-TtDAE were further transformed into E.Coli DH5α.

![](https://static.igem.wiki/teams/5528/engineering4.png)

Figure 4. The plasmid map of pPICZαA-TcDAE

###### Build

We transferred the plasmid pPICZαA-TtDAE into E.coli-DH5α and did single clone verification. Figure 5-A
shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of
the target gene. Figure 5-B, showed the E. coli plates we cultured and the
location of sample for single
clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 5-C
showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to conﬁrm its
sequence ﬁdelity, and the positive recombinant plasmids were named as pPICZαA-TtDAE (Figure 5).

![](https://static.igem.wiki/teams/5528/engineering5.png)

Figure 5. The single clone verification and sequencing comparison of
plasmid pPICZαA-TtDAE ( E.coli DH5α)

###### Part 3 [BBa\_K5528007](https://parts.igem.org/Part%3ABBa_K5528007)(pPICαA-NtDAE)

###### Design

Selection of novel NtDAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its
C-terminus were optimized and synthesized.The plasmid map was constructed by snapgene software (Figure 6).
and NtDAE inserted into the expression vector pPICZαA between restriction endonucleases EcoRI and SalI
sites, the recombinant plasmids pPICZαA-NtDAE were further transformed into E.Coli DH5α.

![](https://static.igem.wiki/teams/5528/engineering6.png)

Figure 6. The plasmid map of pPICZαA-NtDAE

###### Build

The results plasmid pPICZαA-NtDAE (Figure 7) are consistent with pPICZαA-TcDAE,
which proves that the
plasmid is also successfully constructed.

![](https://static.igem.wiki/teams/5528/engineering7.png)

Figure 7. The single clone verification and sequencing comparison of
plasmid pPICZαA-NtDAE ( E.coli DH5α)

###### Part 4 [BBa\_K5528008](https://parts.igem.org/Part%3ABBa_K5528008)(pPICαA-DsDAE)

###### Design

Selection of novel DAEs was conducted in NCBI database. The genes encoding DAE fused with 6×His-tag at its
C-terminus were optimized , and chemically synthesized.The plasmid map was constructed by snapgene software
(Figure 8). And DsDAE inserted into the expression vector pPICZαA between
restriction endonucleases EcoRI
and SalI sites, the recombinant plasmids pPICZαA-DsDAE were further transformed into E.Coli DH5α.

![](https://static.igem.wiki/teams/5528/engineering8.png)

Figure 8. The plasmid map of pPICZαA-DsDAE

###### Build

We transferred the plasmid pPICZαA-DsDAE into E.coli-DH5α and did single clone verification. Figure 9-A
shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of
the target gene. Figure 9-B, showed the E. coli plates we cultured and the
location of sample for single
clone verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 9-C
showed the DNA sequence has no mutation. The recombinant construct was analysed by sequencing to conﬁrm its
sequence ﬁdelity, and the positive recombinant plasmids were named as pPICZαA-DsDAE (Figure 9).

![](https://static.igem.wiki/teams/5528/engineering9.png)

Figure 9. The single clone verification and sequencing comparison of
plasmid pPICZαA-DsDAE ( E.coli DH5α )

###### Part 5 Test

###### 5.1 Protein expression

###### 5.1.1 The transformed plate colony diagram(Pichia Pastories GS-115)

The recombinant plasmids were transformed into Pichia Pastoris GS115 for protein expression. Each kind of
plasmid was cultured in three separate petri dishes. All 12 plate successfully developed colonies in Figure
10, which verified the plasmid (pPICZαA-TtDAE, pPICZαA-NtDAE, pPICZαA-DsDAE and pPICZαA-TcDAE) have
successfully transformed into Pichia Pastories GS115.

![](https://static.igem.wiki/teams/5528/engineering10.png)

Figure 10. The transformed plate colony diagram of pPICZαA-TtDAE,
pPICZαA-TcDAE, pPICZαA-NtDAE, pPICZαA-TcDAE(Pichia pastoris GS115)

###### 5.1.2 The colony PCR identification(Pichia Pastories GS115)

A single colony containing the recombinant plasmid was cultured in medium containing antibiotics (100 mg/mL
Zeocin) at 30oC.Next, verify the transformation status using monoclonal antibodies.The gene represents a
clear band falling between 750 and 1500 bp, which corresponds to the length of the target gene in the Figure
11.The result indicates successful transformation of Pichia Pastories GS115.

![](https://static.igem.wiki/teams/5528/imageengineering11.jpeg)

Figure 11. The colony PCR identification of pPICZαA-TtDAE/pPICZαA-TcDAE/pPICZαA-NtDAE/pPICZαA-TcDAE(Pichia
pastoris GS115)

###### 5.1.3 SDA-PAGE detection of the products

When the cultures reached optical density (OD600) about 0.6, methanol was added to the broth for induction
of the recombinant protein. After the induction,we induced sampling at 24 and 98 hours the bacterial cells
were lysed by sonication in phosphate buffer.The recombinant His6-fused TtDAE/TcDAE/NtDAE/TcDAE were
purified by Ni2+ at ﬁnity chromatography.The protein was detected by 15% sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

In both figure 12 A and B, there’s protein falling between 34kDa to 43kDa. The
coherence between target
protein size and the observed bands indicates successful protein expression in the 24h.

![](https://static.igem.wiki/teams/5528/engineering12.png)

Figure 12. The SDA-PAGE detection of the products in the24h. A.SDA-PAGE detection of crude protein extract.
B Purification of the recombinant His6-tagged protein
Note: TcDAE:37.8kDa, TtDAE:37.3kDa, NtDAE:37.9kDa, DsDAE:37.8kDa

Next, the same steps were taken to process the 96 hour protein sample. In both figure
13A , the purified
protein protein falling between 34 kDa to 43 kDa. The coherence between target protein size and the observed
bands indicates successful protein expression in the 96h.

The expression level of proteins can be characterized by intden values. The bands of the target protein was
cutted , and used ImageJ software to calculate the intden values of the protein bands (Figure 13B). The
intden values were established by GraphPad Prism with five gray values in each group. The abscissa was
TcDAE, TtDAE, NtDAE,DsDAE and CK(GS115), and the ordinate was intden value of protein. In Figure 13C, the
intden value of NtDAE was significantly higher than that of CK (GS115) TcDAE, TtCAE, and DsDAE. It indicates
that the protein expression level of NtDAE is higher than that of other groups.The protein expression of
TcDAE and TtCAE was similar. The intden value of DsDAE compared with TcDAE and TtDAE, and the protein
expression level of DsDAE is the lowest. But the intden value of DsDAE,TcDAE,TtDAE and NtDAE significantly
higher than those in control group(GS115). It shows that the protein expression of DsDAE,TcDAE,TtDAE and
NtDAE is successful.

![](https://static.igem.wiki/teams/5528/engineering13.png)

Figure 13. The protein expression of TcDAE, TtSAE, NtDAE, and DsDAE at 96 hours. A represents the SDS-PAGE
gel image of the purified protein after 96 hours of induction. B represents the intden value analysis image
of Image J. C represents the intden value of different proteins, GS115 represents Pichia pastoris GS115.

###### 5.2 Production of D-psicose

###### 5.2.1 Production of D-psicose

Using 10mg/ml D-fructose as the substrate,we added 0.3 μmol of purified recombinase
TcDAE,TtDAE,NtDAE,DsDAEand CK-GS115, 1 mmolL CoCl2.Then each recombinant DAE was reacted at 40,50,60,70
and80 degrees for 10min, and boiled for 10 min. After the reaction, the product was centrifuged and diluted
to a certain concentration, and the content of D-psicose was detected by HPLC.

We used High performance liquid chromatography(HPLC) to detect the content of D-psicose by of weipu
biological company, and the confidence interval was 0.05 %. HPLC detection conditions: fixed phase is 2695
HPLC Waters Sugar-PakI sugar column, and mobile phase is ultrapure water, The flow rate is 0.4 m /min, and
column temperature is 85 °C.

Table 1 shows the detection data of D-psicose, which includes the average of
three replicates for each
sample group. The results indicate that D-psicose production was not detected in the blank control group and
negative control group(Pichia pastoris GS115), while TcDAE, TtAE, NtDAE, and DsDAE proteins can catalyze
D-fructose to generate D-psicos.

###### Table 1. Production of D-psicose at different temperatures

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Temperature/D-psicose production | 40° | 50° | 60° | 70° | 80° |
| Blank control | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| Pichia pastoris GS115 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| TcDAE | 1.988 | 2.038 | 2.144 | 0.829 | 0.000 |
| TtDAE | 1.174 | 2.666 | 2.317 | 0.017 | 1.585 |
| NtDAE | 2.557 | 2.656 | 2.860 | 3.470 | 2.608 |
| DsDAE | 1.108 | 1.365 | 1.930 | 2.017 | 1.165 |

Next, the production of D-psicose was analyzed using Graphpad software. The X axis represents temperature,
and the Y axis represents the yield of D-psicose. The production of D-psicose, increasing with temperature,
shows a trend of first increasing and then decreasing in figure 14A.Moreover,
the TcDAE and TtDAE groups at
60 degrees showed the highest production of D-psicose, which indicating the enzyme activity of TcDAE and
TtDAE are highest at 60 degrees.

In figure 14B, the production of D-alloulose in the DsDAE and NtDAE groups showed a trend of first increasing and then decreasing. The NtDAE and DsDAE groups at 70 degrees had the highest D-psicose production. It indicating that DsDAE and NtDAE proteins are active and have the highest activity at 70 degrees.

![](https://static.igem.wiki/teams/5528/engineering14.png)

Figure 14. Line graph of D-psicose production at different temperatures. A represents the D-psicose
production in the TcDAE and TtSAE groups. B indicate the D-psicose production of Nt DAE and TcDAE groups.

Comparing the D-psicose production of TcDAE, TtAE, NtDAE, and DsDAE groups, it was found that the D-psicose
productivity of the NtDAE group was significantly higher than that of the TcDAE, TtAE, and DsDAE
groups(Figure 15). There was no significant difference in the maximum yield
among the TcDAE, TtAE, and DsDAE
groups. Therefore, the NtDAE group has the highest yield and the highest thermal stability

![](https://static.igem.wiki/teams/5528/engineering15.png)

Figure 15. Production of D-psicose at different temperatures

###### 5.2.2 Conversion rate of D-psicose

###### 5.2.2.1 Conversion rate of D-psicose in the TcDAE, TtAE, NtDAE, and DsDAE

Using D-fructose as a substrate, TcDAE, TtAE, NtDAE, and DsDAE proteins were added to measure the yield of D-psicose, and the conversion rate of DAE could be calculated. Table 2 shows that the conversion rates of TcDAE, TtAE, NtDAE, and DsDAE groups are 21.4%, 23.2%, 34.7%, and 20.2%, respectively.

###### Table 2. Maximum conversion rate of D-alloulose in TcDAE, TtAE, NtDAE,and DsDAE group

|  |  |  |  |
| --- | --- | --- | --- |
| Group | D-Fructose(mg) | D-psicose(mg) | Maximum Conversion rate(100%) |
| Blank control | 10 | 0 | 0% |
| Pichia pastoris GS115 | 10 | 0 | 0% |
| TcDAE | 10 | 2.144 | 21.4% |
| TtDAE | 10 | 2.317 | 23.2% |
| NtDAE | 10 | 3.470 | 34.7% |
| DsDAE | 10 | 2.017 | 20.2% |

###### 5.2.2.2 PTVA method for screening high copy NtDAE and D-psicose production

Inoculate the expression strain of D-alloulose-3-isomerase from Novibacillus thermophilus into 3mL YPD
medium with an initial concentration of Zeocin 500 μg/mL. Next, we centrifuge every 2 days, remove the
supernatant, and inoculate in YPD medium with higher concentrations of antibiotics. The concentration
gradients of Zeocin were 1 mg/ml, 2 mg/ml, 3 mg/ml, and 4 mg/ml, respectively. Finally, they were inoculated
onto a 4mg/ml Zeocin YPD medium, and the yield of D-alloulose was measured using the same method by HPLC.

In figure 16 A, the growth of NtDAE group at various concentrations of Zeocin
showed that there were more colonies at 500 μg/mL, and the number of colonies decreased with increasing
concentration.

NtDAE-op stands for selecting high-yield NtDAE. In figure 16B, the D-psicose
production of NtDAE group is
lower than that of NtDAE-op group. And the table in figure 16B shows that the
conversion rate of NtDAE group
is 34.7%, and the conversion rate of NtDAE op group is 37.0%.

![](https://static.igem.wiki/teams/5528/engineering16.png)

Figure 16. PTVA method for screening high copy NtDAE and D-psicose production

###### Part 6 Learn

At present, there are still some aspects of our subject to be further studied. Due to the particularity of
Pichia pastoris, only a series of vectors such as pPICZαA can be selected. The promoter of the plasmid is
the alcohol oxidase promoter AOX1, so it needs methanol induction. Considering the safety problem, we will
replace the promoter in the future. For example, replaced with PGK1 promoter, GAP promoter, etc. [1-2],
there have been related studies, we can further optimize the vector, the use of fusion promoter [2].

At present, we only selected four sources of DAE, namely Thermoclostridium caenicola ( TcDAE ), Novibacillus
thermophilus ( NtDAE ), Thermogutta terrifontis ( TtDAE ), Dorea sp. CAG317(DsDAE).In the future, we will
choose more sources of DAE, such as D-psicose-3-epimerase ( SfDAE ) from Sinorhizobium fedii [3], and
Thermotogamaritima [4].At the same time, site-directed mutagenesis can be used to optimize DAE from
different sources to increase the yield of D-psicose. Let our products have more advantages.

Reference

[ 1 ] Zhang Huijie, Liao Simin, Ling Xiaocui, etc. P.pastoris truncated PGK1 promoter combined with
different terminators to regulate exogenous gene expression [ J ].Microbiology, 2022,62 ( 07 ) :
2642-2657.DOI : 10.13343 / j.cnki.wsxb.20210653.

[ 2 ] Qian Kai, Zhang Jingjing, Wu Suping, etc. Expression and purification of GLP-1 analogues in Pichia
pastoris using GAP promoter [ J ]. Chinese Journal of Bioengineering, 2015,35 ( 05 ) : 66-73.DOI : 10.13523
/ j.cb.20150510.

[ 3 ] Wu Mian. Study on the efficient secretory expression of D-psicose 3-epimerase by Bacillus subtilis to
produce D-psicose [ D ].Tianjin University of Science and Technology, 2022.DOI : 10.27359 /
d.cnki.gtqgu.2022.000764.

[4] Qi Z, Zhu Z,Wang J-W , et al. Biochemical analysis and the preliminarycrystallographic characterization
of D-tagatose 3-epimerase from Rhodobactersphaeroides[J]. Microb Cell Fact．2017, 16．

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![](https://static.igem.wiki/teams/5528/papercrane.png)
Experiments

[1.Disposition of medium](https://static.igem.wiki/teams/5528/1.pdf)
[2.The construction of plasmid](https://static.igem.wiki/teams/5528/experiments2.pdf)

###### 2.1 TcDAE, TtDAE, NtDAE, DsDAE

###### 2.2 pPIcZαA

###### 2.3 pPIcZαA-TcDAE

###### 2.4 Transformation E.Coli

###### 2.5 Monoclonal colony verification

###### 2.6 plasmid extraction

###### 2.7 Transformation Pichia pastoris

###### 2.8 Monoclonal colony verification

![](https://static.igem.wiki/teams/5528/experiments1.jpeg)
[3.Protein expression](https://static.igem.wiki/teams/5528/experiments3.pdf)

###### 3.1 Amplification culture

###### 3.2 Protein extraction

###### 3.3 Protein purification

###### 3.4 Protein identification-SDS-PAGE

![](https://static.igem.wiki/teams/5528/experiments2.jpeg)
[4. Functional Test](https://static.igem.wiki/teams/5528/experiments4.pdf)

###### 4.1Production of D-psicose

![](https://static.igem.wiki/teams/5528/experiments3.jpeg)

###### 4.2 Screening high-yield strains by PTVA

![](https://static.igem.wiki/teams/5528/experiments4.jpeg)

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![](https://static.igem.wiki/teams/5528/candys.png)
![](https://static.igem.wiki/teams/5528/papercrane.png)
Results

![](https://static.igem.wiki/teams/5528/logo.png)

* [OVERVIEW](#content-1)
* [Result of our work](#content-2)
* [Reference](#content-3)

OVERVIEW

D-psicose-3-epimerase (DAE) plays an important role in the biotransformation of D-psicose, but the thermal
stability of D-psicose-3-epimerase is poor and the expression level is low, which cannot meet the
requirements of industrial production. In this study, we choosed four origin of DAE, is respectively
Thermoclostridium caenicola(TcDAE)，Novibacillus thermophilus(NtDAE), Thermogutta terrifontis(TtDAE), and
Dorea sp. CAG 317(DsDAE). Then we ligated the coding genes into the expression vectors, and transformed them
into Pichia pastoris GS115 strain. The target protein was induced and purified, and tested the yield of
D-psicose (Figure 1). Based on the yield of D-alloulose, it can be screened which source of DAE has a higher
yield and higher thermal stability.

On this basis, the liquid PTVA method was used to obtain high-copy strains by Zeocin antibiotic screening to
further improve the expression level of D-psicose-3-epimerase. This study will provide a new strategy for
the industrial production of D-psicose.

![](https://static.igem.wiki/teams/5528/result1.png)

Figure 1.The engineering schematic diagram of the project
design

Result of our work

###### 1.Plasmid Construction

###### 1.1 PCR amplification(target gene)

At present, we choose four origin of DAEs, is respectively Thermoclostridium caenicola(TcDAE)，Novibacillus
thermophilus(NtDAE), Thermogutta terrifontis(TtDAE), and Dorea sp.CAG 317(DsDAE). The DNA encoding
TcDAE,TtDAE,NtDAE and DsDAE were optimised according to the codon preference of E.coli and synthesised. To
construct the expression plasmid for TcDAE,TtDAE,NtDAE and DsDAE, ﬁrstly, the gene encoding
TcDAE,TtDAE,NtDAE and DsDAE was ampliﬁed using polymerase chain reaction (PCR) with primer. The obtained PCR
product was digested with EcoRI and SalI and inserted into pPICZαA plasmids.

The results are showed in [figure 2](javascript%280%29), all 4 target
genes(TcDAE,TtDAE,NtDAE and DsDAE) are highlighted between
750 and 1000bp markers, which accurately matched with theoretical length and indicate the succeed of
amplification.

![](https://static.igem.wiki/teams/5528/result2.png)

Figure 2. The electrophoresis verification of the target fragment TcDAE,TtDAE,NtDAE and DsDAE.
A.TtDAE: 873 bp B.NtDAE: 855bp C.TcDAE:870 bp D.DsDAE: 870 bp

###### 1.2:Double Digestion(plasmid)

The plasmid pPICZαA was digested with EcoRI and SalI, and used the unoperated pPICZaA as control group.The
electrophoresis verify the results of enzyme digestion, the results are shown in [Figure 3](javascript%280%29). The double
digestion pPICZαA are highlighted between 3000 and 5000bp, and the length is greater than the control group
to verify the digestion was very successful.But the control group are already became a super-helical
structure, so the electrophoretic migration speed is fast, so it is less than the actual length. turn to
liner from curcle.

![](https://static.igem.wiki/teams/5528/result3.png)

Figure 3 . The electrophoresis verification of digestion vector pPICZαA
Note: 1)pPICZαA: 3593 bp 2)Double Digestion pPICZαA:3487bp

###### 1.3:The Single Clone Verification and Sequencing Comparison(E.coli DH5α )

We transferred the plasmid pPICZαA-TcDAE into E.coli-DH5α and did single clone verification. Figure 4-A
shows the PCR products were all highlighted between 750 and 1000bp, which was consistent with the length of
the target gene. Figure 4-B, showed the E. coli plates and the location of
sample for single clone
verification. We compared the plasmid sequencing results with the target DNA sequence. Figure 4-C showed the
DNA sequence has no mutation. The recombinant construct was analysed by sequencing to conﬁrm its sequence
ﬁdelity, and the positive recombinant plasmids were named as pPICZαA-TcDAE.

![](https://static.igem.wiki/teams/5528/results44.png)

Figure 4. The single clone verification and sequencing comparison of plasmid pPICZαA-TcDAE ( E.coli DH5 )

![](https://static.igem.wiki/teams/5528/result5.png)

Figure 5. The single clone verification and sequencing comparison of plasmid pPICZαA-TtDAE ( E.coli DH5 )

The results plasmid pPICZαA-TtDAE(Figure 5), pPICZαA-NtDAE (Figure 6), and pPICZαA-DsDAE (Figure 7) are
consistent with pPICZαA-TcDAE, which proves that the plasmid is also successfully constructed.

![](https://static.igem.wiki/teams/5528/result6.png)

Figure 6. The single clone verification and sequencing comparison of plasmid pPICZαA-NtDAE ( E.coli DH5 )

![](https://static.igem.wiki/teams/5528/result7.png)

Figure 7. The single clone verification and sequencing comparison of plasmid pPICZαA-DsDAE ( E.coli DH5 )

###### 2.Protein expression

###### 2.1:The transformed plate colony diagram(Pichia Pastories GS-115)

The recombinant plasmids were transformed into Pichia Pastoris GS115 for expression. Each kind of plasmid
was cultured in three separate petri dishes. All 12 plate successfully developed colonies in Figure 8, which
verified the plasmid (pPICZαA-TtDAE, pPICZαA-NtDAE, pPICZαA-DsDAE and pPICZαA-TcDAE) have successfully
transformed into Pichia Pastories GS115.

![](https://static.igem.wiki/teams/5528/result8.png)

Figure 8. The transformed plate colony diagram of pPICZαA-TtDAE,pPICZαA-TcDAE,pPICZαA-NtDAE and
pPICZαA-TcDAE(Pichia pastoris GS115)

###### 2.2:The colony PCR identification(Pichia Pastories GS115)

A single colony containing the recombinant plasmid was cultured in medium containing antibiotics (100 mg/mL
Zeocin) at 30oC.Next, verify the transformation status using monoclonal antibodies.The gene represents a
clear band falling between 750 and 1500 bp, which corresponds to the length of the target gene in theFigure
9.The result indicates successful transformation of Pichia Pastories GS115.

![](https://static.igem.wiki/teams/5528/result9.jpg)

Figure 9. The colony PCR identification of pPICZαA-TtDAE/pPICZαA-TcDAE/pPICZαA-NtDAE/pPICZαA-TcDAE(Pichia
pastoris GS115)

###### 2.3: SDA-PAGE detection

When the cultures reached optical density (OD600) about 0.6-0.8, methanol was added to the BMMY for
induction of the recombinant protein. After the induction,we induced sampling at 24 and 98 hours the
bacterial cells were lysed by sonication in phosphate buffer.The recombinant His6-fused TtDAE,TcDAE,NtDAE
and TcDAE were purified by Ni2+ at ﬁnity chromatography.The protein was detected by 15% sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE).

In both figure 10 A and B, there’s protein falling between 34kDa to 43kDa.
The coherence between target
protein size and the observed bands indicates successful protein expression in the 24h.

![](https://static.igem.wiki/teams/5528/result10.png)

Figure10. The SDA-PAGE detection of the productsin the24h. A.SDA-PAGE detection of crude protein extract. B
Purification of the recombinant His6-tagged protein
Note: TcDAE:37.8kDa, TtDAE:37.3kDa, NtDAE:37.9kDa, DsDAE:37.8kDa

Next, the same steps were taken to process the 96 hour protein sample. In both figure 11A , the purified
protein protein falling between 34 kDa to 43 kDa. The coherence between target protein size and the observed
bands indicates successful protein expression in the 96h.

The expression level of proteins can be characterized by intden values. The bands of the target protein was
cutted , and used ImageJ software to calculate the intden values of the protein bands (Figure 11B). The
intden values were established by GraphPad Prism with five gray values in each group. The abscissa was
TcDAE, TtDAE, NtDAE,DsDAE and CK(GS115), and the ordinate was intden value of protein. In Figure 11C, the
intden value of NtDAE was significantly higher than that of CK (GS115) TcDAE, TtCAE, and DsDAE. It indicates
that the protein expression level of NtDAE is higher than that of other groups.The protein expression of
TcDAE and TtCAE was similar. The intden value of DsDAE compared with TcDAE and TtDAE, and the protein
expression level of DsDAE is the lowest. But the intden value of DsDAE,TcDAE,TtDAE and NtDAE significantly
higher than those in control group(GS115). It shows that the protein expression of DsDAE,TcDAE,TtDAE and
NtDAE is successful.

![](https://static.igem.wiki/teams/5528/result11.png)

Figure 11. The protein expression of TcDAE, TtSAE, NtDAE, and DsDAE at 96 hours. A represents the SDS-PAGE
gel image of the purified protein after 96 hours of induction. B represents the intden value analysis image
of Image J. C represents the intden value of different proteins, GS115 represents Pichia pastoris GS115.

###### 3.Test

###### 3.1 Production of D-psicose

Using 10mg/ml D-fructose as the substrate,we added 0.3 μmol of purified recombinase
TcDAE,TtDAE,NtDAE,DsDAEand CK-GS115, 1 mmolL CoCl2.Then each recombinant DAE was reacted at 40,50,60,70
and80 degrees for 10min, and boiled for 10 min. After the reaction, the product was centrifuged and diluted
to a certain concentration, and the content of D-psicose was detected by HPLC.

We used High performance liquid chromatography(HPLC) to detect the content of D-psicose by of weipu
biological company, and the confidence interval was 0.05 %. HPLC detection conditions: fixed phase is 2695
HPLC Waters Sugar-PakI sugar column, and mobile phase is ultrapure water, The flow rate is 0.4 m /min, and
column temperature is 85 °C.

Table 1 shows the detection data of D-psicose, which includes the average of
three replicates for each
sample group. The results indicate that D-psicose production was not detected in the blank control group and
negative control group(Pichia pastoris GS115), while TcDAE, TtAE, NtDAE, and DsDAE proteins can catalyze
D-fructose to generate D-psicos.

Table 1. Production of D-psicose at different temperatures

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| Temperature/D-psicose production | 40° | 50° | 60° | 70° | 80° |
| Blank control | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| Pichia pastoris GS115 | 0.000 | 0.000 | 0.000 | 0.000 | 0.000 |
| TcDAE | 1.988 | 2.038 | 2.144 | 0.829 | 0.000 |
| TtDAE | 1.174 | 2.666 | 2.317 | 0.017 | 1.585 |
| NtDAE | 2.557 | 2.656 | 2.860 | 3.470 | 2.608 |
| DsDAE | 1.108 | 1.365 | 1.930 | 2.017 | 1.165 |

Next, the production of D-psicose was analyzed using Graphpad software. The X axis represents temperature,
and the Y axis represents the yield of D-psicose. The production of D-psicose, increasing with temperature,
shows a trend of first increasing and then decreasing in figure
12A.Moreover, the TcDAE and TtDAE groups at
60 degrees showed the highest production of D-psicose, which indicating the enzyme activity of TcDAE and
TtDAE are highest at 60 degrees.

In figure 12B, the production of D-alloulose in the NtDAE and DsDAE groups showed a trend of first increasing and then decreasing. The NtDAE and DsDAE groups at 70 degrees had the highest D-psicose production. It indicating that NtDAE and DsDAE proteins are active and have the highest activity at 70 degrees.

![](https://static.igem.wiki/teams/5528/result12.png)

Figure 12. Line graph of D-psicose production at different temperatures. A represents the D-psicose
production in the TcDAE and TtSAE groups. B indicate the D-psicose production of Nt DAE and TcDAE groups.

Comparing the D-psicose production of TcDAE, TtAE, NtDAE, and DsDAE groups, it was found that the D-psicose
productivity of the NtDAE group was significantly higher than that of the TcDAE, TtAE, and DsDAE
groups(Figure 13). There was no significant difference in the maximum yield
among the TcDAE, TtAE, and DsDAE
groups. Therefore, the NtDAE group has the highest yield and the highest thermal stability

![](https://static.igem.wiki/teams/5528/result13.png)

Figure 13. Production of D-psicose at different temperatures

###### 3.2 Conversion rate of D-psicose

###### 3.2.1 Conversion rate of D-psicose in the TcDAE, TtAE, NtDAE, and DsDAE

Using D-fructose as a substrate, TcDAE, TtDAE, NtDAE, and DsDAE proteins were added to measure the yield of D-psicose, and the conversion rate of DAE could be calculated. Table 2 shows that the conversion rates of TcDAE, TtDAE, NtDAE, and DsDAE groups are 21.4%, 23.2%, 34.7%, and 20.2%, respectively.

Table 2. Maximum conversion rate of D-alloulose in TcDAE, TtAE, NtDAE,and DsDAE group

|  |  |  |  |
| --- | --- | --- | --- |
| Group | D-Fructose(mg) | D-psicose(mg) | Maximum Conversion rate(100%) |
| Blank control | 10 | 0 | 0% |
| Pichia pastoris GS115 | 10 | 0 | 0% |
| TcDAE | 10 | 2.144 | 21.4% |
| TtDAE | 10 | 2.317 | 23.2% |
| NtDAE | 10 | 3.470 | 34.7% |
| DsDAE | 10 | 2.017 | 20.2% |

###### 3.2.2. PTVA method for screening high copy NtDAE and D-psicose production

Inoculate the expression strain of D-alloulose-3-isomerase from Novibacillus thermophilus into 3mL YPD
medium with an initial concentration of Zeocin 500 μg/mL. Next, we centrifuge every 2 days, remove the
supernatant, and inoculate in YPD medium with higher concentrations of antibiotics. The concentration
gradients of Zeocin were 1 mg/ml, 2 mg/ml, 3 mg/ml, and 4 mg/ml, respectively. Finally, they were inoculated
onto a 4mg/ml Zeocin YPD medium, and the yield of D-alloulose was measured using the same method by HPLC.

In figure 14 A, the growth of NtDAE group at various concentrations of Zeocin showed that there were more
colonies at 500 μg/mL, and the number of colonies decreased with increasing concentration.

NtDAE-op stands for selecting high-yield NtDAE. In figure 14B, the D-psicose production of NtDAE group is
lower than that of NtDAE-op group. And the table in figure 14B shows that the conversion rate of NtDAE group
is 34.7%, and the conversion rate of NtDAE op group is 37.0%.

![](https://static.igem.wiki/teams/5528/result14.png)

Figure 14. PTVA method for screening high copy NtDAE and D-psicose production

Reference

[ 1 ] Zhang Huijie, Liao Simin, Ling Xiaocui, etc. P.pastoris truncated PGK1 promoter combined with
different terminators to regulate exogenous gene expression [ J ].Microbiology, 2022,62 ( 07 ) :
2642-2657.DOI : 10.13343 / j.cnki.wsxb.20210653.

[2] Qian Kai, Zhang Jingjing, Wu Suping, etc. Expression and purification of GLP-1 analogues in Pichia
pastoris using GAP promoter [ J ]. Chinese Journal of Bioengineering, 2015,35 ( 05 ) : 66-73.DOI : 10.13523
/ j.cb.20150510.

[ 3 ] Wu Mian. Study on the efficient secretory expression of D-psicose 3-epimerase by Bacillus subtilis to
produce D-psicose [ D ].Tianjin University of Science and Technology, 2022.DOI : 10.27359 /
d.cnki.gtqgu.2022.000764.

[4] Qi Z, Zhu Z,Wang J-W , et al. Biochemical analysis and the preliminarycrystallographic characterization
of D-tagatose 3-epimerase from Rhodobactersphaeroides[J]. Microb Cell Fact．2017, 16．

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