iGEM 2022 — Team tongji-china

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Notebook

[Forewords](#forewords)
[Week 1: 9.3-9.9](#week1)
[Week 2: 9.10-9.16](#week2)
[Week 3: 9.17-9.23](#week3)
[Week 4: 9.24-9.30](#week4)
[Week 5: 10.1-10.7](#week5)

![](https://static.igem.wiki/teams/4228/wiki/page-pet.png)

Forewords

For the severe epidemic in Shanghai in the first half of the year and our school policy, our wet experiments
stared very late. Therefore, the content of our experiment was greatly reduced and the experiment was carried
out in a hurry. Our experiment was divided into three groups: wild-type (group 1), single mutant (group 2),
double mutant (group 3). You can see what we have done below.

Week 1: 9.3-9.9

The plasmid containing the target gene of double mutant was transformed into *E.coli BL21*.

Week 2: 9.10-9.16

Rejuvenation, amplification and preservation of *B.subtilis WB800*.
Liquid and solid LB medium were prepared.
The plasmid containing the target gene of single mutant and the wild-type were transformed into *E.coli
BL21*.

Week 3: 9.17-9.23

The plasmids were transformed into *E.coli BL21* again.
The double mutant monoclonal strain was selected and cultured.
The double mutant strain was induced to express bromelain by IPTG.

Week 4: 9.24-9.30

The monoclonal strains of single mutant and wild-type were selected and cultured.
Both single mutant and wild-type strains were induced to express bromelain by IPTG.
The bacterial solution induced by IPTG was collected, and the bacteria were cleaved by ultrasound.
Bromelain was isolated and purified.
The expression of bromelain was confirmed by SDS-PAGE.

Week 5: 10.1-10.7

Characterized the part of the 2021 team of Tongji\_China: BBa\_K3823001 SQR (Sulfide:QuinoneOxidoreductase) from
Acidithiobacillus spp.
Detected the activity and stability of double mutant bromelain.
Single factor induction experiment of IPTG.
Characterization experiment of soybean meal.
Preparation of competent cells of *B.subtilis WB800* and plasmid transformation.

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Engineering

[Selection of target segments](#segments)
[Selection of Chassis microbes](#microbes)

[Early Stage: Common Chassis microorganism](#microbes-microorganism)
[Later stage: *Bacillus subtilis*
comes into
view](#microbes-view)
[Selection of specific strains](#strains)

[Selection of *Bacillus subtilis*
strains
commonly
used in the laboratory](#strains-selection)
[Strains for feed vs. Strains
commonly used in
the laboratory](#laboratory-strains)
[Optimisation of strains](#laboratory)
[Construction and selection of
plasmid](#plasmid)

[Plasmid](#plasmid-plasmid)
[Promoter](#plasmid-promoter)
[Terminator](#plasmid-terminator)
[Signal peptide](#plasmid-peptide)
[References](#reference)

![](https://static.igem.wiki/teams/4228/wiki/page-pet.png)

Selection of target segments

The AlphaFold2 platform was used to search for bromelain. Two different bromelain sequences were found, one with
212 amino acids and the other with 291 amino acids.

So there is a question to solve. Which one to choose?

The global protein sequence alignments of these two sequences were performed by Clustal W, and the results were
as follows.

![](https://static.igem.wiki/teams/4228/wiki/engineering-segments-1.png)

![](https://static.igem.wiki/teams/4228/wiki/engineering-segments-2.png)

The 291-amino acid stem bromelain sequence had extra 122 amino acids at the beginning and the 212-amino acid
stem bromelain sequence had extra amino acids at the end. However, there was a length of constant amino acids in
the middle of both sequences that are highly similar. Clustal W showed that the amino acids in this region are
extremely conserved. At the same time, 291 and 212 structure comparison analysis was conducted. The RMSD value
was 0.42. It could be seen that they were highly similar in structure, which also indicated that both of them
were stem bromelain. Their structure was shown below.（the left is 291 and right is 212）( Part:
[BBa\_K4228000](http://parts.igem.org/Part%3ABBa_K4228000) )

![](https://static.igem.wiki/teams/4228/wiki/engineering-segments-3.png)
![](https://static.igem.wiki/teams/4228/wiki/engineering-segments-4.png)

Selection of Chassis microbes

- Early Stage: Common Chassis microorganism

In the early stages of selection, we considered two sump microorganisms commonly used for experiments: *E.
coli* and *B. bicolor*, and compared them.

Advantages and disadvantages of *Bilobacteria*

*Bilobacterial* yeast endogenous secreted protein production is limited and easy to purify. And as a
eukaryote, it is capable of post-translational modification and glycosylation of bromelain, which is superior
to Saccharomyces cerevisiae and *E. coli*. Despite these advantages, the disadvantages of *B.
bicolor* are more obvious and difficult to address. Firstly bromelain is toxic/inhibitory to the growth
of B. aeruginosa. Secondly the studies on localisation signals in *B. birchii* are not clear. Although it
has been shown that STB3 has 88% homology to proteins with lysosomal localisation and that the lysosomal
sequence of this protein is conserved, there are no clear studies to demonstrate or find a mechanism for
co-localisation, to the detriment of design production. Finally, methanol induction is generally indicated for
the expression of recombinant proteins in *B. birchii*. A minimum concentration of 0.5% methanol is
required for the production of recombinant proteins and a maximum concentration of 2-2.5% (wt/vol) is
necessary for full protein expression. In addition, high concentrations of methanol (above 5%) are toxic to
cells, leading to the accumulation of formaldehyde and hydrogen peroxide, which can lead to cell death. On the
other hand, low levels of methanol trigger the proteolytic degradation of heterologous proteins, leading to
reduced productivity. On the one hand methanol is difficult to remove completely when extracting recombinant
proteins, and on the other hand the adverse effects of products containing methanol added to feed are
difficult to assess in feed animals.

![](https://static.igem.wiki/teams/4228/wiki/engineering-microbes-1.png)

Advantages and disadvantages of *E. coli*

*E. coli* has been studied in rich detail and the modification and utilisation of *E. coli* is
relatively common. Heterologous expression of pineapple protease has been performed in *E. coli* in the
literature. In the literature codon optimisation of pineapple protease has been carried out and the protein
has been modified. The yield and activity of the recombinantly expressed pineapple protease were very
promising. However, *E. coli* has a periplasmic space and heterologous expression of recombinant proteins
in *E. coli* tends to form inclusion bodies, which makes subsequent isolation and purification more
difficult. Secondly, our ultimate goal is the characterisation of pineapple protease, which cannot be
glycosylated by *E. coli*, which is detrimental to our subsequent work. Moreover, *E. coli* is a
conditional pathogen and recombinant *E. coli* for industrial production has potential adverse effects on
animal and human health when complete removal of the chassis microorganism cannot be guaranteed.

![](https://static.igem.wiki/teams/4228/wiki/engineering-microbes-2.png)

Comparison

Table 1: Comparison

| Chassis microbes | Advantages | Disadvantages |
| --- | --- | --- |
| Pichia pastoris | Highly active;  structurally intact; | High cost;  autolytic expression;  low total yield |
| BL21-CodonPlus (DE3) | Many studies available;  easy to operate | Low total yield;  not safe compared to yeast |

Initially we planned to use *E. coli* as the sump microorganism, taking into account ease of handling,
yield and safety.

- Later stage: *Bacillus subtilis* comes into view

After the initial selection of *E. coli* as the chassis microorganism, we communicated and exchanged
ideas with Associate Professor Mengjie Zhang from Tongji University to ask her opinion.

Listening carefully to the pros and cons of both *E. coli* and B. ricinus, Prof. Zhang recommended that
we choose *E. coli*. On learning that we wanted to apply pineapple protease in feed production, she
mentioned a probiotic, *Bacillus subtilis*, and suggested that we learn a little about *Bacillus
subtilis* before choosing between *E. coli* and *Bacillus subtilis*.

After gaining some understanding of *Bacillus subtilis*, we made a comparison.

Advantages and disadvantages of *Bacillus subtilis*

*Bacillus subtilis* is often used as a model strain for bacterial genetics and cellular metabolism
studies due to the clear sequencing of its genome and the resolution of essential genes. In addition,
*Bacillus subtilis* has a high safety profile and is recognised as generally recognized as safe (GRAS) by
the US Food and Drug Administration (FDA). Because of its clear physiological and biochemical characteristics,
simple genetic manipulation, high secretion and expression capacity and ease of culture and fermentation,
*Bacillus subtilis* has also been transformed into a microbial cell factory for the production of
industrial enzymes, vitamins, functional sugars, nutraceuticals and drug precursors and other target products,
showing strong industrial applications. Compared to Gram-negative bacteria represented by *E. coli*, the
monolayer membrane structure of *Bacillus subtilis* makes the secretion of heterologous proteins easier,
and because *B.subtilis*' own protein expression and secretion systems are well documented and more
comprehensively studied, *B.subtilis* is widely used in the expression of heterologous protein-based
target products. Specifically, the following.

1. Non-pathogenicity. *Bacillus subtilis* is widely found in the animal gut in nature and is considered to
   be an intestinal probiotic with a history of preparing fermented foods that do not produce heat-sensitive
   proteins or various toxins and are safe up to food grade.
2. *Bacillus subtilis* cells have a strong capacity for extracellular secretion of proteins, with
   approximately 300 proteins being secreted directly into the extracellular compartment, simplifying and even
   removing some of the purification steps for the target proteins compared to the commonly used industrial
   host bacterium, *E. coli*.
3. Protein products expressed in the *Bacillus subtilis* expression system do not readily form inclusion
   bodies. In the *E. coli* expression system, if the coding sequence of the exogenous protein to be
   expressed contains a large number of consecutive rare codons, the expression level is usually low or even
   translation is terminated prematurely; inclusion bodies lead to insoluble proteins, which makes it difficult
   to isolate and purify the target protein, but the *Bacillus subtilis* expression system does not have
   these problems and shows significant advantages in these aspects.
4. *Bacillus subtilis* is an aerobic, thermophilic bacterium that does not require an anaerobic
   environment or special media, with a short culture cycle and fast growth rate.
5. *Bacillus subtilis* is a Gram-positive bacterium and there are many components present in the lysed
   system, such as peptidoglycan, phosphopeptide and protein. The type and content of phospholipids in
   *Bacillus subtilis* are well defined and the ratio between components is little affected by the culture
   conditions, which is conducive to the design of subsequent isolation and purification experiments.

In addition, unlike the Gram-negative expressing host *E. coli*, *B.subtilis* lacks an outer
membrane and associated periplasmic space, which gives it a greater capacity for protein secretion.
*Bacillus subtilis* heterologously expressed proteins were more productive compared to other chassis
microorganisms, but varied considerably between proteins. Compared to other strains already commercially
available there is a gap but it can meet the production requirements. There is no literature available on the
heterologous expression of pineapple protease by *Bacillus subtilis* at this time, there is some
literature relating to the expression of other proteases by *Bacillus subtilis* with more promising
yields.

![](https://static.igem.wiki/teams/4228/wiki/engineering-microbes-3.png)

Comparison

*Bacillus subtilis* has three possible advantages as a chassis compared to *E. coli*, as follows.

1. potentially higher expression levels than *E. coli*
   Since protein products expressed by the *Bacillus subtilis* expression system do not readily form
   inclusion bodies and *Bacillus subtilis* lacks an outer membrane and associated periplasmic space, it
   has a greater capacity for protein secretion. Therefore, *Bacillus subtilis* may express recombinant
   proteases at a higher level than *E. coli* and is more conducive to enzyme isolation and
   purification.
2. Biological chassis residues
   As any transgenic chassis must not be used as a feed additive, the final output must be sterile. However,
   it is not possible for the final output enzyme preparation to be completely free of chassis residues,
   whereas *Bacillus subtilis* is a probiotic and *E. coli* is a pathogenic bacterium, compared to
   the more harmless residues of *Bacillus subtilis*. The Chinese feed hygiene standard requires a
   residual sump dose of bacteria for biological enzyme preparations of <2 x 104 CFU/g. The optimal dose
   of the common *Bacillus subtilis* feeding strain PB6 is around 1 x 105 CFU/g or 1 x 104 CFU/g, and
   its safety as a commonly used *Bacillus subtilis* preparation has been certified by the European Food
   Safety Authority. Therefore, it is a win-win situation for *Bacillus subtilis* as a chassis
   microorganism as it can still be used for feeding purposes within the standard chassis residue count.
3. Beneficial protein secretion
   While *E. coli* secretes harmful toxins as it reproduces and grows, *Bacillus subtilis* produces
   a number of beneficial proteins. These beneficial proteins include antimicrobial active substances
   (chymotrypsin, polymyxin, myclobactin, short bacillus peptides), digestive enzymes (alpha-amylase,
   protease, lipase, cellulase) and B vitamins (B1, B2, B6, niacin).
   After comparing *Bacillus subtilis* and *E. coli*, we finally chose *Bacillus subtilis* as
   the chassis microorganism due to all these factors.

Selection of specific strains

- Selection of *Bacillus subtilis* strains commonly used in the laboratory

After choosing *Bacillus subtilis* as the general direction, we have been informed about the various
strains of *Bacillus subtilis* with the aim of identifying a safe and commonly used strain of *Bacillus
subtilis*. Considering that *Bacillus subtilis* is currently added and used in feeds, we wanted to
find a forage strain of *Bacillus subtilis* and modify him to produce our target product, bromelain, in a
safe and efficient manner. However, in order to keep the experiments on schedule, we split up into two strain
types of *Bacillus subtilis* for feeding and *Bacillus subtilis* for the common laboratory chassis.

*Bacillus subtilis* is a Gram-positive model bacterium that has been widely used for metabolic mechanism
studies and metabolic engineering. *Bacillus subtilis* has a well-defined genetic background and a range
of genome engineering tools that make *Bacillus subtilis* an important chassis for biological production.
The rapid growth of *Bacillus subtilis* chassis facilitates shorter fermentation cycles and higher yields
of target products. However, engineered *Bacillus subtilis* typically has a reduced growth rate compared
to the initial strain, especially after intensive rewiring of metabolic pathways, which severely hinders its
use in bioproduction.

In the section on strains of *Bacillus subtilis* commonly used in laboratory chassis, we focus on
*Bacillus subtilis* strain 168.

Whereas it has been shown that recombinant expression plasmids constructed in *Bacillus subtilis* suffer
from instability problems, our focus was on an improved strain of *Bacillus subtilis* strain 168 that
performed well in terms of both yield and hardware.

However there is no clear literature proposing an improved strain of *Bacillus subtilis* 168 that solves,
and alleviates, the problem of unstable recombinant expression plasmids in *Bacillus subtilis*, but we
did borrow three genes, oppD, hag and flgD, based on modification methods in the literature for use in
subsequent modifications of our strain. We therefore thought of strains of *Bacillus subtilis* for
addition to feed that might be able to be modified for protein production. We have therefore investigated and
compared strains for use in feed.

- Strains for feed vs. Strains commonly used in the laboratory

Expression strains have been chosen over feed strains for the following reasons.

1. Neither GM strains nor their recombinant DNA are allowed to be present in fermented products placed on the
   EU market as food or feed additives. Chinese feed hygiene standards also limit the residual presence of GM
   chassis. For this reason, feed strains cannot be retained in enzyme preparations as feed preparations.
2. (As most of the forage strains are isolated from animal faeces (i.e. intestinal parasitic strains of
   livestock), while the expressive strains are mostly derived from indigenous microorganisms in the soil)
   several common forage strains could not be found in the literature for expressing heterologous proteins, so
   the final preference was to choose the wild type strain 168 and its modified strains with high expression
   yield.

However, there are still many similarities between the expressive strains and the feeder strains, with the
greatest commonality between them being the production of bacteriocins. The commonly used feeder strain PB6
(ATCC-PTA 6737) has been shown to produce antimicrobial substances and has broad in vitro activity against a
variety of strains, including Campylobacter spp. and Clostridium spp. *Bacillus subtilis* strain 168 can
also produce antimicrobial substances.

- Optimisation of strains

One of the main factors limiting the application of *Bacillus subtilis* as an expression host is its
production of at least eight extracellular proteases. *Bacillus subtilis* produces at least eight
extracellular proteases (listed below) that have a variety of functions, including degrading proteins in
bioorganisms for nutrient supply and proteolytic processing of other proteins. However, extracellular
proteases
have a high propensity to degrade heterologous secreted proteins, resulting in significant loss of target
products.

In order to reduce the effect of endogenous proteases on the heterologous protein pineapple protease, several
protease-deficient strains were considered for comparison.

|  |  |
| --- | --- |
| Neutral protease | NprE NprB |
| Serine protease | Epr Bpr Vpr |
| Alkaline protease | AprE |
| Metalloprotease | Mpr |
| Cell wall protease | WprA |

Nevertheless, researchers have also noted that sometimes certain proteases favour the secretion of exogenous
proteins. Therefore, in order to maximise the production of exogenous proteins, proteases should be
selectively
inactivated. Our original plan was to use an evaluation system containing nine *Bacillus subtilis* 168
mutant strains to identify favourable and unfavourable proteases for target proteins (Zhao L, Ye B, Zhang Q,
Cheng D, Zhou C, Cheng S, Yan X. Construction of second generation protease -deficient hosts of *Bacillus
subtilis* for secretion of foreign proteins. Biotechnol Bioeng. 2019 Aug;116(8):2052-2060. doi:
10.1002/bit.26992. (Epub 2019 Apr 24. PMID: 30989640.). The mutant strain PD8 is inactivated for all eight
proteases, while each of the other eight mutant strains expresses only one of these eight proteases. The
target
protein is secreted in these nine mutant strains, and if the yield of the target protein is higher in the
mutant
strain than in the PD8 strain, the corresponding protease is considered favourable. Thus, an optimal
protease-deficient host was constructed by inactivating the unfavourable protease.

Unfortunately, however, due to the Shanghai epidemic, we were unable to return to school early in the summer
and
the experiment started late, so our original plan could not be implemented due to time constraints and we had
to
screen various protease-deficient hosts through literature research. Currently the common protease-deficient
strains are WB600 (ΔnprE,ΔaprE,Δepr, Δ bpr , mpr::ble , nprB::bsr), WB700 (Δ nprE,Δ aprE,Δ epr,Δ bpr,
mpr::ble,
nprB::bsr,Δ vpr) and WB800 (Δ nprE,Δ aprE,Δ epr,Δ bpr, mpr::ble, nprB::bsr,Δ vpr, wprA::hyg).

As there is no literature on recombinant expression of bromelain in *Bacillus subtilis*, we wanted to
investigate the literature on recombinant expression of these three strains in order to find an entry point
and
finally identify the strains. The recombinant enzymes expressed in WB600 had little to no correlation with
pineapple protease, while WB700 had little information available and was difficult to obtain, so these two
strains were not chosen. WB800 has been applied to produce recombinant enzymes that have a strong correlation
with pineapple protease.

In summary, WB800 was chosen as the pineapple protease expressing strain. (Note: All the above
protease-deficient hosts are modified strains of the 168 wild strain)

Construction and selection of plasmid

Considering that our ultimate goal is to express the recombinant protein in *Bacillus subtilis*, we mainly focus
on the design and construction of expression vector in *Bacillus subtilis*.

- Plasmid

We selected the common shuttle plasmid of *Bacillus subtilis*: pBE2R, about 7kb.

*E. coli*-*B.subtilis* shuttle plasmid is convenient for us to complete the operation in *E.
coli* and then transfer it into *Bacillus subtilis* for expression. pBE2R has low copy number,
simple structure and low price. We chose this plasmid without special requirements.

![](https://static.igem.wiki/teams/4228/wiki/engineering-plasmid-1.jpg)

- Promoter

Promoter is one of the many key factors for the expression efficiency, and the expression level of proteins
could be regulated by controlling transcript production at the promoter level. Generally, promoters used for
the expression of recombinant proteins in *B.subtilis* mainly include inducible promoters,
stress-specific promoters, auto-inducible (self-inducible) promoters, and constitutive promoters.So far, many
native, artificial, and engineered promoters have successfully been used for the expression of recombinant
proteins in *B.subtilis*, they are listed as follows.

Table Properties and
advantages of promoters[1]
(Blue
for disadvantages and orange for advantages)

| Promoters | Properties | Expression level | Features | References |
| --- | --- | --- | --- | --- |
| Pgrac212 | IPTG-inducible promoter. | Concentration of recombinant HRV3C reached 16% of total cellular proteins in *B.subtilis.* | IPTG is very expensive and toxic for cells to inhibit growth of hosts. | Le et al. [2019](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR99) |
| A cumate-inducible promoter | A synthetic expression module under control of PserA and a synthetic expression cassette with Pveg, CuO operator, and an optimal RBS; | Concentration of recombinant sfGFP with 50 μM cumate reached 81.1 mg/L. | Inducer is cheap, not utilized as a carbon source, efficient, and highly controllable. | Seo et al. [2019](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR169) |
| PspoVG | Middle-log and early stationary phases; Class II. | The expression level of eGFP was 1.9-fold of that of P43; PspoVG was also preferred for expression of keratinase in *B.subtilis.* | Strong. | Yang et al. [2017](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR218) |
| PabrB | pH inducible promoter; Class I. | The expression level of eGFP was highest at pH 6.0; it was also preferred for expression of esterase in *B.subtilis.* | Strong. | Yang et al. [2017](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR218) |
| PBH4 | Mutant of PsrfA after consecutive two-step evolution; a highly evolved synthetic promoter. | The strength of PBH4 was higher approximately threefold than that of PsrfA. | Strong. | Han et al. [2019](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR63) |
| P-HA-1 | Under stressed or normal conditions, it can be recognized by some sigma factors and produce strong transcriptional strength. | The transcription activity of promoter P-HA-1 for GFP was 18.6-fold that of P43 in *B.subtilis*. | Strong;sometimes need stressed conditions. | Wang et al. [2019](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR199) |
| PsrfA | Quorum-sensing related promoter; autoinducible promoter and inducer-free activity correlated with cell density. | The strength of PsrfA for GFP expression in *B.subtilis* was approximately 1.3-fold higher than that of P43. | Strong. | Cheng et al. [2016](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR22) |
| Pveg | Including a strong consensus signature [(-35)TTGACA-N14-tgn(-10)TATAAT] for house-keeping sigma factor σA of *B.subtilis*; | The activity of Cellulomonas fimi endoglucanase in *B.subtilis* is > four fold higher than that of the best previously reported. | Strong. | Guiziou et al. 2016; Lam et al. [1998](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR61) |
| PserA | Including a strong consensus signature [(-35)TTGACA-N14-tgn(-10)TATAAT] for house-keeping sigma factor σA of *B.subtilis.* | Expression level is high, and similar with Pveg. | Strong. | Guiziou et al. [2016](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR61) |
| P43 | Expression during early stationary phase than during growth; the arrangement of overlapping − 35 and − 10 regions reported for promoters in ctc was similar with that of P43. | Methyl parathion hydrolase can be continuously expressed during exponential growth phase and late stationary phase. | Strong. | Wang and Doi [1984](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR196);  Zhang et al. [2005](https://link.springer.com/article/10.1007/s00253-021-11533-2#ref-CR232) |

Given that there is no need for us to express the protein at a particular time, constitutive promoters may be
a decent option in consideration of time and cost. Thus, we chose the P43 promoter at first because it’s
easiest to obtain and can meet our fundamental requirements.

- Terminator

The terminators of *Bacillus subtilis* are rich in GC inverted repeats followed by several A(T)s which
are similar to the structures of terminators in *E. coli*, so we take the T7 terminator, the most
frequently used terminator for recombinant protein expression, as our first choice. Besides, we found that
artificially designed T31 terminator had the highest stop efficiency among most
frequently used known terminators[2] and had not been used by the iGEM teams
before. Thus, we take it into consideration as well. Unfortunately, we were unable to test T31 terminator’s
stop efficiency indeed because of the pandemic.

- Signal peptide

Tat pathway and Sec pathway are two important pathways for *Bacillus subtilis* to secrete extracellular
proteins. Sec pathway is the main exocrine pathway, which secretes proteins that are not well folded; Tat
pathway secretes folded extracellular proteins and has higher secretion efficiency for eukaryotic proteins,
providing an alternative for proteins that cannot pass through Sec pathway. Proteins secreted through Tat
pathway have two characteristics: they fold in the cell and have Tat signal peptide sequence. PhoD is strictly
secreted by Tat pathway in *Bacillus subtilis*, so we found the signal peptide of PhoD through the
literature and used it to construct plasmids. After reading the literature, we also found that using different
Tat signal peptides will make the exocrine efficiency of the same protein different. If time permits, we also
hope to screen the most efficient signal peptide through experiments.

References

[1] Kolkman, M. A. B. , René Van Der Ploeg, # Michael Bertels, Dijk, M. V. , Laan, J. V. D. , & Dijl, J. M. V. ,
et al. (2019). The twin-arginine signal peptide of *Bacillus subtilis* ywbn can direct either tat-or
sec-dependent secretion of different cargo proteins: secretion of active subtilisin via the *B.subtilis*
tat pathway. Applied and Environmental Microbiology, 74(24).

[2] 江南大学. 一种双精氨酸途径蛋白质分泌载体的构建方法及其应用:CN200810180274.8[P]. 2009-04-15.

[3] Kolkman, M. A. B. , René Van Der Ploeg, # Michael Bertels, Dijk, M. V. , Laan, J. V. D. , & Dijl, J.

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Protocols

[*E. coli*](#e)

[Transformation of *E. coli*](#e-transformation)
[Determining the optimum
 concentration of iptg](#e-determining)
[Induction of iptg](#e-induction)
[Crude protein extraction](#e-crude)
[SDS-PAGE](#e-sds)
[Enzyme activity determination](#e-enzyme)
[Soybean meal experiment](#e-soybean)
[*Bacillus subtilis*](#b)

[Resuscitation of *Bacillus subtilis*](#b-resuscitation)
[Preservation of *Bacillus subtilis*](#b-preservation)
[Transformation of *Bacillus subtilis*](#b-transformation)

![](../asset/img/page-pet.png)

*E. coli*

Transformation of *E. coli*

1. Take 100 µL of melted receptor cells on ice, add 10 μL of the target plasmid, mix gently and leave on ice
   for 30 min.
2. Heat stimulate at 42°C for 45-60 s in a water bath, transfer to an ice bath and leave for 2 min.
3. Add 700 µL of sterile liquid LB medium containing the antibiotic Amp to the centrifuge tube, mix well and
   recover at 37°C for 60 min at 220 rpm.
4. Pipette 20 μL of the recovery solution onto the LB solid medium containing Amp and place the plate upside
   down in an incubator at 37°C for overnight incubation.

Determining the optimum concentration of iptg

1. Rejuvenation of the bacterial broth
   Take out the bacterial broth from the fridge and incubate it in a small volume for re-strengthening
   Overnight at 37°C and 220rpm.
2. Expand the culture
   Inoculate the re-strengthened bacterium into 25 mL of LB (amp+) and incubate in a shaker at 37 degrees and
   220rpm.
3. Induction of iptg
   After step 2, the obtained bacterial broth was divided into 5 groups of about 5 mL each and numbered. 1
   group continued to be incubated on a shaker at 37 degrees, 220rpm. The OD600 of the bacterial broth was
   measured and iptg (final concentration 0.5mM) was added to groups 2, 3, 4 and 5 at OD600=0.6, 0.8, 1 and 1.2
   respectively. 3-4h incubation at 37 degrees, 220rpm.
4. SDS-PAGE

Induction of iptg

1. Pick a single colony from each plate into 4 LB (4 mL) tubes corresponding to the resistance, numbered "0"
   "1" "2" "3 ".
   Place the tubes in a shaker (37°C; 220r), incubate for a period of time and measure the OD value.
2. Add 700 μL of suspension from tube "0" to 100 μL (C=80%) of seed preserving glycerol, shake well and store
   in the freezer (-20°C).
3. Add 2 μL of IPTG to tubes "1", "2" and "3" of each group of bacteria, at a final concentration of 0.5 mM.
4. Expression at 37°C for 3h to 4h.

Crude protein extraction

1. Transfer *E. coli* to 2 mL centrifuge tubes (50 mL), 4°C, 12000rpm, 25min, discard supernatant and
   collect
   bacteriophage.
2. 400 μL (2 mL of bacteriophage premise) PBS was used to wash the bacteriophage, followed by centrifugation
   at 4°C, 4000rpm, 5min, discard the PBS supernatant and collect the bacteriophage.
3. Resuspend the bacterium using 1 mL PBS.
4. The bacterial solution was placed in an ultrasonic cell crusher with 150W power, 1s working, 2s gap,
   temperature alarm set to 30°C and ultrasonic crushing in an ice bath. The total working time is 10-20 min,
   depending on the volume.
5. After crushing, the sample is centrifuged at 4°C for 25min at 12000rpm and the supernatant is discarded
   and stored as the crude product.

SDS-PAGE

1. Cleaning the glass plate
   Hold the glass plate firmly with one hand and gently scrub it with the other hand with a little washing
   powder. Rinse with tap water after scrubbing both sides, then rinse with distilled water and leave to dry in
   the basket.
2. Gluing and sampling
   1. Align the glass panels and place them in the clamps to be stuck tightly. Then snap vertically onto the
      shelf ready for gluing. (The operation should be done so that the two glasses are aligned so as not to
      leak glue.)
   2. Preparation of the separating glue and glue concentrate: Configure 12% of the separating glue and 5%
      of the glue concentrate in the order and proportion of the solutions in the table below.

      ![](https://static.igem.wiki/teams/4228/wiki/protocol-e-1.png)
      ![](https://static.igem.wiki/teams/4228/wiki/protocol-e-2.png)

      When there is a fold line between the water and the glue, the glue has set. Wait 3 min for the gum to
      solidify before pouring off the top layer of water and blotting up the water with absorbent paper.
   3. Rinse the gel concentrate with water and place it in the electrophoresis bath. (Small glass plate
      facing inwards, large glass plate facing outwards. If you are running only one piece of glue, the other
      side of the tank should be padded with a plastic plate and the side with the letters should face
      outwards.)
3. Electrophoresis
   After the sample is added, cover the top lid, connect the electrophoresis instrument, turn on the switch of
   the electrophoresis instrument, control the voltage at 100-200V before the sample enters the glue, about
   15-20min; after the bromophenol blue indicator in the sample reaches the separation glue, the voltage rises
   to 200V, keep the voltage stable during electrophoresis. The electrophoresis was stopped when the
   bromophenol blue indicator migrated to 1~2cm from the front, about 0.5-1hour.
4. Staining and decolorization
   After electrophoresis, turn off the power, take out the glass plate, pry it gently with a knife in the gap
   between the lower corners of the short and long glass plates to separate the adhesive surface from one glass
   plate, then gently lift the film, insert a copper wire in the center of the indicator zone as a marker and
   put it into a large petri dish for staining, using 0.25% Kormas Brilliant Blue staining solution, stain for
   2-4h, or overnight if necessary.
   Discard the staining solution and rinse the gel surface several times with distilled water, then add
   decolorizing solution and perform diffusion decolorization, changing the decolorizing solution frequently
   until the protein band is clear.

Enzyme activity determination

1. Standard curve preparation
   1. L-Tyrosine Standard Solution: This can be prepared by referring to the table below. (Note: L-Tyrosine
      dilutions should be measured immediately after dilution)
      No. Concentration of tyrosine standard solution/(μg/mL) Volume of tyrosine standard stock solution/mL
      Volume of water/mL

      | Group | Concentration of tyrosine standard solutions /(μg/mL) | Volume of tyrosine standard stock solution /mL | H2O/mL |
      | --- | --- | --- | --- |
      | 0 | 0 | 0 | 10 |
      | 1 | 10 | 1 | 9 |
      | 2 | 20 | 2 | 8 |
      | 3 | 30 | 3 | 7 |
      | 4 | 40 | 4 | 6 |
      | 5 | 50 | 5 | 5 |
   2. Take 1.00mL of each of the above solutions (parallel test must be done), add 5.00mL of sodium carbonate
      solution and 1.00mL of forint reagent solution, shake well, place in a water bath at 40℃±0.2℃ for 20min,
      remove and measure the absorbance of each solution with a spectrophotometer at a wavelength of 680nm,
      10mm cuvette, using a 0 tube without tyrosine as a blank. The absorbance A was used as the vertical
      coordinate and the concentration c of tyrosine was used as the horizontal coordinate to draw the
      standard curve.
   3. Using the regression equation, calculate the amount of tyrosine when the absorbance is 1, which is the
      value of the absorbance constant K. The K value should be in the range of 95 to 100. (If it is not, the
      reagents need to be reconstituted and the test carried out.)
2. Sample determination
   1. Preparation of the enzyme solution to be tested
      Weigh 1g~2g of enzyme sample, accurate to 0.0002g, then dissolve and dilute to a certain concentration
      with the appropriate buffer, the recommended concentration range is 10 U/mL~15 U/mL. For powdered
      samples, dissolve with the appropriate buffer, then take slow qualitative filter paper and dilute to the
      appropriate concentration.
   2. Assay procedure (for reference only)
      The casein solution is first placed in a constant temperature water bath at 40°C ± 0.2°C and preheated
      for 5 min.

      ![](https://static.igem.wiki/teams/4228/wiki/protocol-e-3.png)
3. Activity calculation
   Read the enzyme activity of the final dilution of the sample from the standard curve in U/mL. The enzyme
   activity of the sample is calculated according to the following formula:
   * X=(A\_1×V\_1×V\_0×n)/m\_0 ×1/10In the formula:
   * X → enzyme activity of the sample U/g;
   * A1 → the activity U/mL of the final dilution of the sample derived from the standard curve;
   * V1 → volume of the volumetric flask used to dissolve the sample mL;
   * V0 → the total volume of reaction reagent mL;
   * n → the number of dilutions of the sample;
   * m0 → the mass of the sample g;
   * 1/10 → reaction time of 10 min, measured in 1 min.The results shown are taken approximately to the nearest whole number.

Soybean meal experiment

1. Preparation of pineapple protease treated feed samples
   1. Experimental group:
      Soya bean meal powder (4 g) was added with 50 mL (0.5%) of pineapple protease and incubated in a shaker
      water bath at 50°C for 3 hours. After protein hydrolysis, 20% (w/v) TCA (trichloroacetic acid) at a
      volume ratio of 1:1 was added to the protein hydrolysate to give 10% TCA soluble material. The mixture
      was allowed to stand for 20 minutes to allow precipitation and then diluted to a volume of 100 mL. The
      sample (35 mL) was placed in a conical tube and the supernatant (1.5 mL) was analysed for protein by
      centrifugation for 20 minutes.
   2. Control group:
      Soya bean meal powder (4 g) was added with 50 mL of distilled water and incubated for 3 hours at 50°C in
      a shaker water bath.
      The W5 solution was not used as the soybean meal had destroyed the cell structure.
      A 1.5 mL sample was mixed before analysis.
2. SDS-PAGE

*Bacillus subtilis*

Resuscitation of *Bacillus subtilis*

1. Wipe the outer tube with cotton moistened with 70% alcohol and heat the tip of the tube over a flame.
2. Apply a few drops of sterile water to the heated area to break the outer tube and then crack the tip with a
   stiff stick.
3. Remove the insulating paper and the inner tube and use sterilised forceps to remove the cotton plug from the
   inner tube.
4. Pipette 0.3-0.5 ml of the specified liquid medium into the inner tube and mix well. After the dry powder has
   dissolved, pipette with a sterile tip into a tube containing approximately 5 ml of sterile LB medium and
   incubate for 24 h.

Preservation of *Bacillus subtilis*

1. Add glycerol 1:1 to the fully heated and mixed *Bacillus subtilis* solution and dispense into EP tubes
   (2 mL or so).

Transformation of *Bacillus subtilis*

1. Prepare a fresh 168 monoclonal plate, take a LB plate full of *Bacillus subtilis* glycerol and incubate
   for 12 h at 37°C.
2. The day before the transformation, pick a single colony in the evening and incubate it in 3 ml LB medium at
   37°C, 250 r/min overnight.
3. The next morning, transfer 160 μL of culture to 8 ml of SPI medium and incubate at 37°C, 250 r/min until the
   end of logarithmic growth (168 for about 4-5 h).
4. Transfer 0.2 ml of the end of logarithmic growth to 2 ml of SPII medium and incubate at 37 °C for 90 min at
   100 r/min.
5. Add 20 μL of 10mmol/L EGTA to the SPII medium and incubate at 37°C for 10 minutes at 100 r/min.
6. Add 5 μL of DNA to each 0.5 ml tube (the amount of DNA should not exceed 5 ug) and incubate at 37 ℃, 250
   r/min for 90 minutes.

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{% extends "layout.html" %}
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Results

[Search for mutable sites to obtain superior variants](#search)
[Exploration of upper relationship
between residues of stem bromelain](#exploration)
[Substrate docking of stem bromelain
and its variant](#substrate)
[Molecular Dynamics Simulation
Analysis](#molecular)
[Expression of bromelain in *E. coli*](#expression)
[Enzyme activity assay](#assay)
[Enzyme stability assay](#enzyme)
[Determining the optimum
concentration of iptg](#determining)
[Expression of bromelain in *Bacillus
subtilis*](#subtilis)

![](https://static.igem.wiki/teams/4228/wiki/page-pet.png)

Search for mutable sites to obtain superior variants

Table 1 and 2 show the point mutation sites provided by Hotspot Wizard. In the search for point mutations, reference was made to the original analysis of residue 291. All conserved residues in enzymes are composed of polar, nonpolar, acidic and basic groups. Most of the conserved residues, such as Tyr, Asn, Gly, Thr, Gln and Cys, belong to polar groups, followed by non-polar groups (Phe, Trp, Ala, Val and Pro), acidic groups (Glu and Asp) and basic groups (Arg and His). So we evaluated some functional residues as well as other residues.

Table 1: Mutable structural residues

| # | Site | Original residues | Mutational residues |
| --- | --- | --- | --- |
| 1 | 18 | Lys | Arg or Gln |
| 2 | 20 | Gln | Arg |
| 3 | 51 | Gln | Ala |
| 4 | 159 | Val | Phe or Leu |

Table 2: Mutable nonstructural resideus

| # | Site | Original residues | Mutational residues |
| --- | --- | --- | --- |
| 1 | 16 | Ser | Gly or Asp |
| 2 | 161 | Thr | Phe or Leu |
| 3 | 172 | Tyr | Ile |

Rosseta's Relax module was used to score these point mutation models. The initial model energy was calculated as -607.691. The energy of No. 18 Arg mutation was -332.407 and the structure was disorganized（Figure 1）, so the mutation scheme has been abandoned. The possible reason was that site 18 was mutated into Arg, and the surrounding area was positively charged, while Arg was negatively charged in the past, and the energy was too high, so the structure appeared to fall apart.

![](https://static.igem.wiki/teams/4228/wiki/model-search-1.png)

Figure 1

The energy of the Gln mutation 18 was -605.077. Although its structure did not fall apart, its energy increased and thus it was abandoned. The structure of Arg 20 was disintegrated after mutation, so it was abandoned too. (Figure 2)

![](https://static.igem.wiki/teams/4228/wiki/model-search-2.png)

Figure 2

However, the energy of Gly 16 was -610.561 after mutation. Meanwhile, compared with the structure before mutation, its RMSD value was 0.683, so it was selected for further mutation.

![](https://static.igem.wiki/teams/4228/wiki/model-search-3.png)

Figure 2

Finally, we screened the model with Leu mutation at site 67, and its energy was -614.826. Compared with the original model, the RMSD value was 0.97. Its reduced energy and relatively stable structure suggested that it might be a better variant of bromelain, as the experiments would prove.

Exploration of upper relationship between residues of stem bromelain

The definition of upper relationship means that some single mutations of A are unfavorable, but the addition of B mutation will have better performance than single B mutation, which means that B has upper relationship with A. After calculating by Relax, we found that the Gly single mutation at site 16 had an upper relationship with the Leu single mutation at site 67: when Leu single mutation was detected, the structure was disordered, RM value was 1.4, and energy was -348. But with the addition of Gly, the structure stabilizes and the energy drops to -614.82. At the same time, the single mutation of Gly at site 16 has an upper relationship with the single mutation of Val at site 64: the energy of single mutation of Val is -599, while the energy of mutation of both Gly and Val decreases to -608.

Substrate docking of stem bromelain and its variant

After literature review and network search, BAEE was finally determined as the reaction substrate of bromelain, and its docking and energy calculation were performed with bromelain and mutated variants. The calculations were done online by Hdock.

Finally, the binding energy of bromelain in wild-type was -125.09, while that in mutant was -126.26, which decreased. This also proves that the stem bromelain variant we developed is more easily bound to the substrate and its reactivity is enhanced（Figure 4, 5, 6 and 7）

![](https://static.igem.wiki/teams/4228/wiki/model-substrate-1.png)

Figure 4

![](https://static.igem.wiki/teams/4228/wiki/model-substrate-2.png)

Figure 5

![](https://static.igem.wiki/teams/4228/wiki/model-substrate-3.png)

Figure 6

![](https://static.igem.wiki/teams/4228/wiki/model-substrate-4.png)

Figure 7

Molecular Dynamics Simulation Analysis

A molecular dynamics simulation was conducted to analyze the binding stability of Stem Bromelain(212aa) and BAEE complexes, where multiple descriptors were analyzed to understand the flexible and stable nature of the complexes. The system has been balanced in advance (the result of temperature and pressure balance is shown in below(Figure 8,9).

![](https://static.igem.wiki/teams/4228/wiki/model-molecular-1.png)

Figure 8: the result of temperature balance

![](https://static.igem.wiki/teams/4228/wiki/model-molecular-2.png)

Figure 9: the result of pressure balance

After the whole system was balanced, the molecular simulation started to run, which took 10h (1ns) in total.

RMSD and RMSF analysis were performed on the simulated results, and the results were shown in the figure below. It can be seen that Stem bromelain(212) has good reactivity with BAEE in neutral environment. The RMSD value was less than 0.4, and the RMSD value structure did not change much before and after simulation, indicating that the reaction between the complex was very stable. At the same time, the RMSF value changed greatly, which reflected that the atomic motion of Stem bromelain (212) was relatively free when it reacted with BAEE.

![](https://static.igem.wiki/teams/4228/wiki/model-molecular-3.png)

Figure 10: the RMSD of Stem Bromelain(212) and BAEE complex

![](https://static.igem.wiki/teams/4228/wiki/model-molecular-4.png)

Figure 11: the comparing RMSD of Stem Bromelain(212) and BAEE complex

![](https://static.igem.wiki/teams/4228/wiki/model-molecular-5.png)

Figure 12: the RMSF of Stem Bromelain(212) and BAEE complex

As for the mutant, since it can also dock with BAEE and its binding energy is better than that of Stem Bromelain (212), we predict that its RMSD map fluctuation should be smaller, and the data will be made up in the later molecular simulation.

Expression of bromelain in *E. coli*

Bromelain wild type, bromelain variant 1 and bromelain variant 2 were synthesized at 37°C, pH=7 and induced by iptg (50mg/mL, Sangon Biotech). After 3-4 hours all sluices containing the three proteins were collected and sonicated to lyse the bacteria. Correctly transformed and induced *E. coli* expressed bromelain wild type, bromelain variant 1 and bromelain variant 2 (Fig 13). We can see the bromelain (variant) bands around 25-35 kDa. By isolating and purifying pineapple protease variant 1 and variant 2 by Ni columns, we obtained a protein solution containing all three of these proteins.

![](https://static.igem.wiki/teams/4228/wiki/results-expression-1.png)

Fig 13: Electrophoresis of proteins before and after transformation

Enzyme activity assay

In the experiment of formulating the working curve, we configured several standard L-tyrosine with gradient concentrations of 0, 10, 20, 30 and 40 respectively μg/ml; After a thorough reaction with Folin reagent for 20min, microplate reader was applied to measure the absorbance of the system after reaction at the wavelenth680nm. The A values obtained are 0.001, 0.436, 0.801, 1.096 and 1.293 respectively. From this series of data, the following working curve can be obtained:

![](https://static.igem.wiki/teams/4228/wiki/proof-of-concept-enzyme-1.png)

The formula we applied to is: A=0.03246c+0.076, and the reaction is carried out at 40℃ then the absorbance of the system is measured:

| 40℃ | A1 | A2 | A3 |
| --- | --- | --- | --- |
| Wild type | 0.556 | 0.549 | 0.550 |
| p.(S16G) | 0.772 | 0.794 | 0.689 |
| p.(S16G,W67L) | 0.802 | 0.789 | 0.796 |

Taking the wild stem bromelain for an example. The average value of the data is 0.552, and the concentration of L-tyrosine can be calculated as 14.66μg/ml according to the function. The amount of substance consumed in the reaction time is 0.08091μmol per minute. According to the formula provided in GB/T23527-2009:

![](https://static.igem.wiki/teams/4228/wiki/proof-of-concept-formula-1.png)

Where X is the enzyme activity of the sample, A1 is the enzyme activity of the reaction diluent obtained from the working curve (0.08091μmol here), V1 is the volume of reaction vessel (15ml here), V0 is the volume of the reaction system (10ml here), n is the dilution multiple of the sample (2 here), m0 is the mass of the sample taken (0.5g here), and T is the time consumed (10min here). It can be calculated that the activity of our p.(S16G), p.(S16G,W67L) and wild type are respectively 6900U, 7344U, 4854U per gram of coarse cell extracts in the buffer.

Enzyme stability assay

——Only p.(S16G,W67L)

The experimental process is basically consistent with the contents above. The reaction temperature is raised to 50, 55 and 60℃, and three parallel groups are still set at each temperature. After reacting with Folin reagent, the absorbance of the system is measured at 680nm:

|  | A1 | A2 | A3 |
| --- | --- | --- | --- |
| 50℃ | 0.548 | 0.518 | 0.466 |
| 55℃ | 0.214 | 0.307 | 0.279 |
| 60℃ | 0.085 | 0.079 | 0.096 |

The calculation process will not be repeated. Therefore, the average absorbance values at three temperatures are 0.510, 0.266 and 0.087, and the enzyme activities at these three temperatures are 4412U, 1915U and 1541U per gram of coarse cell extracts in the buffer respectively. The enzyme activity-temperature diagram can be plotted from the above data:

![](https://static.igem.wiki/teams/4228/wiki/proof-of-concept-enzyme-2.png)

Overall, our enzyme activity data confirms the success of the initial mutant as well as the model. We produced a bromelain variant with a 1.5-fold increase in activity in our final design.

Determining the optimum concentration of iptg

In order to determine the optimal induction concentration of iptg, we prepare to add iptg to induce expression when the od values of *E. coli* are 0.6, 0.8, 1.0, 1.2, and study the expression results. However, after many times of measurement with the microplate reader, we found that the od value of the bacterial fluid always fluctuate around 0.3, and no matter how we adjusted the culture time, this value would not increase too much. We speculate that this is because the T7 expression system which we selected has leaked expression. However, the leaked expression of bromelain decomposes the endogenous protein of *E. coli*, causing it to be unable to continue to increase in value ---the bands showed at 25-35KD in the sample which was not induced can prove this hypothesis to a certain extent.

![](https://static.igem.wiki/teams/4228/wiki/results-determining-1.png)

![](https://static.igem.wiki/teams/4228/wiki/results-determining-2.png)

Expression of bromelain in *Bacillus subtilis*

We have transformed the bromelain variant into *Bacillus subtilis*, but due to the epidemic, we have not fully completed our experiments with *Bacillus subtilis* as the chassis for bromelain production and believe that our mutant will perform better after undergoing glycosylation in a eukaryotic chassis.

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