iGEM 2022 — Team whu-china

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Notebook

2022 WHU-China

# Notebook

# Week1(4.25-5.1)

## Wet

1. Experiment design and check the protocol.

## Dry

1. Discuss how to use modeling method in our project and bring up several potential
modeling ideas.

## HP

1. Human practice program planning and hand out questionnaires.

# Week2(5.2-5.8)

## Wet

1. Purchase reagents, prepare consumables.
2. Experiment design.

## Dry

1. Design physical models in conjunction with enteral drug simulation.

## HP

1. Interaction with High School Teenagers.

# Week3(5.9-5.15)

## Wet

1. Synthesize the target fragments and amplify XylR from Bacillus subtilis.
2. Search for articles containing protocols on the Internet.
3. Prepare each fragment and try overlap extension PCR.
4. Amplify segments: Chem, Temp, EGFP, Holin; extract plasmid: pBE2.

## Dry

1. Design the first version device for better enteral drug simulation.

## HP

1. Visit professor Yu Yi in college of pharmacy of Wuhan University.

# Week4(5.16-5.22)

## Wet

1. Use molecular tools like restriction enzyme and ligase to construct recombined
plasmid pHT-Sam2 and pBE-PET8; transfer into Escherichia coli DH5α.
2. Try out overlap extension PCR.
3. Digest using restriction enzyme and construct recombined plasmids: pBE2-Chem, pBE2-Temp.
4. Collate the protocols we will use.

## HP

1. Communication with DO & GO.

# Week5(5.23-5.29)

## Wet

1. Identify the positive strains transfered by pHT-sam2 and pBE-XylR and extract
plasmids to be sequenced.
2. Redesign protocol based on new vector.
3. Prepare competent cells: E. coli-DH5α; transform recombined plasmids and spread
plates.
4. Order enzymes, primers and synthesize fragments.

## HP

1. Communication with NJMU-iGEM.

# Week6(5.30-6.5)

## Wet

1. Identify the positive strains transfered by pBE-XylR and extract plasmids to be
sequenced; extract the pHT-Sam2 prepared to transfer Bacillus subtilis.
2. Colony PCR to select positive recombinants for sequencing.
3. Purchase reagents and refine our protocols.

## HP

1. Conversation with the freshmen of College of Life sciences in WHU.

# Week7(6.6-6.12)

## Wet

1. Identify the positive strains transfered by pBE-XylR and extract plasmids to be
sequenced; use Two-step Chemical Method to transfer Bacillus subtilis.
2. Colony PCR to select positive recombinants for sequencing.
3. Prepare each fragment and try overlap extension PCR.
4. Prepare experimental consumables such as chemically competent cells.

## HP

1. Seek the help of professor Gan Fei from the School of Life Sciences.

# Week8(6.13-6.19)

## Wet

1. Identify the positive strains transfered by pBE-XylR-PET8 and extract plasmids to
be sequenced; use Two-step Chemical Method to transfer Bacillus subtilis.
2. Use the fragment that comes with Gibson Assembly Kit as an control, and do the whole experiment (PCR, gel electrophoresis, gel extraction, Gibson assembly, transformation, colony PCR), to serve as an positive control, to make sure that this whole system is working.
3. Extract plasmids: pBE2-Chem, pBE2-Temp; digest and then construct recombined plasmids: pBE2-Chem-EGFP, pBE2-Temp-EGFP, pBE2-Chem-Holin, pBE2-Temp-Holin.
4. Try out overlap extension PCR.

## HP

1. Hold a series of popular science activities with NJMU-iGEM and DO & GO.

# Week9(6.20-6.26)

## Wet

1. Change the method of coloning pBE-XylR to homologous recombination; use Two-step
Chemical Method to transfer Bacillus subtilis.
2. Finish the control experiment and start to do the formal experiment after verification. Obtain and purified linearized fragments and vector, use Gibson Assembly to link the fragment and the vector together and then transform DH5α chemically competent cells.
3. Redesign protocols and separate the construction process into two.
4. Prepare competent cells: E. coli-DH5α; transform recombined plasmids and spread plates.

## Dry

1. Purchase parts of enteral drug simulation device and assemble.

## HP

1. Communication with DO & GO.

# Week10(6.27-7.3)

## Wet

1. Use homologous recombination to construct pBE-XylR-PET8; use Two-step Chemical
Method to transfer Bacillus subtilis.

2. Link the PCR products again using Gibson Assembly, transform and colony PCR.
3. Colony PCR to select positive recombinants for sequencing.
4. Purchase reagents.

## HP

1. Take part in an online meetup.

# Week11(7.4-7.10)

## Wet

1. Identify the positive strains of pBE-XylR-PET8 and send them to be sequenced; use
Two-step Chemical Method to transfer pHT-Sam2 Bacillus subtilis.

2. Order commercial competent cells and prepare our own chemical competent cells once more.
3. Check fragments and construct the first plasmid intermediate.
4. Colony PCR to select positive recombinants for sequencing and preserve bacterial strains.

# Week12(7.11-7.17)

## Wet

1. Identify the positive strains of pBE-XylR-PET8 and send them to be sequenced;
change the method to electrotransformation to transfer pHT-Sam2 Bacillus subtilis.

2. Try the whole experiment again using PCR purification as an alternative way around gel extraction.
3. Construct the final plasmid, colony PCR and sequence to identify.
4. Amplify segments: CinI; extract plasmid: pET-28a.

## Dry

1. Design the second version device for better enteral drug simulation.

## HP

1. Organize a training for the students in the Elite Program.

# Week13(7.18-7.24)

## Wet

1. Use electrotransformation to transfer pBE-XylR-PET8 into Bacillus subtilis.
2. Identify the positive Bacillus subtilis of pHT-Sam2 and send them to be sequenced.
3. Analyze the problems and redo the construction.
4. Digest using restriction enzymes and construct recombined plasmids: pET-CinI.

## Dry

1. Purchase parts of the second version enteral drug simulation equipment and
assemble.

# Week14(7.25-7.31)

## Wet

1. Explore the induction condition of Sam2 B. subtilis through SDS-PAGE;use
electrotransformation to transfer pBE-XylR-PET8 into Bacillus subtilis.

2. Try one more time, from PCR amplificaiton of products to Gibson and colony PCR, using PCR purification rather than gel extraction.
3. Build the variant library and sequence the mutagenesis.
4. Prepare competent cells: E. coli-DH5α, E. coli-BL21; transform recombined plasmids and spread plates.

## HP

1. Hold the Central China iGEM Communication.

# Week15(8.1-8.7)

## Wet

1. Use Western Blot to verify the expression of Sam2; identify the positive strains
of pBE-XylR-PET8 and send them to be sequenced.

2. Look up online publications and make modifications to our protocols by assembling multiple fragments for one single time.
3. Build the variant library and sequence the mutagenesis.
4. Colony PCR to select positive recombinants for sequencing.

## HP

1. Participate in ICII.

# Week16(8.8-8.14)

## Wet

1. Explore the induction condition of PET8 B. subtilis through SDS-PAGE
2. Use Western Blot to verify the expression of pHT-Sam2; identify the positive strains of pBE-XylR-PET8 and send them to be sequenced.

3. Separate the desired variant through selection culture.
4. Extract plasmids: pET-CinI; transform recombined plasmids and spread plates.

## Dry

1. Design portable dual port filter.

## HP

1. Participate in CCiC.

# Week17(8.15-8.21)

## Wet

1. Use Western Blot to verify the expression of pHT-Sam2; identify the positive
strains of PET8 and send them to be sequenced.

2. Do another round of molecular cloning and amplify the fragments that we will ligate together; design new primers.
3. Separate the desired variant through selection culture.
4. Colony PCR to select positive recombinant for sequencing; preserve bacterial strains.

## Dry

1. Purchase parts of portable dual port filter and assemble.

## HP

1. iGEM introduction for 2022 freshmen.

# Week18(8.22-8.28)

## Wet

1. Purchase new antibodies and repeat the Western Blot to verify the expression of
Sam2 and PET8.
2. Amplify the fragments to be ligated again successfully and do PCR amplification and Gibson Assembly.
3. New cycles of selection and sequencing identification.
4. Transform plasmids: pBE2-Chem-Holin, pBE2-Temp-Holin into B. subtilis and spread plates.

## HP

1. Investigate and survey in MGI.

# Week19(8.29-9.4)

## Wet

1. Repeat Western Blot to verify the expression of Sam2 and PET8;
2. Use fluorescence microscopy to verify the localizaiton of PET8 through its fused sfGFP.
3. Colony PCR to select positive recombinants for sequencing and construct the intact repressilator in E. coli and B. subtilis.
4. New cycles of selection and sequencing identification.

## Dry

1. Complete enteral drug simulation experiment.

# Week20(9.5-9.11)

## Wet

1. Use fluorescence microscopy to verify the localizaiton of PET8 through its fused
sfGFP.
2. Design colony PCR primers for the detection of co-transformation; extract the Repressilator plasmid and the reporter plasmid; prepare for the competent cells using E. coli that already contains one of the plasmids.
3. Develop the procedures and conduct a new round of selection.
4. Colony PCR to select positive recombinants for sequencing; preserve bacterial strains.

## HP

1. Hand out questionnaires.

# Week21(9.13-9.18)

## Wet

1. Identify the positive strains of pBE-XylR-PET8 and send them to be sequenced;
change the method to electrotransformation to transfer pHT-Sam2 Bacillus subtilis.

2. Try the whole experiment again using PCR purification as an alternative way around gel extraction.
3. Construct the final plasmid, colony PCR and sequence to identify.
4. Amplify segments: CinI; extract plasmid: pET-28a.

## Dry

1. Complete the test experiment of the portable dual-port filter.

## HP

1. Participate in ICII.

# Week22(9.19-9.25)

## Wet

1. According to the result, redesign the biobricks about Sam2 and PET8p.
2. Using the co-transformed strain, we test the fluorescence under microplate reader and under the fluorescence microscope.

3. Develop the procedures and conduct a new round of selection.
4. Quantify fluorescence in E. coli and concentration of B. subtilis to verify.

## Dry

1. Revise our hardware articles and translate them into English.

[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)[Protocols](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27protocol%27%29%20%7D%7D)

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Engineering Success

2022 WHU-China

# Engineering Success

# Overview

    We have accomplished Design-Build-Test-Learn (DBTL) cycles in Production Module, Oscillator Module, Directed Evolution Module, Safety Switch Module and Hardware.

    In Production Module, we used DBTL cycle to improve the enzymatic activity of SAMe synthetase opSam2 (BBa\_K4144011) and to improve membrane localization of SAMe transporter opPet8p (BBa\_K4144004) by designing a new part (BBa\_4144010) considering the structural disruption caused by sfGFP.

    In the Oscillator Module, we used the parts of Repressilator (BBa\_K3482025) to test their function. And we failed many times but finally made it to observe green fluorescence and explored the suitable way of testing the oscillation.

    In the Directed Evolution Module, to develop a lactose-tolerant LacI protein is our goal. After several rounds of selection, we have gained a colony with expected LacI in a certain way.

    In the Safety Switch Module, we struggled to construct double switches using Holin and EGFP. We met great difficulties during the process, but we successfully got positive results in a part of test experiments in the end.

    In hardware, we improved efficiency of Portable Dual Port Filter based on results of previous experiments.
We have characterized several new basic parts in engineering this year: BBa\_4144004, BBa\_K4144011, and BBa\_K4144012.

# DBTL cycle of SAMe Production Module

## Design of the First Cycle

    Sam2 is a S-adenosyl methionine (SAMe) synthetase from
Saccharomyces cerevisiae which won’t be inhibited by its enzymatic reaction
product SAMe [1]. We chose it as the enzyme to our produce SAMe in our
engineered bacteria. In past iGEM competitions, there are few teams constructing
such protein to generate SAMe. They all worked on gram-negative bacteria
Escherichia coli (E. coli), while we tend to express such protein in
gram-positive bacteria model bacteria Bacillus subtilis (B. subtilis) firstly
and then in Bifidobacterium longum (B. longum). Thus, we used a codon
optimization tool ExpOptimizer to optimize the coding sequencing, and the
optimized sequence is called opSam2 (BBa\_K4144011). Then to activate its
expression, we utilized a lactose-inducible promoter Pgrac which is endogenous
for B. subtilis. We expect that with induction of IPTG, opSam2 can be expressed
successfully and catalyze SAMe synthesis in B. subtilis.

## Build of the First Cycle

    We used a shuttle vector pHT01K to insert our fragment
opSam2 to construct recombined plasmid pHT-opSam2. Through molecular clone
procedure, like PCR, enzyme cut, ligation, and transformation, we have
successfully transferred our pHT-opSam2 into E. coli and B. subtilis. We have
demonstrated the results in Result Page.

## Test of the First Cycle

    In order to verify opSam2 expression in B. subtilis, we
utilized the 6xHis tag fused to N-terminal of opSam2 to perform Western Blot.
Since the opSam2 is downstream of Pgrac, a
lactose induced promoter, we used IPTG to induce its expression in E. coli
first. To enlarge the expression of opSam2, we choose the induction condition as
1mM IPTG and 37˚c according to the literature. Then, we prepared loading sample
at different time after induction. We firstly ran SDS-PAGE and used Coomassie
staining to verify the lysis of bacteria and protein input (Fig. 1.1A). Then to
compare the induction efficiency and detect opSam2 expression, we used BCA
quantification method to measure the concentration of total protein (Fig. 1.1B).
Then after the concentration measurement, we diluted samples into almost the
same concentration, and used such diluted solution to perform western blot using
antibody against His tag (Fig. 1.1C and D). In this case, we can correspond the
band brightness of protein of interest to the concentration directly.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-1.png)

Figure. 1.1 Expression verification of opSam2 in E. coli
and B. subtilis through WB.

     A. Coomassie staining to verify the lysis
efficiency and protein input.

     B. BCA quantification standard curve before WB.

    C. Western Blot in E. coli DH5a. Lane 1-2: Protein
pre-stained marker, Lane 3-8:
lysate pellet induced for 0h (negative control), 1h, 2h, 4,h, 8h, 24h,
respectively.

     D. Western Blot in B. subtilis. Lane 1: Protein
marker, lane 2, 3, 4: lysate
pellet induced for 0h (negative control), 1h, and 4h, respectively.

    As the result shows, we can figure out that the band
between 40 and 55 displays the existence of 6xHis::opSam2 whose molecular weight
can reach 45.80 kDa. As the induction during increases, the band brightness
becomes darker, which means that the expression of 6xHis::opSam2 is increased.
Meanwhile, we may find some shifting phenomenon in rear lanes like lane 6, 7 and
8. This could be caused by the degradation of opSam2 due to the much longer
induction. The similar result can be found in B. subtilis (Fig. 1.1D). The target
band obviously becomes darker when induction time increases. So we can conclude
that the expression of opSam2 in B. subtilis succeeded.

![](https://static.igem.wiki/teams/4144/wiki/engineering/f.png)

Figure 1.2 Enzymatic activity verification of opSam2 in
B. subtilis

     A. ELISA principle depiction

     B. Standard cruve of SAMe quantification ELISA
kit.

     C. Absolute concentration change of SAMe versus
induction time.

     D. Relative concentration change of SAMe versus
induction time.

    Since we have verified the expression of opSam2, we next
measured its catalytic capacity of synthesizing SAMe from ATP and methionine. We
induced opSam2 expression in B. subtilis, then aspirated the supernatant, and
took the pellet to measure the SAMe concentration through SAMe quantification
ELISA kit (Fig. 1.2A). The standard curve demonstrated our result was credible.
In this experiment, we set a control group with empty vector (EV), which did not
contain the recombined protein gene but contain the primary plasmid pHT01K, so
we can use this group to standardize the concentration change of experiment
group (Fig. 1.2C and D).

    We can figure out that the after induction, the
concentration of SAMe definitely
increased compared to EV group, so we can conclude that opSam2 can function
normally in gram-positive bacteria B. subtilis, which means that it would be
greatly possible to work in our final target probiotic B. longum.

## Learn of the First Cycle

     In the first cycle, we can conclude that we have
achieved the engineering success since we have verified the expression and
function of opSam2.

    As we can see, IPTG induction may cause the
toxicity to bacteria and high
strength of induction expression also brings metabolic stress. So we can observe
the shark declines of SAMe concentration in both EV group and pHT-opSam2 group.
So in the final version of design, we tend to put opSam2 downstream of pTet
promoter of Oscillator Device, we will not need toxic IPTG to induce, so the
decline existing in above experiments might not disrupt our primary design.

    However, we still have several to improve. To have
a better understanding of
opSam2 and to improve its activity, we used Alphafold2 and SWISS-Model to
analyze this homodimer’s structure (Fig 1.3). We can easily find that the
C-terminus remains unordered and free, while the N-terminus is hid inside. So in
the first version, in the case where we added the 6xHis tag, the high order
structure may be disrupted or the enzymatic activity may be reduced.

    According to the prediction result, we can figure
out the essential domain
adjacent to SAMe. In the next cycle, we can use directed evolution to improve
its enzymatic ability.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-3.png)

Figure 1.3 Structural analysis of opSam2 through
AlphaFold2 and SWISS-Model.

     A. Sequence coverage of opSam2 when searching in
Alphafold2.

     B. Structure prediction of opSam2 by AlphaFold2,
colored by N -> C.

     C. Structure prediction of opSam2 by SWISS-Model,
colored by confidence.

    D. Structure prediction of opSam2 by SWISS-Model,
using Trace model to display
the substrate SAMe binding site.

## Design of the Second Cycle

    According to what we learned in last DBTL cycle, we
decided to improve enzymatic ability of opSam2 and to optimize the catalytic
condition.

### 1. Directed Evolution of opSam2.

    We can figure out that opSam2 has several ligands, such
as magnesium ion, potassium ion, PPK, adenosine and SAM. The binding of such
ion’s residues can be found according to the structural prediction model. For
example, the SAMe is adjacent to 20 residues within 4å. We decided to collect
the relative residue sites and perform point mutation to construct a mutation
library (Fig 1.4A). Through SAMe production ability selection, we can gain a
m-opSam2 with a better enzymatic ability.

### 2. Tag Addition Optimization

     We found that the C-terminus remains unordered and
free, while the N-terminus is hid inside. So in the first version, in the case
where we added the 6xHis tag, the high order structure may be disrupted or the
enzymatic activity may be reduced. In this cycle, we will add the tag into the
free C-terminus which might affect the folding and structure of opSam2 (Fig
1.4B).

### 3. Condition optimization of SAMe production

     Since the reagents of SAMe synthesis reaction are Met
and ATP, and the binding to magnesium and potassium ions are demonstrated, we
will performed orthogonal experiment to explore the best SAMe production
condition for such molecules when induction (Fig 1.4C).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-4.png)

Fig 1.4 Improved design of the Second DBTL cycle of SAMe
production.

    A. All residues adjacent to ligands (Down) and
several sites marked red to be
mutated (Up).

    B. Fused terminus change of His tag

    C. Orthogonal experiment table for exploration of
production condition of opSam2

## Build of the Second Cycle

    We have sent required fragments to synthesize in Tsingke
Biology. However, due to the lack of time, we don’t have enough time to do more
experiments to build in second DBTL cycle.

# DBTL cycle of SAMe Secretion

## Design of the First Cycle

### Purpose

    To secrete the natural depressant SAMe, we need a SAMe
transporter to localize in the membrane of our engineered bacteria. However, we
didn’t find such transporter in prokaryotic cells since they almost don’t have
any organelles. In eukaryotic cells, we search SAMe transporter in yeast, and
found Pet8p.

     Pet8p is one transporter of mitochondrial carrier
protein family of
Saccharomyces cerevisiae. It is also called Sam5p, since it functions to
transport S-adenosyl methionine (SAMe). We planed to express it in gram-positive
model bacteria Bacillus subtilis firstly and then in Bifidobacterium longum, and
then to verify its transportation function.However, we need to consider lots of
problems since we plan to insert such eukaryotic multi-span transmembrane
protein into bacterial plasma membrane.

     Firstly, some researches have reported that they
had successfully expressed
Pet8p in E. coli [2].

### Design

    In our first-version design, to ensure the expression
efficiency in Bacillus subtilis (B. subtilis), we use a codon optimization tool
to optimize its coding sequence, and the optimized coding sequence is named as
6xHis::opMistic::opPet8p::sfGFP (Fig 1.5A). Then to verify its expression, we
added a 6xHis tag to its N-terminus. Since it is a transmembrane protein, we
have to observe its membrane localization, so we fused a sfGFP into its
C-terminus. Meanwhile, to facilitate the membrane localization, we insert a
short peptide Mistic derived from B. subtilis to the N-terminus, which is
reported to help transmembrane protein target at bacterial plasma membrane. With
a high-magnification fluorescent microscope, we can figure out whether the
recombined proteins are enriched at the “peripheral” compared to cytosol.

## Build of the First Cycle

    Then we constructed such part in a shuttle vector pBE2
for cloning in Escherichia coli DH5α, and for expression in B. subtilis. We
firstly inserted a P43-promoted XylR fragment into the plasmid with PstI/KpnI
double cut, and then we inserted another fragment Pxyl-promoted opPet8p into the
first recombined plasmid using KpnI/EcoRI double cut. XylR expressed
onstitutively and promoter Pxyl constructed a xylose-inducible device to
activate the expression of opPet8p. The XylR fragment was PCR from genome of B.
subtilis, and the other fragments are synthesized. pBE-XylR-opPet8p (10046bp)
has been constructed and transferred into E. coli and B. subtilis (Fig 1.5 B, C,
D, E).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-5.png)

Figure 1.5 Successful cloning result of pBE-XylR-opPet8p in E. coli and B.
subtilis.

     A. Graph description of plasmid construction from
pBE2 to pBE2-XylR, and then to
pBE2-XylR-opPetp.

     B. Enzyme cut of XylR fragment and pBE2. Lane1:
GL DNA 5000 marker, Lane2, 4:
pBE2, Lane3, 5: pBE cut by PstI/KpnI, Lane6: XylR PCR fragment, Lane7: XylR cut
by PstI/KpnI, Lane 8: GL DNA 2000 marker.

     C. Enzyme cut of pBE-XylR and PCR Pxyl-opPet8p.
Lane1: pBE-XylR, Lane2, 3:
pBE-XylR cut by KpnI/EcoRII, Lane4, 5, 6: Pxyl-opPet8p PCR fragment, Lane7: GL
DNA 2000 marker.

     D. Colony PCR of pBE-XylR-opPet8p in B. subtilis.

     E. Partial opPet8p sequencing result in E. coli.

     F. Partial opPet8p sequencing result in B.
subtilis.

## Test of the First Cycle

     And the expression of E. coli and B. subtilis are
verified by green fluorescence (Fig 1.6A, B), fluorescent image (Fig 1.6C),
Western Blot (Fig 1.6D, E, F). We can concluded that we have successfully
expressed recombined protein 6xHis::Mistic::opPet8p::sfGFP, since the N-terminus
tag and C-terminus tag are both verified (Fig 1.6G).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-6.png)
![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-7.png)
![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-8.png)

Figure. 1.6 Expression verification of opPet8p in E. coli and B. subtilis.

     A. Plate spread with E. coli containing plasmid
pBE-Pxyl::6xHis::opMistic::opPet8p::sfGFP can emit green fluorescence when
excited by ultraviolet light (UV).

     B. Visual green shows the expression of opPet8p
in E. coli after 1%xylose
induction for different periods of time.

     C. Fluorescent image of E. coli and B. subtilis
with pBE-XylR-opPet8p::sfGFP
after induction.

     D. BCA quantification standard curve before WB.

     E. Western blot of opPet8p using anti-His-HRP.
The front 5 lanes show the
induction expression of assumed fusion protein in E. coli, and the rear 5 lanes
show those in B .subtilis, induced for 0h, 1h, 2h, 4h and 8h, respectively.

     F. Western blot of opPet8p using anti-GFP. The
front 5 lanes show the induction
expression of assumed fusion protein in E. coli, and the rear 5 lanes show those
in B .subtilis, induced for 0h, 1h, 2h, 4h and 8h, respectively.

     G. Graph demonstrates that we used anti-His
antibody to target the N-terminus of
opPet8p and uesd anti-GFP antibody to target the C-terminus of opPet8p as well
in WB, whose results are showed in E and F above.

    Meanwhile, we also verified the membrane localization of
opPet8p (Fig 1.7). According to the distribution of fluorescent signal, we can
conclude that the recombined proteins can target to the bacterial membrane.
However, if the expression rate is too high to conduct the proper folding so
that they might form the particle-like inclusion body, they might gather
together to precipitate. To better understand the membrane integration of our
recombined protein opMistic::opPet8p::sfGFP, we used a prediction tool Phobius
to predict. The results are showed below (Fig. 1.7A).

## Learn of the First Cycle

     As we can see in Fig 1.7A, the addition of Mistic can
result in the addition of transmembrane segment which can facilitate the
membrane insertion of opPet8p, while the sfGFP does the opposite thing. So after
using sfGFP to verify its expression and membrane localization, if we want to
improve its membrane localization efficiency, we need to replace the sfGFP with
other tag, like some small particular fluorescent group. According to the
integration pattern prediction, we draw the model graph to display the
difference (Fig 1.8A).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-9.png)
![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-10.png)

Figure 1.7 Membrane localization verification of opPet8p in E. coli and B.
subtilis.

     A. Membrane integration prediction of primary
opPet8p (Left) and recombined
fusion protein opMistic::opPet8p::sfGFP (Right)

    B. Fluorescent image of Bacillus subtilis induced
by different concentration of
xylose and a constitutive expression strain.

## Design of the Second Cycle

     In above results, we found that there are some particles
enriched with fluorescent signals in bacteria, except for the average
distribution. We supposed that the formation of the particle could be due to
wrong protein folding. So we performed membrane insertion prediction of coding
sequence of opPet8p and the recombined protein, and we found that the addition
of sfGFP induce a large hydrophilic segment which is unfriendly to the membrane
insertion (Fig 1.7A). It could be possible that sfGFP disrupts the folding of
opPet8p, so to eliminate such particles, we designed a new part in which a
linker (Gly–Gly–Gly–Gly–Ser) was inserted between the opPet8p and sfGFP (Fig.
1.8A, B). With such a flexible linker, sfGFP is supposed to keep a distance
from the opPet8p so that the folding of opPet8p won’t be less disrupted and the
membrane insertion could be better.

![](https://static.igem.wiki/teams/4144/wiki/engineering/66.jpg)

Figure 1.8 Graph description of different recombined proteins

## Build of the Second Cycle

    We have sent required fragments to synthesize in Tsingke
Biology.

## Test of the Second Cycle

    Then we performed the same operation as the last cycle to
observe the membrane localization of 6xHis::opMistic::opPet8p::linker::sfGFP, and
set the old strain who expressed opPet8p without fusing with linker. We can
obviously found that strains expressing opPet8p fused with linker have absolutely
fewer particle-like inclusion bodies in cytosol compared with strains expressing
opPet8p without linker (Fig 1.9). So we can conclude that the linker can work to
facilitate the folding of proteins and reduce the formation of inclusion body.
However, as the large copy number of vector and decrease of protein precipitation,
large amount of proteins are distributed in cells whose fluorescent signal is too
large to observe the membrane localization. Due to the lack of time, we haven’t
explored more proper condition to observe, but we are optimistic to observe its
membrane localization after decreasing the induction time.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-aaa.png)

Figure 1.9 Comparison of fluorescent signals in strains expressing opPet8p fused
with linker and without linker.

# DBTL cycle of Oscillator model

## Design of the First Cycle

    During the time when we assembled our project, we decided
that it would be a great idea to simulate the normal sicario of drug uptake, to
be more precise, we want our engineered bacteria to give off drug at due time
intervals rather than a constant secretion that would last the whole time, in
hope that this can achieve fluctuation of the serum drug concentration to aid in
the avoidance of adverse effects led to by elevated serum drug concentration
while still maintaining a minimum concentration that can have a curing effect.

    To simulate this sicario of drug uptake, we
decided to introduce an oscillator
that can be exploited to control the production, and subsequent secretion of the
drug SAMe, by controlling the expression of the enzyme sam5p that is responsible
for the conversion of Met into SAMe and which is the only step in the pathway
leading to the synthesis of this compound.

     There have already been many mature solutions in
E. coli for constructing an
oscillator, but when it comes to Gram positive bacteria, we found none that is
sure to work. So, after looking up previous publication and works done by
previous iGEM teams, we decided on changing an existing design that has already
shown to be working robustly in E. coli, namely, the Repressilator. Published in
2001 on Nature, the Repressilator consists of three transcription factors and
their corresponding promoters.

![](https://static.igem.wiki/teams/4144/wiki/engineering/233.png)

Figure 2.1 A graphic representation of the Repressilator

## Build of the First Cycle

    And in the experimental design of our group, we focused
on determining whether these three components can function as expected in B.
subtilis as has been characterized in E. coli, respectively.

     In order to determine the function of these
transcription factors, we cloned
these transcription factors and their promoters onto shuttle vectors that can
self-replicate and express in B. subtilis as well as in E. coli.

## Test of the First Cycle

    And we intended on using microplate reader to obtain data
on the fluorescence intensity of GFP, whether is driven by the promoter, or is
combined with the expression of the corresponding transcription factor.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-13.png)

Figure 2.2 The plasmid we are planning on constructing

## Learn of the First Cycle

    However, due to failure in the process of
molecular
cloning, we didn’t obtain these plasmids, and as a result, no further
experiments were carried out.

    But nevertheless, we do believe results strongly
in favor of our hypothesis will
result due to the fact that the three transcription factors and there
corresponding promoters are thoroughly studied, and the fact that there has been
publication confirming the success of protein expression in the chassis.

    And taking the possibility of failure into
account, we designed another set of
experiments from the beginning, and we think that this can be taken as the
second cycle of the DBTL cycle

## Design of the Second Cycle

     Another thing we would like to try is to
reconstruct the intact Repressilator into our engineered bacteria, and see if it
works as expected. In this way, we can verify the three component that
constitute the Repressilator in one experiment.

## Build of the Second Cycle

     As mentioned, we are planning to reconstruct the
intact Repressilator in B. subtilis, and we first tried to reconstruct it in E.
coli.

    So, we transformed the Repressilator plasmid into
E. coli, and found a reporter
plasmid from iGEM repository that constitute an GFP under the expression of tetR
promoter.

## Test of the Second Cycle

     After co-transformation, we verified the result
via colony PCR, and after synchronizing the bacteria by adding and removing
IPTG, we measured the fluorescence intensity using microplate reader. But the
result showed no significant oscillation.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-ddd.png)

Figure 2.3 Results of colony PCR

## Learn of the Second Cycle

     We think that this is due to some bacteria not
fully synchronized, and due to the fact that the bacteria will gradually become
out of pace in respect of each other. So, we planned on measuring the
fluorescence intensity of single bacteria.

## Design of the Third Cycle

     As mentioned, we planned on measuring the
fluorescence of the single bacteria. So, we first simply planned on videoing the
same bacteria without removing it from the microscope.

    Before we took the video, we took a photo of the bacteria, to see if we can observe fluorescence, and the result showed that there are indeed fluorescence in the bacteria co-transformed the reporter plasmid and the Repressilator plasmid.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-eee.png)

Figure 2.4 Photo of bacteria that contains both the reporter
plasmid and the Repressilator plasmid taken under a fluorescence micrpscope.

## Build of the Third Cycle

     The plasmid and the bacteria strain we used in
this cycle were the same as the last, but the modifications we made is in the
protocol that we used to photograph the bacteria.

     We learnt that we can cover the slide we used to
image with a thin slice of
solid LB medium, in this way, the bacteria can survive for a relative period,
while for the whole duration we were able to image it.

## Test of the Third Cycle

     We set the system up as described, consisting of
the fluorescence microscope that we used for imaging, the special glass slide we
used for supporting the bacteria, and the bacteria itself.

## Learn of the Third Cycle

     But when doing imaging, we found some big issues.
The biggest one being photobleaching, and there are also minor ones such as
difficulty in image processing. So, we started searching for other ways around
this problem. And fortunately, we found one that worked, and the result we
obtained made its way into the result section of our project.

## Design of the Fourth Cycle

     The way around the problem is by photographing the
same region of the slide at given time intervals. So that in this way, we do not
have to place the bacteria under the excitation light for the whole duration,
and instead of processing a video that can last for hours, we only need to
process a few photos.

## Build of the Fourth Cycle

     We set the imaging system up the same way as in
the last cycle, but this time we didn’t take video, but instead, took photo once
every 10 minutes.

## Test of the Fourth Cycle

     Using the procedure described above, we took various photos, as shown down below.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-14.png)

Figure 2.5 Photo of the same region of the slide taken at an
time interval of 10 minutes.

     Then, we measured the light intensity of the whole photo, to serve as the background of the fluorescence intensity (making a hypothesis that the light intensity of the bacteria with fluorescence is so much less than the background that the light intensity of the bacteria can be neglected, and as can be seen below, this hypothesis is correct)

![](https://static.igem.wiki/teams/4144/wiki/engineering/lllll.png)

Figure 2.6 The measurement of the light intensity of the
whole photo, to see the background.

    The first box indicates the average light intensity per pixel
of the whole photo, and the second box indicates the number of pixels of the image.
The whole light intensity can be obtained by multiplying the two numbers together.

    Then, we cycled out the same bacteria of each photo, making sure that they are all
at the same region in each photograph, numbered them 1-10, as shown in Fig 2.7a, and
measured the light intensity in each of the small regions, the result is represented
in Fig 2.7b, and the data obtained is shown below in the table.

![](https://static.igem.wiki/teams/4144/wiki/engineering/fjhjfjgf.png)

Figure 2.7 The numbering of each bacterium, and the
measurement of the light intensity in regions around each bacterium.

![](	https://static.igem.wiki/teams/4144/wiki/engineering/dfghdfh.png)

Total number of pixels around each bacterium and the whole picture.

![](https://static.igem.wiki/teams/4144/wiki/engineering/shtrhtr.png)

Average light intensity around each bacterium and the whole picture.

![](https://static.igem.wiki/teams/4144/wiki/engineering/dfhtrdht.png)

Total light intensity around each bacterium and the whole
picture without background.
Figure 2.8 Analysis workflow of light intensity around each bacterium and the whole
picture.

    To visualize these data, we imported them into Prism, and performed non-linear regression using sine waves as the model, to find out the expected oscillation cycle of the Repressilator plasmid. The result is shown below.

![](https://static.igem.wiki/teams/4144/wiki/engineering/dfghdyhrdht.png)
![](https://static.igem.wiki/teams/4144/wiki/engineering/fghrthtrhthsthrf.png)

Figure 2.9 The analyzed result of the oscillation cycle of the
co-transformed bacteria.

    But as can be felt from the regression curve, the data point
at 30 minutes violated the whole trend, and the goodness of fit is quite low. So, we
decided to delete this data point, and do the regression once more.

![](https://static.igem.wiki/teams/4144/wiki/engineering/dgerrt.png)
![](https://static.igem.wiki/teams/4144/wiki/engineering/srthtrhth.png)

Figure 2.10 The analyzed result of the oscillation cycle of
the co-transformed bacteria after deleting the data obtained from the photo taken at
30 minutes.

    And as can be seen, the result is much better, and the
oscillation cycle settled
around 60 minutes, which agrees with the result shown in the original article.

## Learn of the Fourth Cycle

     Through these experiments, we found the mostly
likely oscillation cycle of the Repressilator, and we think that this can wrap
up the experiments we need to do in E. coli. Then, we think that its time to
move on to the verification of the function of the Repressilator plasmid in B.
subtilis.

## Design of the Fifth Cycle

     In this cycle, we planned to reconstruct the
Repressilator plasmid in B. subtilis.

     The protocol we decide to follow and the plasmid
we decided to transform into
the bacteria strain is the same as the last cycle.

## Build of the Fifth Cycle

     Due to the lack of time, we didn’t manage to get
into this part of the experiments, but we do think that positive result will
come into being if we get our hands on this.

# DBTL cycle of selection plasmid construction

    We set up the Directed Evolution module to develop our
LacI protein, making it only responsive to high lactose in a way gut environment
would hardly contain. By lifting the threshold value of lactose toleration, our
engineered bacteria can stably produce periodical SAMe, achieving better
therapeutic effect. According to the previous research, we chose three
residues as our mutagenesis positions - I79, F161 and L296 - since they have
been proved to function in substance binding.

## Design of the First Cycle

    At first, we planned to utilize fluorescence intensity as
result instruction, using EGFP gene controlled by Lac promoter as reporter (Fig
3.1). However, as we are selecting LacI protein based on one single
quantitative, screening fluorescence could be quite complicated and inaccurate.
So, we developed our design and added two more genes – sacB and KanR – so that
we can conduct the evolution procedure on rounds of selective culture of
engineered bacteria. The positive selector sacB eliminate variants that couldn’t
tolerate present level of lactose, while negative selector making sure the LacI
protein still obtain its function to release from the promoter. More details are
illustrated in Design of the Directed Evolution Module.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-bbb.png)

Figure 3.1 Diagram of plasmid construction. A: Initial design
of selection element; B: Present design of selection element;

## Build of the First Cycle

     During the process, we first used overlap extension PCR
to construct the selection element including mRFP1, sacB and KanR into one giant
fragment. By using flanking primers, we successfully observed the ideal bands at
first. However, the products turned out to be quite unstable and would easily
break into two after a second amplification (Fig 3.2A). Also, the whole PCR
process is quite complicated and time-consuming.

## Test of the First Cycle

    We improved our protocol by dividing the construction into
two steps, connecting 2-3 fragments at a time, using only Gibson Assembly to
construct the entire plasmid. This alteration significantly increased the successful
rate. The final plasmid then underwent MEGAWHOP to build mutagenesis library and was
transformed into BL21 and carried on for further selection. DpnI digestion contrast
experiments proves the mutagenesis being effective (Fig 3.2B).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-ccc.png)

Figure 3.2 Plasmid and mutagenesis library construction. A:
Overlap extension PCR product break in the second amplification; B-C, plasmid
transformation results after DpnI digestion. B: MEGAWHOP without megaprimer; C:
Normal MEGAWHOP;

## Learn of the First Cycle

    In the first cycle we successfully achieved overlap
extension PCR of adjacent fragment. However, perhaps it’s because the fragments
are rather too long (sacB-1437bp) or too short (terminator+Pc-164bp), the
product isn’t quite stable.

    By concluding the fragments into occlusive plasmid and deriving target ones through antibiotic selection, the percentage of desired plasmid has risen, even though the fragment loss still occasionally occurred. Using transformation to derive the desired product is relatively time-consuming, while the results are also more promising.

# DBTL cycle of variant selection

## Design of the First Cycle

    If the LacI responds to certain level of lactose and release
from the promoter, sacB will be expressed and cause bacterial death. By applying
gradually lifted lactose, we were able to establish rounds of selection based on
sucrose or kanamycin plates until the ideal LacI proteins were derived.

## Build of the First Cycle

    We first built up solid medium with IPTG gradient
addition. Containing chloramphenicol, sucrose, and IPTG, theoretically only
desired mutants can grow on such medium. The reason why we chose IPTG is because
natural metabolism of lactose would result in the inconsistency of concentration
during the experiment, and its product is immeasurable. Since IPTG is more
stable and has been widely used in lactose-related research, we used IPTG as an
alternative of lactose to maintain a reliable result.

## Test of the First Cycle

    The first round of selection was carried out on 2%
sucrose plate with IPTG at 0.1mM and 0.2mM respectively, by spreading bacteria
culture in different collections of plates. However, it turned out to have bare
difference between different concentrations of IPTG and fail to separate the
improved variants (Fig 3.3A). We wondered whether the sucrose content wasn’t
high enough, so we lifted the IPTG and sucrose concentration at the same time in
the next round, setting IPTG gradient of 0.2, 0.4, 0.6, 0.8, 1mM and sucrose
content of 8%. Unfortunately, we still failed to observe the expected difference
(Fig 3.3B).

    We instead stepped forward to explore the role of IPTG
induction duration played
in results. Adding gradient of IPTG when OD600 equals to 0.6, we sampled each
group every hour, with the duration reaching to around 6 hours. However, the
results are still unappreciated, with bare difference observed (Fig 3.3C),
either among IPTG levels or among induction duration. Our selection based on
sacB ended in failure.

![](https://static.igem.wiki/teams/4144/wiki/engineering/5.png)

Figure 3.3 Series of exploration toward sacB.

   A: Comparison between different IPTG concentration on 2%
sucrose plate.

   B: Comparison between different IPTG concentration on 8%
sucrose plate.

   C: Comparison between different IPTG concentration and
inducing time on 8%
sucrose plate.

## Learn of the First Cycle

    After investigation, we found that the inefficiency of
sacB is quite common, influenced by many factors such as sucrose content, media
component and even bacteria species. Our followed-up experiment also revealed
the timing to apply IPTG induction or solid media spreading being vitally
important as well. When spreading the culture at OD600=0.2 approximately, that
is when turbidity can just be observed, the killing effect from sacB expression
is remarkably significant (Fig 3.4). On regular antibiotic plate with 15ug/ml
chloramphenicol transformed bacteria grew into lawn, while on Cml plate with 8%
sucrose and 0.8mM IPTG no colony is witnessed. In previous selection, we rather
added the inducer IPTG at OD600=0.8 as in regular protein experiments or spread
the culture when it has grown overnight. This may be the reason why the first
round of our selection failed.

    However, this discovery appeared in late stage of
our experiments when a new
round of selection is not available. Before it we had to change our mind and
establish the selection in a new path. This is how the next round of selection
based on KanR was put forward.

![](https://static.igem.wiki/teams/4144/wiki/engineering/7.png)

Figure 3.4 Contrast between 8% sucrose and non-sucrose
plate.

## Design of the Second Cycle

    Even though KanR gene was first added to exclude the
possibility that mutant LacI cannot release from the promoter, however, since sacB
selection didn’t work out, we decided to utilize KanR to carry out the next round of
selection. Due to its property, the expression of KanR survives the bacteria. Thus,
we need to compare one mutant’s behavior in multiple inducer concentration to make
the conclusion. If the mutant can only growth on high-IPTG plates rather than lower
ones, then it’s still promising to say that the LacI can only respond to high-level
lactose.

## Build of the Second Cycle

    We set up IPTG gradient in kanamycin plates of 0, 0.1,
0.5 and1 mM, and each mutant culture needs to spread plate in such combination
group. In total 30 colonies were cultured and undergo selection.

## Test of the Second Cycle

    Even though the procedure is complicated, the results
turn out to be quite satisfying. Different mutants showed different tolerance to
IPTG, which once again illustrates the successful construction of our lacI
mutagenesis library (Fig 3.5). Among them, we derived the desired variant that
only responds to high-level of IPTG (0.5mM) with little growth at low
concentration (Fig 3.5). More surprising, a few growths also appeared on 0.8mM
IPTG plate, suggesting that the mutant still contains its ability to release
from the promoter and thus can be utilized in oscillator (Fig 3.5).

![](https://static.igem.wiki/teams/4144/wiki/engineering/8.png)

Figure 3.5 Selection results.

     A: Control group 1. Original strain and random
mutant are applied on empty
antibiotic plate with IPTG contrast.

    B: Control group 2. Original strain is applied on
Kan+ plate with IPTG gradient.

    C: Desired mutant, Strain #29. No colony growth is
observed at 0 and 0.1mM IPTG,
suggesting that the LacI can tolerate high-level IPTG.

    D: Strain #27. The mutagenesis causes its binding
to lactose (IPTG) to be
easier, and lawn are witnessed at all concentration, even without IPTG
existence. Its behavior is quite familiar to performance on non-antibiotic
medium.

    E: Strain #9. The mutagenesis slightly reduces the
leakage of the promoter,
however, barely difference occurred between different inductor (IPTG)
concentration.

    F: Strain #24. Little up-regulation of the
promoter takes place as IPTG
concentration lifting. Its response to low-level inductor (IPTG) is still
relatively high.

    G: Strain #2. Few colonies occurs at 0mM IPTG and
no growth at higher level.

    H: Strain #6. No growth at all. The sensation to
IPTG is totally lost.

    I: Strain #13. Expression is activated only at
0.1mM IPTG.

    J: Strain #14. Few colony are witness, expect one
at 0.5mM IPTG, suggesting its
affinity to IPTG has greatly dropped.

    In order to evaluate the behaviors of different
mutants for better judgement, we
designed an algorithm to access the IPTG responsive ability of the strains. We
use 1-7 to measure the intensity of the colonies on each plate, and integrated
them into a heatmap for visualization (Fig 3.6). Our goal is to select a LacI
variant that cannot sense the low concentration IPTG and at the same time that
normally functions at high concentration of IPTG. Thus, reflecting on different
colors, the ideal strain is supposed to have lighter color between darker ones
at high range of IPTG. Comparing the data, Strain #29 is exactly the one that we
are looking for (Fig 3.5 C). Meanwhile, a few growths under 0.8mM IPTG
guarantees that the mutant LacI still keeps the ability to release from promoter
and it could function normally in our oscillatior - “Repressilator”. Selected
mutant also appeared to solve the leakage problem that original LacI contains,
showing a more outstanding performance.

![](https://static.igem.wiki/teams/4144/wiki/engineering/45.png)

Figure 3.6 Visualization of arithmetic LacI evaluation
result. The triangle points out the desired variant strain #29.

## Learn of the Second Cycle

    In the second cycle we managed to achieve our goal of
deriving improved LacI mutant using KanR.
The drawback and advantage of this plan are all distinct. The biggest drawback
is the great amount of works because the experiment requires cultures of single
colony and each one requires groups of spreading. However, this also keeps its
consistency in specificity, with no need to re-culture the post-selective
colony.

     Although we successfully separated the ideal
variant, the experiment still has
several faultiness and could be improved in the future. For example, the
concentration gradient can be divided into more interval, which can identify a
more detailed section that LacI functions in. Besides, such actions with high
labor and time cost can be solved depending on computer using high throughput
equipment, such as micro-fluidic chip or microplate reader. We are looking
forward to such attempt afterwards.

     During the experiment, the leakage of the lac
promoter is still an unignorable
problem (Fig 3.5B). Though the selected variant seems to have improved this
situation, some improvements to solve this problem in the first place are still
highly appreciated and will undoubtedly benefit in selection efficiency. We
believe that methods such as adding a sponge could better improve the
performance of the plasmid and thus of the selection process.

# DBTL cycle of Safety Switch

## Design of the First Cycle

    In our design, we use chemical switch for active
termination. CinR is an autoinducer binding protein found in Rhizobium
leguminosarumis. In complex with O3-C14 HSL, which is synthesized by the
inducer CinI, CinR protein activates transcription of genes downstream the
pCin promoter. Thus, when the bacteria accept this chemical signal and
activate the pCin promoter, the downstream gene of holin will express. Holin
is a protein that will make holes on cell membrane and cause death of
bacteria. Next, we use temperature switch for leakage protection. TlpA36 is
a temperature sensitive protein which is capable of repressing the pTlpA
promoter under temperature of below 36 degrees Celsius, thus repress the
expression of PenI repressor. As a result, holin will express below 36
degrees Celsius and cause the death of bacteria (Fig 4.1).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-18.png)

Figure 4.1: design of safety switch.

## Build of the First Cycle

    We used restriction enzyme and DNA ligase to
construct our recombined plasmids (Fig 4.2). And we transformed the plasmids
into E. coli and B. subtilis for examination.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-19.png)

Figure 4.2: The result of plasmid construction and
sequencing.

    A, B: Construction and sequencing of chemical
switch.

     C, D: Construction and sequencing of
temperature switch. )

## Test of the First Cycle

    We chose EGFP as its downstream reporter. Firstly, we
tested the function of the plasmids in E. coli DH5α. For chemical switch, we
used recombined E. coli BL21 to produce autoinducer and extract the
supernate. We tested the function of chemical switch by inducing the
expression of EGFP with the supernate. For temperature switch, we induced it
under 25℃. As a result, in E. coli the fluorescence of temperature switch is
observed under 25℃, but unfortunately, no fluorescence was observed among
different groups of chemical switch. Also, no significant changes in
fluorescence were observed in both groups in B. subtilis (Fig 4.3).

    Lastly, we tested the function of PBSX holin
by measure the cleavage of the
E.coli. In this construction, the expression of holin is induced by IPTG. We
found that after inducing, the bacteria were killed or inhibited to some
extent though not fully (Fig 4.4).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-20.png)

Figure 4.3: The fluorescence in E.coli with chemical
or temperature switch and the fluorescence in B. subtilis with chemical or
temperature switch.

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-21.png)

Figure 4.4: Inducing the expression of PBSX holin in
E. coli.

## Learn of the First Cycle

    Thus, we still need to improve our design in the
future to make it a complete safety switch. Though by many research and
proof, we have confirmed that most basic parts in our design should be able
to work in B. subtilis, further examination of ever single part is still
needed. For we met difficulties when tested the function of the whole
composite parts. For example, TlpA36 promoter should be tested in B.
subtilis whether it have its normal function. We simplify the construct and
can use these plasmids to test the function of TlpA36 and PenI with their
promoter respectively. Due to the lack of time, we did not carry out the
next round of construction. But we planned to make them more available based
on the lessons of previous experiments.

## Design of the Second Cycle

    What is more, as soon as we find the problems of that
single part and improve the switch part, we should bind the composite part
and holin together to achieve the function of the safety switch. By now,
they are designed as follows: (Fig 4.5).

![](https://static.igem.wiki/teams/4144/wiki/engineering/engineering-22.png)

Figure 4.5: Construction of composite part with
holin.

    Although to fully achieve these composite parts is
challenging, we still believe that some methods can be used to direct our
design and increase the success rate. We still hope to try to construct
these safety switch.

# DBTL cycle of Hardware

## Design of the First Cycle

     For the hardware, we have accomplished the systematical
work of turning it from design into reality. We wanted to explore the specific
parameters of the capsule so that the drug could be degraded in the intestine.
Now we have completed part of the gut simulation.

## Build of the First Cycle

     At the beginning of the experiment, we tried to observe
the dissolution of various substances in a stationary solution of different pH
values, but this was quite different from the real human environment, so we
modified and designed a simple device to achieve intestinal peristalsis and
liquid flow by manual pushing.

![](https://static.igem.wiki/teams/4144/wiki/engineering/99.png)

Figure 5.1

## Test of the First Cycle

     However, we found that various chemicals took a
relatively long time to dissolve, and anthropogenic vibration was not a good
idea. To make matters worse, human operation could easily lead to various
problems such as water leakage or instrument damage.

## Learn of the First Cycle

     Therefore, we needed to design an automatic instrument
that could operate stably for a long time.

## Design of the Second Cycle

     Considering that the dissolution time took about 2 hours, we
modified the device
to
include a motor and a microfluid pump to make the operation of the equipment
more
convenient.

## Build of the Second Cycle

     We repurchased the microfluid pump and motor and
assembled it together.

![](https://static.igem.wiki/teams/4144/wiki/engineering/87.png)

Figure 5.2

     The coarse catheter was so short that it caused the
liquid to flow differently from the real situation, so we lengthened the coarse
catheter. In addition, in order to enable the coarse catheter to be disassembled
repeatedly, we used thermoplastic hoses, which would gradually shrink after
heating, to prevent the deformation of the coarse catheter caused by repeated
disassembly, which would lead to water leakage.

![](https://static.igem.wiki/teams/4144/wiki/engineering/a1.png)

Figure 5.3

## Test of the Second Cycle

     After that, we conducted experiments to explore the
solubility of carboxymethyl cellulose particles of different radii in different
solutions, and the experimental results are as follows.

![](https://static.igem.wiki/teams/4144/wiki/engineering/54.png)

## Learn of the Second Cycle

     In the end, we found that the granular material is made
of carboxymethyl cellulose and the radius should be less than 0.3cm to ensure
that it completely dissolves in the intestine and can stay in the stomach for at
least 2 hours.

     In this DBTL cycle, we used the last device to conduct
the
experiment and
obtained the dissolution rate of different substances. According to the
experimental results and the residence time of the capsule in various parts of
the human body, we designed the parameters of the capsule. Although such a set
of parameters may not be completely in line with the human environment, it
basically provides a reasonable search range, and further confirmation of
parameters requires human clinical trials.

![](https://static.igem.wiki/teams/4144/wiki/engineering/a3.png)

Figure 5.4

## Reference

     [1] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi,
Jennifer R. Keeffe, Pamela J. Bjorkman, Design and characterization of
structured protein linkers with differing flexibilities, Protein Engineering,
Design and Selection, Volume 27, Issue 10, October 2014, Pages 325–330,
https://doi.org/10.1093/protein/gzu043

     [2]Elowitz, M., Leibler, S. A synthetic oscillatory network of transcriptional
regulators. Nature 403, 335–338 (2000). https://doi.org/10.1038/35002125

     [3] Wu J, Jiang P, Chen W, Xiong D, Huang L, Jia J, Chen Y, Jin JM, Tang SY.
Design and application of a lactulose biosensor. Sci Rep. 2017 Apr 7;7:45994.
doi: 10.1038/srep45994. PMID: 28387245; PMCID: PMC5384092.

     [4] Piraner, D., Abedi, M., Moser, B., Lee-Gosselin, A., & Shapiro, M. Tunable
thermal bioswitches for in vivo control of microbial therapeutics. Nature
Chemical Biology, 2016, 13(1), 75-80. http://dx.doi.org/10.1038/nchembio.2233

     [5] Calatrava-Morales, N.; McIntosh, M.; Soto, M.J. Regulation Mediated by
N-Acyl Homoserine Lactone Quorum Sensing Signals in the Rhizobium-Legume
Symbiosis. Genes 2018, 9, 263. https://doi.org/10.3390/genes9050263

     [6] Susanne Krogh, Steen T. Jørgensen, Kevin M. Devine. Lysis Genes of the
Bacillus subtilisDefective Prophage PBSX. Journal of Bacteriology. 1998, Vol.
180, No. 8.
https://doi.org/10.1128/JB.180.8.2110-2117.1998

     [7] Bowtle W J. Materials, process, and manufacturing considerations for
lipid-based hard-capsule formats[M]//Oral Lipid-Based Formulations. CRC Press,
2007: 101-128.

     [8] Duke G E. Alimentary canal: secretion and digestion, special digestive
functions, and absorption[M]//Avian physiology. Springer, New Y

[Measurement](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27measurement%27%29%20%7D%7D)[Results](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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Results

2022 WHU-China

# Results

# Production Module

## Goal

    1. To express exogenous SAMe synthetase opSam2 and SAMe
transporter opPet8p in Bacillus subtilis.

    2. To prove that opSam2 can synthesize S-adenosyl
methionine (SAMe) and that
opPet8p can transport SAMe, respectively in Bacillus subtilis.
.

## Achievement

    1. The expression shuttle vectors of opSam2 and opPet8p
were successfully constructed and transferred into Escherichia coli (E. coli)
DH5α and Bacillus subtilis (B. subtilis) 168.

    2. Successful Expression of opSam2 and opPet8p in
both E. coli DH5α and B.
subtilis 168.

    3. Successful membrane localization verification
of opPet8p in B. subtilis 168

    4. Functional verification of opSam2 in B.
subtilis 168.

## Experiment

### Construction of expression vector of pHT-opSam2 and transformation

    In order to ensure that our exogenous protein opSam2 can
be expressed in gram-positive B. subtilis 168, and deeper in Bifidobacterium
longum (B. longum), we used a E. coli - B. subtilis shuttle vector pHT01K
(8253bp, with Kan resistance), which is derived from original pHT01 (with Cm
resistance). This plasmid gained from our PI Xie’s lab, has an intrinsic
lactose-inducible promoter Pgrac, and this plasmid can express Pgrac repressor
LacI, so the expression of protein of interest inserted can be induced by IPTG,
a lactose analogue. We inserted our synthetic fragment 6xHis::opSam2 into MCS of
pHT01K, using enzyme cut and ligation to construct the recombined plasmid
pHT-opSam2 (9506bp). After transformation, we performed colony PCR and extracted
plasmid to run agarose electrophoresis. The correct band was observed (Fig.
1.1A, B, C and D), and the sequencing results verified the successful insertion
in B. subtilis as well (Fig. 1.1E).

![](https://static.igem.wiki/teams/4144/wiki/result30/result301.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result302.png)

### Construction of expression vector of pBE-XylR-opPet8p and transformation

    In order to ensure that our exogenous protein opPet8p can
be expressed in gram-positive B. subtilis 168, and deeper in Bifidobacterium
longum (B. longum), we used a E. coli - B. subtilis shuttle vector pBE2
(6292bp), which also makes co-transformation with pHT-opSam2 available. This
plasmid was gained from our PI’s lab as well. It has merely a common prokaryotic
S6 promoter, which can’t satisfy our need to verify the inducible expression. So
we firstly inserted a P43-promoted XylR fragment into the plasmid between
PstI/KpnI double cut sites, and then we inserted another fragment Pxyl-promoted
opPet8p into the first recombined plasmid between KpnI/EcoRI double cut sites.
The XylR fragment was amplified through PCR from genome of B. subtilis, and the
other fragment was synthesized. Similar to the procedure done in pHT-Sam2,
pBE-XylR-opPet8p (10046bp) has been constructed and transferred into E. coli and
B. subtilis (Fig. 1.2).

![](https://static.igem.wiki/teams/4144/wiki/result30/result303.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result304.png)

### Expression of opSam2

    In order to verify opSam2 expression, we utilized the
6xHis tag fused to N-terminal of opSam2 to perform Western Blot. Since the
opSam2 is downstream of Pgrac, a lactose-induced promoter, we used IPTG to
induce its expression in E. coli first.

![](https://static.igem.wiki/teams/4144/wiki/result30/result305.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result306.png)

    As the result shows, we can figure out that the band
between 40 and 55 displays the existence of 6xHis::opSam2 whose molecular weight
is predicted to reach about 45.80 kDa. As the induction during increased, the
band brightness became darker, which means that the expression of 6xHis::opSam2
is increased. Meanwhile, we may find some shifting phenomenon in right several
lanes like lane 6, 7 and 8. This could be caused by the degradation of opSam2
due to long time of induction and lysis. So when we performed WB of opSam2 in B.
subtilis, we strickly controled the lysis time and reduce the induction time.
Thus, the result looks clearer and more specific (Fig. 1.3D). The target band
obviously became darker when induction time increased. So we can conclude that
the expression of opSam2 in B. subtilis succeeded.

### Expression of opPet8p

    In order to verify opPet8p expression, we utilized
the 6xHis tag fused to N-terminal of opPet8p and the sfGFP tag fused to
C-terminus to perform Western Blot. Since the opPet8p is downstream of Pxyl, a
xylose induced promoter, we used xylose to activate its expression.

    We synthesized the fragment
Pxyl-6xHis::Mistic::opPet8p::sfGFP from Tsingke
Biology. We found that without XylR, the repressor of Pxyl, bacteria containing
such fragment can constitutively express 6xHis::Mistic::opPet8p::sfGFP, for we
observed the green fluorescence in colonies in the plate when they were excited
by UV light (Fig. 1.4A) and turned green under visual light. However, when
inserted together with P43::XylR, bacteria cannot obviously emit green
fluorescence visible to the naked eyes, so we concluded that the Pxyl could work
and XylR could repress Pxyl as reported.

    To induce the expression of opPet8p in E. coli and
B. subtilis, we tried 0.3%,
0.8% and 1% xylose to induce, and finally we used 1% xylose to induce since its
efficiency is best. When preparing the protein sample after xylose induction, we
can easily figure out the expression of opPet8p::sfGFP in E.coli and B. subtilis
through sfGFP tag. Only the image in E. coli was taken (Fig. 1.4B). Meanwhile,
we performed fluorescent microscopy of E. coli and B. subtilis with
pBE-XylR-opPet8p after induction (Fig. 1.4C). The results shows the successful
expression of opPet8p::sfGFP. After induction and lysis, we performed BCA
quantification (Fig. 1.4D) and dilution to make sure that the loading mass of
total proteins remained the same, then we performed western blot to detect
opPet8p expression level in both E. coli and B. subtilis (Fig. 1.4G). We used
two kinds of antibodies, one against sfGFP (Fig 1.4E) and another against 6xHis
(Fig 1.4F).

![](https://static.igem.wiki/teams/4144/wiki/result30/result307.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result308.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result309.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result3010.png)

    We can figure out that as the induction during
increased, the expression of His-conjugated protein increased. Meanwhile, the
induction or expression efficiency in B. subtilis is higher than in E. coli,
according to the optical density of the lanes. It’s easy to understand, the
promoter Pxyl is endogenous for B. subtilis and it will be more suitable to be
activated in it than in E. coli. However, what confused us is that the molecular
weight observed (about 30kD) is different from what we predicted (73kDa). We
thought it could be some particular mechanism to cut such protein during
maturation so that it became smaller, but the transcription and translation are
working normally since both the N-terminal signal His tag and the C-terminal
sfGFP can be detected.

    For the above reason, we used another antibody
anti-GFP to perform Western Blot
(Fig. 1.4G), and the results shown that there are several truncated proteins
except full-length protein (73kD) (Fig. 1.4E). So further experiments should be
done to learn about the existing state of opPet8p in B. subtilis.
According to above results, we can conclude that B. subtilis can expressed the
recombined protein opPet8p, while the precursor proteins might be cleavaged to
mature proteins.

### Membrane localization of opPet8p

Since the opPet8p is membrane-bound, we tended to find out whether it can
localize in membrane of prokaryotic cells. To better understand the membrane
integration of our recombined protein opMistic::opPet8p::sfGFP, we used a
prediction tool Phobius to predict . The
results are showed below (Fig. 1.5A). As we can see, the addition of Mistic can
result in the addition of transmembrane segment which can facilitate the
membrane insertion of opPet8p, while the sfGFP does the opposite thing. Thus
after using sfGFP to verify its expression and membrane localization, if we want
to improve its membrane localization efficiency, we need to replace the sfGFP
with other tag, like some small particular fluorescent group.
Then we used xylose to induce the expression of opPet8p-sfGFP in B. subtilis,
and then take some bacterial solution to perform fluorescent microscopy (Fig.
1.5B). The group constitutively expressing opPet8p without repressor XylR is on
the right. In groups with XylR, the expression is induced by xylose. Treated
with different concentration of xylose (Fig. 1.5B Left with 0.5% and Middle with
1%), we can briefly observe the density of fluorescence became stronger when
bacteria were induced with higher concentration of xylose. However, the
particle-like distribution became more obvious when bacteria were treated with
more xylose.

    So according to the distribution of fluorescent
signal, we supposed that the
recombined proteins might target to the bacterial membrane. However, if the
expression rate is too high to conduct the proper folding so that they might
form the particle-like inclusion body, they might gather together to
precipitate.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3011.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result3012.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result3013.png)

### Catalytic ability of opSam2

    Since we have verified the expression of SAMe
synthetase opSam2, we next measured its catalytic capacity of synthesizing SAMe
from ATP and methionine. We induced opSam2 expression in B. subtilis, then
aspirated the supernatant, and took the pellet to measure the SAMe concentration
through SAMe quantification ELISA kit (Fig. 1.6A). The standard curve
demonstrated our result was credible. In this experiment, we set a control group
with empty vector (EV), which did not contain the recombined protein gene but
contained the primary plasmid pHT01K, so we can use this group to standardize
the concentration change of experiment group (Fig. 1.6C and D).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3014.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result3015.png)

∆Relative concentration = (Induced Experiment Goup –
Induced Control Group) / (non-induced Experiment Group – non-induced Control
Group)

We can figure out that the after induction, the concentration of SAMe in
Experiment grouo (pHT-opSam2) and Control (Empty Vector) group both definitely
increased. But the increase of Experiment ground is sharper than control group.
Besides when we used the control to standardize the concentration change of
Experiment Group, we found a obvious increase of relative concentration change
in Experiment Groups. we can conclude that opSam2 can function normally in
gram-positive bacteria B. subtilis, which means that it would be greatly
possible to work in our final target probiotic Bifidobacterium longum.

# Oscillator Module

## Goal

    1.To express lacI, tetR, lambda CI in B. subtilis, and
verify their function, as in if they can repress the expression starting from
their corresponding promoter.

    2.To test the function of the plasmid
Repressilator, in E. coli.

## Experiment

### Expression of lacI, tetR, lambda CI

     In order to construct a plasmid that contains both the
transcription factors and a GFP whose expression is driven by the corresponding
promoter, we chose to use a shuttle vector, pBE2, as the chassis vector to
construct our plasmid, for its convenient ability to be able to replicate in E.
coli that allows us to finish most of our experiments without using B. subtilis,
to ensure higher possibility of success.

    First of all, we decided to verify the function of
the constitutive promoter,
P43, that we chose as the promoter that would drive the expression of the
transcription factors (e.g. lacI), so, we first designed the plasmid that used
P43 to drive the expression of GFP.

     We obtained the plasmid backbone of pBE2 from
Zhixiong Xie Lab. GFP sequence was
obtained from part BBa\_E0840 that was given by iGEM foundation, and P43 sequence
was synthesized by Tsingke Biotechnology company. After obtaining the sequences,
we designed primers to amplify the desired part of the sequence, and after gel
electrophoresis, band of the expected size was observed.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3016.png)

Figure 2.1 M: Marker; 1-2: PCR product of plasmid; 3-4:
Unsuccessful attempts to amplify the Vector by PCR; 5-6: PCR product of GFP
fragment; 7-8: PCR product of P43 promoter; 9-10: Unsuccessful attempts to
amplify the promoter by PCR; 11-12: PCR product of P43 promoter

But after we used gel purification to purify the corresponding band, the
concentration of the vector was quite low, so, we performed another round of
PCR, and obtained linearized vector with sufficient concentration.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3017.png)

Figure 2.2

    Left: M: Marker; 1: Unsuccessful attempts to PCR amplify the vector; 2:
Unsuccessful attempts to PCR amplify the vector; 3: PCR amplification of the
vector

    Right: M: Marker; 1-4: PCR amplification of the vector

We performed Gibson assembly, and transformed the ligation product into DH5α
competent cells. And after incubating the plate for 20h, we used colony PCR to
verify the positive transformants. The result is shown below. (We didn’t manage
to culture the bacteria after the first round of colony PCR, so, we did another
one.)

![](https://static.igem.wiki/teams/4144/wiki/result30/result3018.png)

Figure 2.3 Left: First round of colony PCR. Right:
Second round of colony PCR. M: Marker; +: Positive results; -: Negative results

After selecting out the positive transformants and culturing them, we extracted
plasmid, but due to the lack of time, we didn’t manage to get to the part of
functional verification.

### Test the function of the Repressilator plasmid in E. coli

Other than verifying the three transcription factors
one by one, we also planned on testing the function of Repressilator as a
whole. So, we decided to transform the Repressilator plasmid and a reporter
plasmid that consists of GFP whose expression is driven by tetR promoter.

    The Repressilator plasmid was ordered directly
from Addgene, and the
reporter plasmid we chose was obtained from iGEM kit.

    After co-transformation, we used
double-antibiotic plate selection and
colony PCR to pick out the transformants that had both plasmids (Fig 2.4).
And we successful gained co-transformed strain.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3019.png)

Figure 2.4 M: Marker; P.C.: Positive Control

    To test the fluorescence of the co-transformed bacteria,
we incubated the bacteria overnight, diluted the culture for 100 times, and
incubated the diluted culture for another 2 hours. Then, we dropped a drop of
molten solid LB media on top of a glass slide, dropped another drop of bacteria
culture, spread it evenly, and observed under the fluorescence microscopy.
We did observe fluorescence, but we didn’t observe the fluctuation of it. We
hypothesized that this was due to the fact that the oscillation cycle was too
long, and in fear of the quenching of GFP molecule or adverse effects exerted on
our bacteria due to long time exposure of blue excitation light, this
oscillation cycle can’t be observed continuously under the microscope, so, we
decided to take photos of the same area of the slide with the same setting and
at given time intervals. We settled on a time interval of 10 minutes, for a
whole duration of 1 hours, and with support from math models, we managed to
solve the problem of the measurement of the oscillation cycle of the
Repressilator. The solving process is shown below.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3020.png)

Figure 2.5 The original photo of the Repressilator taken
at an time interval of 10 minutes.

Then, we measured the light intensity of the whole
photo, to serve as the background of the fluorescence intensity (making a
hypothesis that the light intensity of the bacteria with fluorescence is so
much less than the background that the light intensity of the bacteria can
be neglected, and as can be seen below, this hypothesis is correct)

![](https://static.igem.wiki/teams/4144/wiki/result30/result3021.png)

Figure 2.6 The measurement of the light
intensity of the whole photo, to see the background.

    The first box indicates the average light intensity per pixel
of the whole photo,
and the second box indicate the number of pixels of the image. The whole light
intensity can be obtained by multiplying the two numbers together.

    Then, we cycled out the same bacteria of each photo, making
sure that they are
all at the same region in each photograph, numbered them 1-10, as shown in
figure a, and measured the light intensity in each of the small regions, the
result is represented in figure b, and the data obtained is shown below in the
table.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3022.png)

Figure 2.7 The numbering of each bacterium, and the
measurement of
the light intensity in regions around each bacterium.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3023.png)

Total number of pixels around each bacterium and the whole
picture

![](https://static.igem.wiki/teams/4144/wiki/result30/result3024.png)

Average light intensity around each bacterium and the whole
picture

![](https://static.igem.wiki/teams/4144/wiki/result30/result3025.png)

Total light intensity around each bacterium and the whole
picture without
background

Figure 2.8 Analysis workflow of light intensity around
each bacterium and the whole picture.

    To visualize these data, we imported them into Prism, and
performed non-linear
regression using sine waves as the model, to find out the expected oscillation
cycle of the Repressilator plasmid. The result is shown below.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3026.png) ![](https://static.igem.wiki/teams/4144/wiki/result30/result3027.png)

But as can be felt from the regression curve, the data point at 30 minutes
violated the whole trend, and the goodness of fit is quite low. So, we decided
to delete this data point, and do the regression once more.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3028.png)
![](https://static.igem.wiki/teams/4144/wiki/result30/result3029.png)

And as can be seen, the result is much better, and the oscillation cycle settled
around 60 minutes, which agrees with the result shown in the original article.

# Directed Evolution Module

## Goal

     1. Construct a plasmid containing reporter genes
for
further selection.

    2. Build mutagenesis library of lacI.

    3. Separate desired LacI protein that can tolerate
relatively high level of
lactose.

## Achievement

1.The plasmid is successfully constructed and site-saturation mutagenesis is
successfully introduced;

2.The mutated plasmids are transformed into E. coli BL21(DE3) and successfully build
up the mutagenesis library.

3.The desired LacI protein is successfully derived through rounds of selection
process.

## Experiment

### Plasmid construction

    Our selection plasmid intends to contain three selection
elements: mRFP1, sacB and KanR (Fig 3.1A). In the beginning, we planned to use
overlap extension PCR to connect each fragment into one and to combine it with
linearized vector (BBa\_J04450) as one composite part. However, the fragment was
quite unstable and was easy to break even once successfully connected (Fig 3.1C).
Thus, we decided to directly use Gibson Assembly for the whole construction by
separating the process into building two intermediate plasmids (Fig. 3.1B) with only
two to three fragments at a time. Even though the success percentage wasn’t 100%, as
fragment loss still occasionally occurred, we successfully constructed the final
plasmid using this principle (Fig. 3.1D).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3030.png)

Figure 3.1 Plasmid construction and Electrophoresis
analysis.

A: Diagram of the final plasmid.

B: The first intermediate of plasmid construction.

C: Overlap extension PCR product of sacB and KanR break up at the next amplification.

D: Colony PCR of random transformant, identifying three fragments respectively.

We also optimized the element as we realized the possibility of lacI
mis-expression in previous design because of the inappropriate distance between
RBS and the stating codon of lacI , by deleting some base pairs between RBS and
lacI gene (Fig 3.2).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3031.png)

Figure 3.2 Sequencing of developed lacI expression section
with expected deletion occurred.

### Building variant library

    We decided to use MEGAWHOP (The Megaprimer PCR of Whole
Plasmids) to introduce mutagenesis into LacI gene. To construct megaprimers with
site-saturated mutagenesis, we used overlap extension PCR. After DpnI digestion,
we transform the variants into E. coli BL21(DE3) and obtain the mutagenesis
libraries on on chloramphenicol-added plate (Fig 3.4a). The colonies were
selected for sequencing, expected mutations were observed at target positions
(Fig. 3.4).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3032.png)

Figure 3.3 Mutagenesis library construction.

 A: No megaprimer in MEGAWHOP;

 B: Megaprimer added in MEGAWHOP. DpnI digestion is applied to both group, and
non-mutated plasmid can’t be transformed into competent cells.

### Selection

Based on sacB and KanR added to our plasmid, our selection can be delivered by selective culture of mutants. Since sacB allows negative selections on mutant libraries, details in Design, we first carried out experiments with sucrose medium, exploring multiple conditions to execute the selection including sucrose content and induction duration etc. However, we failed to separate the ideal mutants in this way. Later test suggests that the reason may be the wrong timing for induction or spreading, as the killing effect being significantly distinguishing when we spread the culture on medium with 8% sucrose and 0.8mM IPTG at OD600 equal around 0.2. For detailed information please check on Engineering.

    Due to the time constraint, we turned to establish comparison experiments using KanR as report gene. Gradient IPTG were added into 50 ug/ml kanamycin solid plate to observe variant’s performance in different induction concentration. Our results also once again showed our success in mutagenesis library construction, with various kinds of behavior occurred in different mutant strains (Fig 3.4 C-J).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3033.png)

Figure 3.4 Partial results of selection.

A: Control group 1. Original strain and random mutant are applied on empty
antibiotic plate with IPTG contrast.

B: Control group 2. Original strain is applied on Kan+ plate with IPTG gradient.

C: Desired mutant, Strain #29. No colony growth is observed at 0 and 0.1mM IPTG,
suggesting that the LacI can tolerate high-level IPTG.

D: Strain #27. The mutagenesis causes its binding to lactose (IPTG) to be easier,
and lawn are witnessed at all concentration, even without IPTG existence. Its
behavior is quite familiar to performance on non-antibiotic medium.

E: Strain #9. The mutagenesis slightly reduces the leakage of the promoter, however,
barely difference occurred between different inductor (IPTG) concentration.

F: Strain #24. Little up-regulation of the promoter takes place as IPTG
concentration lifting. Its response to low-level inductor (IPTG) is still relatively
high.

G: Strain #2. Few colonies occur at 0mM IPTG and no growth at higher level.

H: Strain #6. No growth at all. The sensation to IPTG is totally lost.

I: Strain #13. Expression is activated only at 0.1mM IPTG.

J: Strain #14. Few colonies are witnessed, expect one at 0.5mM IPTG, suggesting its
affinity to IPTG has greatly dropped.

    In order to evaluate the behaviors of different mutants for
better judgement, we
designed a algorithm to access the IPTG responsive ability of the strains. We used
number 1-7 to measure the intensity of the colonies on each plate, and integrated
them into a heatmap for visualization (Fig 3.5). Our goal is to select a LacI
variant that cannot sense the low concentration IPTG and at the same time normally
functions at with concentrated IPTG induction. Thus, reflecting on different colors,
the ideal strain are supposed to have lighter color between darker ones at high
range of IPTG. Comparing the data, Strain #29 is exactly the one that we are looking
for (Fig 3.4 C). Meanwhile, a few growths under 0.8mM IPTG guarantees that the
mutant LacI still keeps the ability to release from promoter and it could function
normally in our oscillatior - “Repressilator”. Selected mutant also appeared to
solve the leakage problem that original LacI contain, showing a more outstanding
performance.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3034.png)

Figure 3.5 Visualization of arithmetic LacI evaluation
result. The triangle points out the desired variant strain #29.

    After sequencing, we are surprising to find that the mutations are taken somewhere
else than the positions we set. This situation could happen as our mutagenesis
library contains the original sequence, and new mutations may be introduced during
multiple amplification reactions during the construction process. The ideal results
however suggest that it’s highly possible that these are other residues that may
contribute to LacI’s substance binding ability. Further clarifications could be
delivered to explore the functions that these residues may hold.

    Limited in time, we weren’t able to measure the characters of this variant. However,
present results are convincing enough to indicate the success in our selection. The
improved LacI repressor is documented as a new part BBa\_K4144041.

# Safety Switch Module

## Goal

    1. To construct two composite plasmids which work as
chemical and temperature switch respectively.

    2. To test the function of these composite parts in
B. subtilis.

    3. To test the lytic strength of bacteria when
PBSX holin expresses.

## Achievement

    1. The temperature switch with EGFP as a reporter
can be induced by low temperature.

    2. The expression of PBSX holin has the ability to
inhibit the growth of the
bacteria to some extent.

## Experiment

    In order to test the function of two composite plasmids
which work as chemical and temperature switch respectively, we first chose EGFP
as their downstream reporter. We have constructed these plasmids successfully
(Fig 4.1).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3035.png)

Figure 4.1: The result of plasmid construction and
sequencing.
A, B: Construction and sequencing of chemical switch.
C, D: Construction and sequencing of temperature switch.

    Firstly, we tested the function of the plasmids in E.
coli DH5α.

    For chemical switch, we treated engineered
bacteria with CinI, a molecule for
quorum sensing which can form a complex with CinR to induce expression of
downstream EGFP or Holin. We used recombined E. coli BL21 to produce autoinducer
and extract the supernatant. We tested the function of chemical switch by
inducing the expression of EGFP with the supernatant.

    For temperature switch, in the environment below
36 degrees Celsius, Temp will work to activate the expression of downstream EGFP
or Holin, while over 36 degrees Celsius, Temp will be shut down and bacteria
can’t emit green fluorescence or be killed. To determine whether Temp can work
smoothly, we incubated E. coli containing pBE2-Temp-EGFP overnight and then
equally divide bacteria into two EP tubes, cultured respectively at 25 and 37
degrees Celsius for one hour.

    As a result, unfortunately, no fluorescence was
observed among different groups
of chemical switch. But the fluorescence of temperature switch induced at 25˚c
was observed (Fig 4.2A and B).

     After that, we transformed the plasmids into B.
subtilis successfully and
quantitively measure the expression of fluorescence (Fig 4.2 C and D).

![](https://static.igem.wiki/teams/4144/wiki/result30/result3036.png)

Figure 4.2: The fluorescence in E. coli with chemical or
temperature switch and the fluorescence in B. subtilis with chemical or
temperature switch.

     A: Chemical switch in E. coli

     B: Temperature switch in E. coli: The short
rod-shaped E. coli cultured at 25
degrees Celsius fluoresces green, while the bacteria cultured at 37 degrees do
not fluoresce. This indicates that Temp induced by low temperature can initiate
the expression of downstream genes, while Temp does not work at high
temperatures. The results are in line with our expectations.

     C: Chemical or temperature switch in B. subtilis:

1) Relative to the control group, values of chemical-induced+ inducer increased
to some extent, but the margin wasn’t significant enough.

2) Chart shows data in the second hour. Values at 37 degrees are benchmarks.
There was no significant increase in the data of the first two groups relative
to the latter two groups, which means EGFP in engineered B. subtilis didn’t
express.

    Lastly, we tested the function of PBSX holin by
measuring the lysis of E. coli. In this construction, we transformed pET-Holin
into E. coli BL21 and the expression of holin is induced by IPTG. We found that
after inducing, the bacteria were killed or inhibited to some extent though not
fully (Fig 4.3).
To qualitatively verify holin’s function, we coated the solid medium with 100mL
of E. coli solution and affixed round paper soaked in water on one side, while
the other side was affixed with round paper soaked in 1mM IPTG. The plates were
incubated at 37℃ overnight and observed for the formation of bacteriostatic
circles.

![](https://static.igem.wiki/teams/4144/wiki/result30/result3037.png)

Figure 4.3: Inducing the expression of PBSX holin in E.
coli BL21.

    A. Induction on culture plate:
After incubation overnight, the bacteria had grown into a moss on the medium. A
large number of bacteria still grew around the water-soaked paper, while the
growth of bacteria near the paper soaked with IPTG was inhibited, with the
number of bacteria decreasing and single colonies appearing.

    B. Change in turbidity:
It can be seen that the bacterial concentration of the group induced by IPTG was
significantly reduced compared with that of the control group, but there was no
significant difference between different induced concentrations. The lytic
efficiency did not increase obviously with the extension of culture time. The
results showed that holin could work in E. coli.

    Thus, we still need to improve our designs in the
future to make them complete safety switches. Though we have proved that the
most basic parts in our design will be able to work in B. subtilis, further
examination of each single part is still needed. For example, TlpA36 promoter
should be tested in B. subtilis to verify whether it has its normal function.
What is more, we can use directed evolution method to improve those parts for
the expression in B. subtilis.

[Engineering](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27engineering%27%29%20%7D%7D)[Notebook](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27notebook%27%29%20%7D%7D)