iGEM 2023 — Team thinker-sc

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Experimental preparation for week 1

Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain

Experimental procedure:

1. Use of ultra-clean table: open the ultra-clean table, place the centrifuge tube plate, test tube rack,
Marker pen, alcohol disinfection and sterilization, and conduct UV irradiation for 30 minutes.

2. Sterilization: Prepare Eppendorf tubes, PCR tubes, and centrifuge tubes, and wrap them with gauze. Fill 2
boxes each of large, medium, small, and gun heads, using Velcro paper to wrap the shell, and using string/rubber
band to bind the gun head box. Using pre-prepared LB medium, divide 20 tubes into 50 mL x 2 bottles of LB liquid
medium. The above items were sterilized using 121 ° C for 15 min (this process was about 1.5 h). An experimental
accident occurred during the filling of liquid medium: the medium was spilled on the ultra-clean table, and it
was cleaned up properly afterwards.

3. Activated strains: 1 tube of E. coli DH5α/ empty vector 1 and 1 tube of DH5α/ pheP gene. One tube of E. coli
DH5α/ empty vector 2 and one tube of DH5α/ carrying PAL gene. Turn on the ultra-clean table, ventilated, put the
sterilized LB test tube into the table, light the alcohol lamp, burn the test tube orifice and plug, put 100 μL
of preserved bacteria solution into each test tube, add 2.5 μL Amp antibiotic mother solution, burn the test
tube orifice and plug again. After sealing the tube, the tube was bundled and incubated on a shaker overnight
(150 rpm, 37℃).

![](https://static.igem.wiki/teams/5015/wiki/notebook/img1.jpg)

2 weeks to extract the templates, began to construct the plasmid

Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR

Experimental procedure:

1. Plasmid extraction: Four kinds of plasmids (DH5α/ empty vector 1, DH5α/ empty vector 2, and 1 tube of DH5α/
carrying iaaH gene) were extracted according to the instruction of plasmid extraction kit. One tube of E. coli
DH5α/iaaM, one tube of DH5α/ carrying galU gene, and one tube of DH5α/ carrying Arabinose promoter and mazF
genes. The bacterial solution was centrifuged for 1min and then the precipitate was removed. p1 was added
separately and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL
BL reagent was added to the adsorption column, the waste solution was poured, the supernatant after
centrifugation as described above was added, and the mixture was rinsed twice using pw rinse solution, and
centrifuged for 1min each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for
2min. The recovered DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this
step, most people successfully extracted the plasmid.

![](https://static.igem.wiki/teams/5015/wiki/notebook/img2.jpg)

2. Diluted primer

The dry powder primer was centrifuged at 12,000 RPM for 1min, and the corresponding amount of distilled water
was added to dilute the primer to the appropriate concentration

3.PCR reaction:

(1) Target genes pheP, PAL and two kinds of vector DNA fragments were amplified by high fidelity enzyme Pfu.
The reaction system is as follows:

The Pfu was 0.5 μL

10xPfu buffer 5 μL

Primer 1 1 μL

Primer 2 1 μL

Template (plasmid extracted above) : 1μL

25μL dNTP Mixture

20 μL of sterilized water

(2) Perform the PCR amplification procedure

At 98 ° C for 5 min

The following 3-step reaction was performed for 30 cycles

98℃ 10s

55℃ 5s

72℃ 1min/kb

72℃ 8 min

3 weeks agarose gel electrophoresis, plastic recycling, double
enzyme, recycling

Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery

Experimental procedure:

1 Agarose gel electrophoresis: The above PCR products were added to a previously prepared agarose gel with 5μL
DL 5000 DNA marker and subjected to electrophoresis (120V, 20 min). At the end of electrophoresis, the gel
imaging system was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and
labeled.

![](https://static.igem.wiki/teams/5015/wiki/notebook/img4.jpg)
![](https://static.igem.wiki/teams/5015/wiki/notebook/img5.jpg)

2. Gel recovery: The PCR product DNA gel was recovered according to the instructions of the gel recovery kit

Add 500 microliters of equilibrium solution BL to the adsorption column CA2 and centrifuge for 1 min, pour out
the waste liquid in the collection tube, and put the adsorption column back into the collection tube. A single
band of target DNA was cut from the agarose gel into a clean centrifuge tube. Add an equal volume of solution PN
to the gel block and place in a water bath at 50 ° C until the gel block is completely dissolved. The solution
obtained in the previous step was added to another adsorption column CA2, placed at room temperature for 2 min,
and centrifuged for 1min. Add 600 microliters of bleach solution PW to the adsorption column CA2 (please check
whether absolute ethanol has been added before use), centrifuge for 1min, repeat rinsing twice, and let dry. Add
an appropriate amount of elution buffer EB to the middle position of the adsorption membrane and place it at
room temperature for 2 min. The DNA solution was collected by centrifugation for 2min. The recovered DNA
concentration was determined using Nanodrop.

3 Double digestion: EcoRI and XhoI were used to double digest the extracted plasmid pET23b and the amplified
target DNA fragment. 5x20 μL reaction. The 20 μL digestion system is as follows:

1 μg of DNA

1 μL of EcoRI

1 μL XhoI

4 μL 10xY buffer

H2O up to 20μL

The enzyme was digested at 37 ° C for 4h

4 Agarose gel electrophoresis: The digested product was added to the agarose gel, 5μL of DL 5000 DNA marker was
added, and electrophoresis was performed (120V, 20 min). At the end of electrophoresis, the gel imaging system
was used for observation. The target DNA gel was cut, placed in a 2 mL centrifuge tube, and labeled. The product
was extracted successfully, but the amount was small. After that, the second gum recovery was carried out.

At week 4, competent cells were prepared

Experimental plan: E. coli DH5α and Rosetta were activated to prepare competent cells

Experimental procedure:

1. Activated strains: including E. coli DH5α and Rosetta. Turn on the ultra-clean table, ventilate, put the
sterilized LB test tube into the table, light the alcohol lamp, burn the test tube mouth and test tube stopper,
connect 100 μL of preserved bacteria solution into each test tube, and burn the test tube mouth and test tube
stopper again. The test tube was closed, bundled, and incubated on a shaker overnight (220 rpm, 37℃).

Experimental procedure:

2. Preparation of competent cells:

(1) Open the ultra-clean stage, put in 1 mL tip, 200 μL tip, 5 mL tip, 2mL and 30 mL centrifuge tubes, and 2 x
50 mL LB medium for UV irradiation. The 0.1 M cacl 2 and 15% 0.1 M cacl 2 solution were pre-cooled in a
refrigerator at 4 ° C.

(2) DH5α and Rosetta competent cells were prepared as follows: pre-activated and repacked DH5α and Rosetta
bacteria were precooled in a centrifuge at 4 ° C, centrifuged at 5000 g for 10 min, the supernatant was discard,
and 800 microliters of 0.1 M CaCl2 solution was added to an ice bath for 40 min. After centrifugation again
(4000 g, 10 min), the supernatant was discarded, and 100 microliters of 15% glycerol 0.1 M CaCl2 were added to
each tube to resuspend the bacteria. Aliquots of competent cells into 2 mL centrifuge tubes of 100μL per tube
were dispensed on ice. After labeling, the competent cells were stored in the refrigerator at -80℃.

At week 5, the cells were connected and transformed

Experimental plan: Prepare solid AGAR plates containing antibiotics, perform ligation reactions, and transform
ligation products

Experimental procedure:

1. Preparation of antibiotic plates: LB solid medium (4.5 g AGAR added to 300 mL LB liquid medium) was prepared
and sterilized at 121 ° C for 15 min by autoclave. After cooling to about 60℃, add 150 μL of 100mg/mL Amp
antibiotic, shake evenly, and pour into the disposable Petri dish (about 10-15 plates).

2. Ligation reaction: double enzyme digestion of the vector and the target fragment was connected. The ligation
system is as follows (Note: This system is an empirical system, and the optimal linking vector/target fragment
volume ratio can also be calculated according to the instructions) :

T4 DNA ligase 1 μL

2 μL of 10 x ligation buffer

1 μL of Linear plasmid

Insert 6 μL of DNA

Connect for 1h at 16 ° C using a PCR apparatus. pET23b-insert was obtained, and the ligation product was
stored at -20 ° C.

3. Transformation of ligation products: 10 μL of the above ligation products were added to 100 μL competent
cells, followed by an ice bath for 30 min, a water bath at 42 ° C for 60 s, and an ice bath for 5 min. Then, 1
mL LB liquid medium was added and incubated for 1 h for recovery (37 ° C, 150 rpm). In an ultra-clean stage, 100
μL of recovery solution was taken and the Amp antibiotic plate was coated with a sterile coating rod.

At the 6th week, three kinds of plasmids were extracted and expanded

Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the
correct plasmid and transformation

Experimental procedure:

1.PCR verification: Six colonies were labeled on each plate. Half of the colonies were picked and used as
templates for colony PCR using Taq enzyme. The primers are TF, TR. The reaction system and procedures were
carried out according to the instructions. Configure a 50 mL agarose gel, using DL 2000 DNA marker, and perform
electrophoresis. The correct size of the band indicates that the plasmid was successfully constructed

2. Expanded culture: The correct colonies were selected and added to 5 mL LB liquid medium, and 2.5 μL Amp
antibiotic was added to expand the culture.

![](https://static.igem.wiki/teams/5015/wiki/notebook/img6.jpg)
![](https://static.igem.wiki/teams/5015/wiki/notebook/img7.jpg)

3. Plasmid extraction: Two kinds of plasmids were extracted according to the instruction of plasmid extraction
kit. The bacterial solution was centrifuged for 1min and then the precipitate was taken. p1 was added separately
and resuspended, p2 was mixed upside down, p3 was mixed upside down, and centrifuged for 10min. 500μL BL reagent
was added to the adsorption column, the waste solution was poured, the supernatant after centrifugation as
described above was added, and the mixture was rinsed twice using pw rinse solution, and centrifuged for 1min
each time. EB solution 50μL was added to the adsorption column membrane and centrifuged for 2min. The recovered
DNA concentration was determined using Nanodrop and the DNA was stored at -20 ° C. In this step, most people
successfully extracted the plasmid.

4. Transformation: Verify the correct plasmid 1 transformation into expression Escherichia coli.

Determination of indole-3-acetic acid (IAA) production at week 7

Experimental plan:

Experimental procedure:

Engineered E. coli Rosetta strains expressing IaaM and IaaH and control strains were grown at 37 ° C in
Luria-Bertani (LB) medium with shaking at 200 rpm. Because IAA was easily decomposed in light, the triangle
flask was wrapped in tin foil and the shaker was shaded with newspaper to achieve light-sheltered conditions.
Every 24 hours, 1.5 mL of the bacterial culture medium was taken, and 1 mL of the supernatant was collected
after centrifugation (10,000 rpm, 1 min). For IAA assay, 1 mL of the supernatant was mixed with 1 mL of
Salkowski reagent (2% of 0.5M FeCl₃, 35% perchloric acid) 1. 1 The mixture was incubated in the dark for 30 min,
after which a pink color indicated the presence of IAA. The intensity of the color, which is proportional to the
concentration of IAA, was measured by spectrophotometry at 530 nm. Standard curves were prepared with known
concentrations of IAA for the quantification of IAA in bacterial cultures. All experiments were repeated three
times, and data are presented as mean ± standard deviation (SD). Statistical significance between the engineered
strain and the control strain was tested using Two-way ANOVA (two-way analysis of variance), with p < 0.05
considered statistically significant.

![](https://static.igem.wiki/teams/5015/wiki/notebook/img8.jpg)

Effect of GalU gene on EPS levels at week 8

Experimental plan:

Experimental procedure:

The growth of bacteria was monitored by measuring OD600 at predetermined time points (4, 8, 16 h). At the same
time, the cells were centrifuged, and the supernatant was used to quantify EPS by the anthrone sulfuric acid
method. In brief, a sample of the supernatant was combined with an anthrone reagent, followed by the addition of
concentrated sulfuric acid. The mixture was subsequently heated and the color development was measured by
spectrophotometry at 620 nm. As the main component of EPS is polysaccharide, a standard curve of glucose was
used to evaluate the EPS concentration in the sample. The EPS concentration was then normalized to OD600 to
obtain EPS production per unit bacterial growth. All experiments were repeated three times, and data are
presented as mean ± standard deviation (SD).

![](https://static.igem.wiki/teams/5015/wiki/notebook/img9.jpg)

Testing of the arabinose promoter at week 9

Experimental plan:

Experimental procedure:

1. We added arabinose to the bacterial solution to induce the expression of mRFP. The fluorescence intensity of
mRFP was measured at 584 nm excitation and 607 nm emission using a microplate reader. This intensity was used as
an indicator of arabinose promoter activity.

2. Fluorescence microscopy verification experiment:

(1) Sample preparation: appropriate amount (about 30 μl) of wild type Rosetta and Rosetta-engineered bacteria
droplets were dropped onto the slide and covered with a cover slip.

(2) Turn on the switch of the power control box of the high-pressure mercury lamp.

(3) Insert the light stopper to interrupt the light path.

(4) Preheat for 5-10 min.

(5) Place the slide with the sample on the stage.

(6) Select the objective lens (in order of first low power, then high power).

(7) Rotate the turntable of the spectroscope assembly to select the spectroscope assembly for observing mRFP
(red fluorescence).

(8) Adjust the focal length through thick and thin screws.

(9) Open the computer connected to the microscope and click on the digital imaging system software to collect
digital images.

![](https://static.igem.wiki/teams/5015/wiki/notebook/img10.jpg)

Dependence of arabinose promoter on substrate concentration and
effect of controlling suicide gene mazF at week 10

Experimental procedure:

When the OD600 of the engineered and control strains was 0.6, 1 mM arabinose was added, and after 4 hours of
induction, the fluorescence intensity was measured using a microplate reader and divided by OD600 to obtain the
relative fluorescence intensity. In order to test the response of arabinose promoter to different concentrations
of arabinose. When the OD600 of the engineered strain was 0.6, 0.1, 0.5, 1, 2, and 4 mM arabinose were added.
The fluorescence intensity was measured after 4 hours of induction and divided by OD600 to obtain the relative
fluorescence intensity.

mazF suicide gene was synthesized and cloned into the downstream of the arabinose promoter of pSB1A3 vector.
The recombinant plasmid was transformed into E. coli Rosetta competent cells by the heat-shock method. Positive
transformants were screened on LB AGAR plates supplemented with ampicillin.

Individual colonies were inoculated into LB broth and grown until the optical density, OD600, reached 0.6.
Different concentrations of arabinose were added to the cultures to induce MazF expression. Bacterial growth was
monitored at specific time intervals, namely 2, 6, and 14 h after induction, to assess the inhibitory effect of
MazF expression. All experiments were repeated three times, and data are presented as mean ± standard deviation
(SD).

![](https://static.igem.wiki/teams/5015/wiki/notebook/img11.jpg)

CONTENT

[Experimental preparation for week 1](#a1)
[2 weeks to extract the templates, began to construct the plasmid](#a2)
[3 weeks agarose gel electrophoresis, plastic recycling, double enzyme, recycling](#a3)
[At week 4, competent cells were prepared](#a4)
[At week 5, the cells were connected and transformed](#a5)
[At the 6th week, three kinds of plasmids were extracted and expanded](#a6)
[Determination of indole-3-acetic acid (IAA) production at week 7](#a7)
[Effect of GalU gene on EPS levels at week 8](#a8)
[Testing of the arabinose promoter at week 9](#a9)
[Dependence of arabinose promoter on substrate concentration and effect of
controlling suicide gene mazF at week 10](#a10)

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Cycle 1: Production of IAA by the IAM pathway

Design

First of all, to promote plant growth, IAA is needed so we researched about several different pathways to IAA.

Our final decision is to use the IAM pathway by converting trypophan into IAA in two steps because it has been shown to work in E. coli in and consists of only two enzymes.[1]Through these two steps, tryptophan is converted via the IAM pathway to indole-3-acetic acid (IAA), a natural plant auxin.Derived from Pseudomonas savastanoi, this pathway involves only two enzymes (IaaM and IaaH) to produce IAA.

![](https://static.igem.wiki/teams/5015/wiki/engi/img1.png)

Figure 1. Constructing the genetic element diagram and schematic diagram.

Build

In detail,the IAM (Indole-3-Acetamide) pathway is a two-step pathway to produce indole-3-acetic acid (IAA) from the precursor tryptophan. In this two-step pathway, tryptophan is first converted to IAM via tryptophan-2-monooxygenase (IaaM), which is encoded by the iaaM gene.In the second step, IAM is converted to IAA by IAM hydrolase (IaaH), which is encoded by iaaH [2].

Test

We used the pET23b vector, which carries the pT7 promoter and is therefore transformed into E. coli Rosetta (the bacterium is engineered to carry a bacteriophage RNA polymerase that recognizes the pT7 promoter). Since pET23b does not have a LacI inhibitory protein, downstream genes can be transcribed without IPTG induction.

The primary goal of our project is to mitigate the issue of soil loss by increasing plant growth. In the natural world, plant growth is typically stimulated by a hormone called Indole-3-Acetic Acid (IAA), and one key pathway for increasing plant growth is to provide it with extra IAA. To achieve this, we choose to genetically modify our E. Coli cells to produce more IAA than the wild type.The engineered E. coli strings are grown in a Luria-Botani (LB) media at 30 degrees Celsius and 200 rpm shaking. Since IAA easily degrades upon light exposure, the flasks are covered in aluminum foil and the shaker is covered with newspaper to prevent light exposure. We take 1.5mL of bacterial culture every 24 hours and mix the post-centrifuge (10000rpm, 1min) supernatant with Salkowski reagent (2% of 0.5M FeCl₃ in 35% perchloric acid). The absorbance was measured spectrophotometrically at 530 nm with a prepared standard curve, and our results are shown below.After converting the absorbance data into direct concentrations, we can clearly see that at every 24-hour checkpoint, there is a statistically significant difference between IAA production of our transformed strains and wild-type strains. The differences at each standpoint remain relatively consistent at about 65 micromolarity. Interestingly, the results also show that without any genetic engineering, the wild-type strains do still produce a significant amount of IAA by themselves.The results of the expression engineering strain are shown in Figure 2, which indicate that the overexpression of both genes significantly increases the production of IAA.

![](https://static.igem.wiki/teams/5015/wiki/engi/img2.png)

Figure 2. Comparison of IAA production.

Learn

According to the experiment, we learnt that the control strain E. coli Rosetta itself also produces IAA, indicating that E. coli itself also has an endogenous IAA production pathway. Howerver, we found out about a new problem--the environment where the seed will be grown may have low soil EPS, therfore EPS is required provides the necessary substrate for polysaccharide synthesis, the survival of microorganisms and adaptation to the soil environment.

Cycle 2: Producing EPS through galU genes

Design

Extracellular polymeric substances (EPS) play a key role in bacterial soil crust, as they can provide microbial adhesion to soil particles, promoting microbial settlement and growth on the soil surface. These aggregates are of great significance for the survival of microorganisms and adaptation to the soil environment.

Therefore, we identified and cloned the galU gene(known to play a key role in enhancing bacterial EPS production), which encodes UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. using the Anthrone-Sulfuric Acid Method in order to to increase EPS production [3].

![](https://static.igem.wiki/teams/5015/wiki/engi/img3.png)

Figure 3 Schematic diagram illustrating the principle of the GalU gene.

Build

The GalU gene is known to play a key role in enhancing bacterial exopolysaccharide (EPS) production, so we cloned it into a pET23b expression vector .The recombinant plasmid was transformed into E. coli in competent cells to investigate the effect of GalU genes on the production of EPS when inside E. coil Rosetta.

The measurements include bacterial growth which is monitored by measuring OD600 and glucosamine standard koji was used to assess the EPS concentration in the sample. The EPS concentration is then normalized to OD600 to obtain EPS yield per unit of bacterial growth.

Test

As the GalU gene plays a crucial role in enhancing bacterial extracellular polysaccharide (EPS) production, we have decided to overexpress galU. EPS plays a key role in bacterial soil crust by providing microbial adhesion to soil particles and promoting microbial colonization and growth on soil surfaces. Additionally, extracellular polysaccharides can form microbial aggregates, which are of great significance for microbial survival and adaptation to the soil environment.The successfully engineered strain will be expanded in culture.At predetermined time points (4, 8, 12, 16 hours), bacterial growth was monitored by measuring the OD600. Concurrently, cells were centrifuged, and the supernatant was used to quantify EPS using the anthrone-sulfuric acid method. In brief, a sample of the supernatant was combined with anthrone reagent, followed by the addition of concentrated sulfuric acid. The mixture was subsequently heated, and the developed color was measured spectrophotometrically at 620 nm. A standard curve, established using known concentrations of a standard polysaccharide, facilitated the determination of the EPS concentration in the samples. The EPS concentration was then normalized to the OD600 to obtain EPS production per unit of bacterial growth. To establish a standard curve, 0-200 mM glucose was used as the standard substance and quantified using the anthrone colorimetric method, with absorbance measured at 620 nm, as shown in Figure 4A. Compared to the control group, the engineered bacteria significantly increased EPS levels, which also increased with time, as shown in Figure 4B. The results indicate that the engineered strain can significantly enhance EPS production.

![](https://static.igem.wiki/teams/5015/wiki/engi/img4.png)

Figure 4. The expression effect of galU.

All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD).

Learn

We have solved the problem of the production of auxin and increased the proportion of EPS in the soil but there is still a major issue of the environmental impact of the exposure of the E. coli which contains genes to produce IAA and EPS, disrupting the balance of the content of the soil.

Cycle 3: The arabinose promoter controling the suicide gene mazF

Design

We decided to use arabinose promoter + mazF suicide gene to generate a suicide system so the E. coli will die after seed germinate which would not contaminate the environment.

![](https://static.igem.wiki/teams/5015/wiki/engi/img5.png)

Figure 5. The design of arabinose promoter and mazF.

The reason we choosed the mazF gene is that mazF is the most widely studied and most clearly identified toxin-antitoxin system (TA system). The mazF gene is a downstream gene of the TA system mazEF and encodes a stable toxin protein. MazF is a ribosomal independent mRNA interferase (endonuclease that cleaves single-stranded mRNA at specific sequence sites and is conserved in most microorganisms and some archaeal species).It could result in cell death by restricting the synthesis of protein.

We decided to use the arabinose promoter after we evaluate the function of it through the following experiments.

Build

We used the plasmid sample pSB1A3, which contains the arabinose promoter sequence, as the basis for the mazF gene. We then transformed these constructs into th E. Coli DH5a strains with the heat shock method. the mazF gene codes for a series of protein toxin enzymes that severs mRNA and hence inhibit cellular protein expression and consequentially kill the cells.

![](https://static.igem.wiki/teams/5015/wiki/engi/img6.jpg)

Figure 6. The schematic of experimental.

Test

![](https://static.igem.wiki/teams/5015/wiki/engi/img7.png)

Figure 7. The effect of MazF.

All experiments were conducted in triplicate, and data were presented as mean ± standard deviation (SD). Statistical significance between induced and non-induced cultures was determined using a Student's t-test, with p < 0.05 considered statistically significant.

The mRFP (monomeric Red Fluorescent Protein) gene was utilized as a reporter to assess the functionality of the arabinose promoter. The intensity served as an indicator of the activity of the arabinose promoter，the result is shown in Figure 7A. When the OD600 of the engineered bacteria and the control strain is 0.6, 1 mM arabic acid is added. After induction for 4 hours, the fluorescence intensity is measured using an microplate reader and divided by OD600 to obtain the relative fluorescence intensity in Figure 7B .To test the response of the arabic acid promoter to different concentrations of arabic acid, as shown in Figure 7C, when the OD600 of the engineered bacteria is 0.6, 0.1, 0.5, 1, 2, and 4 mM arabic acid is added. After induction for 4 hours, the fluorescence intensity is measured and divided by OD600 to obtain the relative fluorescence intensity. To induce the expression of MazF, varying concentrations of arabinose were added to the cultures. Bacterial growth was monitored at specific time intervals (i.e., 2, 4, 6 hours post-induction) to assess the inhibitory effects of MazF expression，

The results are shown in Figure 7D. In conclusion, it can be concluded that MazF induction has a significant inhibitory effect on bacterial growth compared to the uninduced control group. After 6 hours of induction, bacterial growth was significantly inhibited, and after 14 hours, growth almost completely stopped.

Learn

By using an Arabidopsis promoter and suicide gene expression, we have successfully addressed the biosafety concern.

Cycle 4: Assessing Engineered E. coli's Influence on Seed Germination via IAA Production

Design

We plan to evaluate the potential promotive effect of IAA-producing engineered E. coli on the germination of various plant seeds. We hypothesize that the IAA produced by the modified E. coli would enhance the germination rates of the selected seeds compared to a control group.

Build

E. coli, engineered to overexpress IAAM and IAAH, were inoculated into 100 mL of M9 medium at a concentration of 100 μg/mL. The culture was incubated on a shaker incubator at 150 rpm for 72 hours at a consistent temperature of 37℃. Following incubation, the culture was centrifuged at 10,000g for 2 minutes. The bacterial pellet was discarded, and the IAA-rich supernatant was collected for seed treatment.

Test

Plant seeds were divided into two groups. The test group seeds were soaked overnight in the supernatant from the engineered E. coli, while the control group seeds were soaked in distilled water. Post-soaking, seeds were placed in cultivation trays, ensuring proper contact and moisture. After three days, germination was observed, and the number of germinated seeds was recorded for both groups. The cumulative germination rates for both groups were calculated and compared to deduce the impact of the engineered E. coli's IAA production on seed germination.

Table 1. Plant seed germination rate statistics.

![](https://static.igem.wiki/teams/5015/wiki/engi/img8.png)

Learn

For pine willow seeds, the test group exhibited an 88% germination rate, while the control group had a 76% rate. Similarly, the soybean seeds in the test group had a 90% germination rate, in contrast to the control group's 74%. These findings underscored the positive influence of IAA from engineered E. coli on seed germination. However, challenges arose with wheat, barley, and triticale seeds, as neither group showed the expected germination. This anomaly indicated potential issues in the cultivation protocol. To refine the process and ensure more accurate outcomes in future experiments, a comprehensive optimization of the plant cultivation protocol is planned. Every step, from growth medium selection to optimal cultivation conditions, will be re-evaluated to guarantee improved results in subsequent trials.

Cycle 5: Effect of IAA-producing engineered E. coli on Arabidopsis thaliana Growth

Design

In this study, we try to assess the impact of E. coli, specifically engineered to overexpress IAAH and IAAM, on the growth patterns of Arabidopsis thaliana. We posited that the supernatant from this modified bacterial strain could potentially bolster the growth of Arabidopsis thaliana when compared to control group.

Build

After autoclaving, the MS agar medium was cooled to approximately 50°C. Subsequently, 10% (v/v) of the supernatant from the IAA-producing E. coli was incorporated to prepare the MS solid medium enriched with the bacterial supernatant. Under sterile conditions, disinfected Arabidopsis thaliana seeds were evenly sown on the MS solid growth medium containing the supernatant using 1 mL pipette (test group). Simultaneously, a control group (CK group) was established, where seeds were sown on standard MS solid growth medium.

Test

The sown plates were incubated at room temperature, adhering to a 12-hour light-dark cycle. To ensure the robustness of the experimental results, each treatment was replicated thrice. On the 7th day, seedlings were isolated using sterile tweezers, and their root lengths were measured with precision using a calibrated ruler. All measurements were systematically documented. The acquired data was processed using Graphpad Prism software, represented visually as bar graphs. An unpaired student's t-test was employed to discern the statistical significance between the test and CK groups.

Table 2. Arabidopsis photos and root length statistics

![](https://static.igem.wiki/teams/5015/wiki/engi/img9.png)

Learn

The experimental results showed that the root length of the experimental group increased by 2.54 cm on average, while that of the CK group was 1.72 cm, which indicated that the supernatant of the engineered strain had a positive effect on the growth of Arabidopsis thaliana. hese findings pave the way for further exploration and optimization of the use of IAA-producing bacteriA in enhancing plant growth. In the future, we consider extending the experiment to study the long-term effects of IAA producing E. coli on plant growth. Further research into the mechanism of action of IAA and its practical applications in areas such as agriculture and ecological restoration will help to better understand the potential impact of this research.

Reference

[1]Spaepen S et al. (2007) Indole-3-acetic acid in microbial and microorganism-plant signaling. Federation of European Microbiological Societies Microbiology Reviews 31: 425–448.

[2]Gang, S., Sharma, S., Saraf, M., Buck, M., & Schumacher, J. (2019). Analysis of Indole-3-acetic Acid (IAA) Production in Klebsiellaby LC-MS/MS and the Salkowski Method. Bio Protoc, 9(9), e3230. https://doi.org/10.21769/BioProtoc.3230

[3]https://journals.asm.org/doi/10.1128/AEM.68.2.784-790.2002

CONTENT

[Cycle 1: Production of IAA by the IAM pathway](#a1)
[Design](#b1)
[Build](#b2)
[Test](#b3)
[Learn](#b4)
[Cycle 2: Producing EPS through galU genes](#a2)
[Design](#b5)
[Build](#b6)
[Test](#b7)
[Learn](#b8)
[Cycle 3: The arabinose promoter controling the suicide gene mazF](#a3)
[Design](#b9)
[Build](#b10)
[Test](#b11)
[Learn](#b12)
[Cycle 4: Assessing Engineered E. coli's Influence on Seed Germination via IAA Production](#a4)
[Design](#b13)
[Build](#b14)
[Test](#b15)
[Table 1. Plant seed germination rate statistics.](#b16)
[Learn](#b17)
[Cycle 5: Effect of IAA-producing engineered E. coli on Arabidopsis thaliana Growth](#a5)
[Design](#b18)
[Build](#b19)
[Test](#b20)
[Learn](#b21)
[Reference](#a6)

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