iGEM 2024 — Team szsais-shenzhen

Notebook

Notebook

Since March, after conducting our Human Practices, we identified the biosynthesis of tagatose as our
research focus. Following this, we embarked on an in-depth review of the literature and studied
relevant biological theories. From July 15th to August 25th, we engaged in continuous experimental
research and held discussions with experts. Ultimately, we successfully synthesized tagatose in the
lab, a milestone achievement that has filled us with confidence about the future prospects of our
team's efforts in synthetic biology. “Smart Sweets, Healthy treats”, we will make it!

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Engineering

![](https://static.igem.wiki/teams/5453/szsais-shenzhen.png)

# Menu

* [##### ⚪ Overview](#one)
* [##### ⚪ Cycle 1: Construction of the D-Tagatose synthesis pathway in Escherichia coli](#two)
* [##### ⚪ Cycle 2: Knocking down competitive pathways](#three)
* [##### ⚪ Cycle 3: Designing a Hardware for E.coli Fermentation](#four)

- Overview -

We consistently adhere to the Design-Build-Test-Learn cycles to advance the project. This year's
project can be broadly categorized into three cycles. In the first cycle, we completed the
construction and testing of the tagatose synthesis pathway within an *E. coli* chassis. In
the
second cycle, we successfully knocked down the competing pathway involved in tagatose synthesis.
Finally, in the third cycle, we designed various bioreactors to optimize the performance of the
engineered strain for tagatose production.

![engineering1](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-17.png)

 *Figure 1: Engineering cycle diagram of our project*

Cycle 1:

- Construction of the D-Tagatose synthesis pathway in Escherichia coli -

· Design

Since *Escherichia coli* lacks the pathway for synthesizing D-tagatose, we synthesized the
*gatz*
gene from *Caldilinea aerophila* and the *pgp* gene from  *Archaeoglobus
profundus*, and constructed
the *gatz* and *pgp* genes into the pYB1c vector to obtain the pYB1c-Gatz-PGP plasmid.
Considering
that enhancing the conversion from G6P to F6P might be beneficial for D-tagatose production, we
also overexpressed the *pgi* gene from *Thermus thermophilus* and constructed the
pYB1c-Gatz-PGP-PGI
plasmid.

![engineering2](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-2.png)

*Figure 2: Schematic diagrams of plasmids. A: Schematic diagram of the pYB1c-Gatz-PGP
plasmid. B: Schematic diagram of the pYB1c-Gatz-PGP-PGI plasmid.*

· Build

We used in step in fusion clone technology. In this way, two or three genes could be assembled
into plasmed efficiently.

### pYB1c-Gatz-PGP:

A、PCR amplification of target genes

B、Colony PCR verification

C、Plasmid digestion verification

D、DNA sequencing verification

![engineering3](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-3.png)

*Figure 3:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for pYB1c,
Gatz, and PGP. B: Gel electrophoresis image of colony PCR performed on 10 randomly picked
colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI and
EcoRI enzymes. D: Sequencing of the correct plasmids verified by colony PCR and enzyme
digestion.*

### pYB1c-Gatz-PGP-PGI：

A、PCR amplification of target genes

B、Colony PCR verification

C、Plasmid digestion verification

D、DNA sequencing verification

![engineering4](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-5.png)

*Figure 4:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for pYB1c,
Gatz, PGI,and PGP. B: Gel electrophoresis image of colony PCR performed on 10 randomly
picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion with KpnI
and EcoRI enzymes.D: Sequencing of the correct plasmids verified by colony PCR and enzyme
digestion.*

· Test

1、Mapping the D-tagatose standard curve

First, we developed a tagatose assay method. By preparing different concentrations of tagatose
standards and mixing them with resorcinol solution, the mixture was incubated at 100°C for 20
minutes. The absorbance was then measured using a microplate reader. A standard curve was
constructed by plotting the absorbance on the Y-axis against the concentration of tagatose
standards on the X-axis.The resulting standard curve equation is y = 0.0023x + 0.058. The
regression constant R²=0.9961. This indicates that our detection method is very effective and
highly sensitive for the detection of tagatose.

![engineering5](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-5.png)

*Figure 5:Determine the color change and absorbance values after the reaction of different
concentrations of tagatose with resorcinol.*

2、D-Tagatose concentration assay

The plasmids pYB1c-Gatz-PGP and pYB1c-Gatz-PGP-PGI were transformed into competent BW25113 cells
to obtain the strains pYB1c-Gatz-PGP/BW25113 and pYB1c-Gatz-PGP-PGI/BW25113. Then, after the
addition of 0.2% arabinose, protein expression was induced at 30°C, and 12 hours later, the
induced bacterial culture was transferred into M9 fermentation medium containing 20 g/L glucose
for the biosynthesis of tagatose. The D-tagatose yield in the fermentation broth was measured.
The yield detection results show that the BW strain did not produce D-tagatose. The strain
overexpressing PGP and GatZ reached a tagatose concentration of 191.01 mg/L after 12 hours of
fermentation. Subsequently, on this basis, we further overexpressed the *pgi* gene, and its
tagatose production reached 197.33 mg/L, which is a 197.3-fold increase in yield compared to the
BW25113 strain, indicating that our construction and strengthening of the pathway play an
important role in the synthesis of tagatose. It is worth noting that further enhancement of PGI
on the basis of GatZ and PGP does not significantly improve the yield of tagatose.

![engineering6](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-6.png)

*Figure 6:Changes in D-tagatose production in strains overexpressing gatz, pgp and PGI genes
compared to strain BW25113*

· Learn

A D-tagatose biosynthesis pathway was successfully established in *E. coli*. Although the
overexpression of the pgi gene did not significantly improve the yield compared to the
non-overexpressing strain, the pYB1c-Gatz-PGP-PGI/BW25113 strain was selected for subsequent
experiments. The D-tagatose concentration in this strain increased by 197.3 -fold compared to
the wild-type BW25113.

Cycle 2:

- Knocking down competitive pathways -

· Design

G6P is generated in the second catalytic reaction of the D-tagatose biosynthesis pathway. In
recombinant *E. coli*, G6P can be converted by glucose-6-phosphate dehydrogenase (G6PDH),
encoded
by the zwf gene, into 6-phosphoglucono-δ-lactone, diverting part of the G6P flow into the
pentose phosphate pathway. F6P and T6P are produced in the third and fourth catalytic steps of
the D-tagatose biosynthesis pathway, respectively. In recombinant *E. coli*, F6P may be
converted
by 6-phosphofructokinase I, encoded by the pfkA gene, and the resulting product flows into the
tricarboxylic acid (TCA) cycle. To reduce the loss of intermediate products in the D-tagatose
biosynthesis pathway during the metabolic processes in recombinant *E. coli*, without
causing
growth defects, we utilized CRISPRi technology to modify the competitive pathways in recombinant
*E. coli*, thereby reducing the consumption of intermediate products by competitive
pathways and
improving the D-tagatose production yield.

First, we verified the D-tagatose yield of strains with individual inhibition of *pfkA* or
*zwf*:

![engineering7](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-7.png)

*Figure 7:To assess the effect of CRISPRi-mediated silencing of the zwf and pfkA genes on
D-tagatose production.*

It was found that individually inhibiting zwf or pfkA could increase D-tagatose production;
however, it is worth noting that bacterial growth was severely affected. Therefore, in the next
step, we simultaneously inhibited pfkA and zwf, and introduced mutations at the 7th and 8th bp
of their sgRNAs to generate a mutant library. From this mutant library, we screened for a mutant
that did not affect bacterial growth but could increase the yield of D-tagatose. The
R6k-dcas9-pfkA-zwf-M plasmid was constructed.

![engineering8](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-8.png)

*Figure 8:Schematic diagram of the R6k-dcas9-pfkA-zwf-M plasmid.*

· Build

We used Golden Gate to assemble sgRNA-pfkA and sgRNA-zwf with a R6K-dCas9 backboen.

1、PCR amplification of target genes

2、Colony PCR verification

3、Validation of plasmid digestion

4、Send Sequencing Verification

![engineering9](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-9.png)

*Figure 9:PCR Results Figure. A: Gel electrophoresis image of PCR amplification for
sgRNA-pfkA-M, sgRNA-zwf-M. B: Gel electrophoresis image of colony PCR performed on 10
randomly picked colonies from the plate. C: Gel electrophoresis image of plasmid digestion
with KpnI and XhoI enzymes.D: Sequencing of the correct plasmids verified by colony PCR and
enzyme digestion.*

· Test

Subsequently, the plasmids R6k-dcas9-pfkA-zwf-M and pYB1c-Gatz-PGP-PGI were co-transformed into
BW25113. Ninety-five colonies were randomly selected for fermentation experiments using glucose
as the substrate, and their growth and production yields were screened.

![engineering10](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-10.png)

*Figure 10:The fluorescence value detected by the Microplate reader.*

![engineering11](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-11.png)

*Figure 11:The blue bar indicates the yield of Tagatose; the yellow circle indicates OD600*

![engineering12](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-12.png)

*Figure 12: Comparative yield graph after rescreening*

A good mutant was identified and named DT-D9. Compared to the non-inhibited strain DT1, the
tagatose yield of DT-D9 increased by 3.34-fold, reaching a D-tagatose yield of 634.66 mg/L.

· Learn

Through sequencing the mutant, we found that the 7th and 8th base pairs of sgRNA-pfkA mutated
from GG to TG, and the 7th and 8th base pairs of sgRNA-zwf mutated from AT to CT. By altering
the 7th and 8th base pairs of the sgRNA, the binding efficiency of dcas9 to the target gene can
be modified, thereby achieving varying degrees of inhibition. The best mutant, DT-D9, showed
good growth and higher yield, proving that our strategy was effective.

![engineering13](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-13.png)

*Figure 13: The sequencing results of the mutations at the 7、8bp position in sgRNA7 of DT-D9*

Cycle 3:

- Designing a Hardware for E.coli Fermentation -

· Design

We aim to create a small, simple bioreactor using inexpensive and readily available materials
which high school students could be easily used. This bioreactor should have features such as
stirring, temperature control, feeding, and pH adjustment, with a capacity for 1.8 L of culture.

· Build

For temperature control, we placed the bioreactor on a heating plate combined with a magnetic
stirrer, which could be used for temperature control and blending the culture mixature. At the
same time, we used an electrode controller that can automatically control the timing of glucose,
acid, and base additions. It is also equipped with an air pump to regulate the airflow into the
reactor. Additionally, we purchased a pH electrode to measure the pH value inside the reactor
and display accurate readings on the controller's screen.

![engineering14](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-14.png)

*Figure 14: Exhibition of Various Components of a Small-Scale Fermentation bioreactors.*

· Test

We cultured the optimal strain DT-D9 through primary and secondary seed cultures, eventually
inoculating it into the 1.8L bioreactor. After 8 hours of cultivation, the culture became
noticeably turbid, proving that the device is functional. We adjusted the bioreactor pH to
around 7, temperature to 37°C (However, it had a very bad temperature control), and stirring
speed to 450 rpm.

![engineering15](https://static.igem.wiki/teams/5453/engineering-success/engineering-success-15.png)

*Figure 15: The growth condition of the strain at two hours and eight hours during
fermentation.*

After adding the final concentration of 0.1mMol/L IPTG and 0.2% arabinose, take a sample every
two hours. Centrifuge samples and place them in an microplate reader to detect tagatose
concentration. The final tagatose concentration reached 1083.01 mg/L after 26h fermentation.

![engineering16](https://static.igem.wiki/teams/5453/engineering-success/data-1.jpg)

*Figure 16: Time carve of tagetose concentration in fermentation.*

· Learn

By designing a small, simple bioreactor, we successfully scaled up the cultivation of the
optimal strain and achieved D-tagatose production in a simple fermentation device. In future
experiments, we can further optimize this small, simple device, such as increasing the stirring
speed, enhancing airflow, and using 3D printing technology to further improve the controller.

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