iGEM 2023 — Team thinker-guangdong

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{% block title %}Notebook{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4987/wiki/cover/note.jpg{% endblock %}
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{% block detail\_title\_a1 %}Experimental preparation for week 1{% endblock %}
{% block detail\_content2 %}Experimental plan: Prepare the medium and experimental consumables and activate the plasmid-carrying strain
Experimental procedure:
1. Guide to the use of the ultra-clean table: Open the ultra-clean table, place the centrifuge tube plate, test tube rack and Marker pen, and conduct alcohol disinfection and sterilization. Next, UV irradiation was performed for 30 minutes.
2. Sterilization process: Prepare the Eppendorf tubes, PCR tubes, and centrifuge tubes and wrap them in gauze. Pack 2 boxes each of the large, medium, and small gun heads, then wrap the shell in brown paper and secure the gun head box in place using string or rubber bands. With the aid of pre-prepared LB medium, aliquots were made into 20 tubes and configured with 50 mL x 2 bottles of LB liquid medium. Place the items in a sterilizer and sterilize them at 121 ° C for 15 minutes (about 1.5 hours). However, an experimental accident occurred in the process of filling the liquid medium: the medium was spilled on the ultra-clean table, but it had been properly cleaned up.
3. Strain activation steps: 1 tube of E. coli DH5α/ empty vector, 1 tube of E. coli DH5α/ pCspA gene, 1 tube of E. coli DH5α/ureABC and 1 tube of E. coli DH5α/ galU gene. On the ventilated ultra-clean table, the LB test tube was put into the table after sterilization. The alcohol lamp was lit, and the test tube orifice and plug were burned. 100 μL of preserved bacterial solution was added to each test tube, followed by 2.5 μL of Amp antibiotic mother solution, and the test tube orifice and plug were burned again. The orifice of the tube was sealed tightly, the tube was bundled, and the tube was placed on a shaker at 150 rpm and 37 ° C for overnight culture.
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{% block detail\_img1 %}https://static.igem.wiki/teams/4987/wiki/notebook/img1.jpg{% endblock %}
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{% block detail\_title\_a2 %}At week 2, the template was extracted and plasmid construction was started{% endblock %}
{% block detail\_content4 %}Experimental plan: Plasmid was extracted and the target fragment was amplified by PCR
Experimental procedure:
1. Extract plasmid:
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{% block detail\_img2 %}https://static.igem.wiki/teams/4987/wiki/notebook/img2.jpg{% endblock %}
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2 Dilute the primers
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) Three target DNA fragments were amplified using high fidelity enzyme Pfu. The reaction system is as follows:
Pfu 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
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{% block detail\_title\_a3 %}3 weeks agarose gel electrophoresis, plastic recycling, double enzyme, recycling{% endblock %}
{% block detail\_content6 %}Experimental plan: Agarose gel electrophoresis, gel recovery, double enzyme digestion, recovery
Experimental procedure:
1. Agarose gel electrophoresis: The above PCR products were added to the prepared agarose gel macrowell, together with 5μL DL 5000 DNA marker, and separated by electrophoresis in the electrophoresis tank (voltage set at 120V for 20 minutes). After the electrophoresis, the gel imaging system was used to observe the gel results, and then the target DNA was cut out and placed in a 2 mL centrifuge tube for labeling. The gel diagram of three gene fragments is shown below.
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{% block detail\_img3 %}https://static.igem.wiki/teams/4987/wiki/notebook/img3.png{% endblock %}
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2. Gel recovery: The DNA gel was recovered according to the instructions of the gel recovery kit. First, 500 microliters of equilibrium solution BL was added to the adsorption column CA2, the waste solution was poured out after centrifugation for 1 minute, and the adsorption column was put back into the collection tube. Then, the single band of target DNA was cut out of the agarose gel and placed into a clean centrifuge tube. Next, add an equal amount of solution PN to the gel block and place it 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 and left at room temperature for 2 minutes before centrifugation for 1 minute. Then, add 600 microliters of bleach solution PW to the adsorption column CA2 (please check if absolute ethanol has been added before use), centrifuge for 1 minute, and repeat the rinsing step twice before drying. Next, an appropriate amount of elution buffer EB was added to the middle of the adsorption membrane in a hanging drop and left at room temperature for 2 minutes. After 2 min of centrifugation, the DNA solution was collected. Finally, the concentration of recovered DNA was determined using Nanodrop.
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{% block detail\_img4 %}https://static.igem.wiki/teams/4987/wiki/notebook/img4.jpg{% endblock %}
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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.
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{% block detail\_title\_a4 %}At week 4, competent cells were prepared{% endblock %}
{% block detail\_content9 %}Experimental plan: E. coli DH5α and Rosetta were activated to prepare competent cells
Experimental procedure:
1. Activated strains: including Escherichia 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) The ultra-clean stage was opened, and 1 mL gun tip, 200 μL gun tip, 5 mL gun tip, 2mL and 30 mL centrifuge tubes, and 2 x 50 mL LB medium were added 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℃.
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{% block detail\_title\_a5 %}At week 5, the cells were connected and transformed{% endblock %}
{% block detail\_content10 %}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.
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{% block detail\_title\_a6 %}At the 6th week, the successful plasmid was extracted and expanded{% endblock %}
{% block detail\_content11 %}Experimental plan: colony PCR verification, expansion of positive clones, extraction and verification of the correct plasmid and transformation
Experimental procedure:
1. PCR validation: Six colonies per plate were labeled. 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.
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.
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{% block detail\_title\_a7 %}The activity of UreABC and the production of CaCO3 were measured at week 7{% endblock %}
{% block detail\_content12 %}Experimental plan: The activity of UreABC and the production of CaCO3 were measured
Experimental procedure:
1. The seed liquid carrying the plasmid was cultured in LB medium supplemented with 100 μg/mL ampicillin for 16 hours, 10 mL of bacteria were collected, centrifuged (10000g, 5 min, 4°C), the medium supernatant was discarded, and the bacteria were resuspended in 20 mM Tris-HCl (pH 7.0). Ultrasonic fragmentation (500W, 1s sonication, 3s interval, 20min) was carried out under ice precondition. Crude enzyme extracts were collected and protein concentrations were determined using the Bradford method. UreABC activity was measured using the urease activity assay kit (ml076473, mlbio). Production of 1μg NH3-N per minute per mg of protein was defined as one unit of enzyme activity. The results are shown below in panel a
2 Cells of each test strain were seeded into medium containing 10 mL urea-cacl, adjusted to OD600 of 0.1, and incubated at 25 ° C for 4 hours. The culture medium of the recombinant strain was supplemented with NiCl2 (1 mM) and appropriate antibiotics. All samples were pre-washed with 6 M HCl. The CaCO3 concentration was subsequently determined using EDTA titration as shown in Figure b.
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{% block detail\_img5 %}https://static.igem.wiki/teams/4987/wiki/notebook/img5.png{% endblock %}
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{% block detail\_title\_a8 %}Ni2+ dependence of UreABC active site was measured at week 8{% endblock %}
{% block detail\_content14 %}Experimental plan: Ni2+ dependence of UreABC active site of urease was measured
Experimental procedure:
1. The seed liquid carrying the plasmid was cultured in LB medium supplemented with 100 μg/mL ampicillin and different concentrations of NiCl2 for 16 hours, 10 mL of bacteria were collected, and urease activity was measured as described previously. The results showed that UreABC activity was Ni2+ concentration dependent, but greater than 5 mM resulted in cytotoxicity.
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{% block detail\_img6 %}https://static.igem.wiki/teams/4987/wiki/notebook/img6.png{% endblock %}
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{% block detail\_title\_a9 %}Testing of cold-induced promoter cspA and EPS production at week 10{% endblock %}
{% block detail\_content16 %}Experimental plan: The mRFP gene was cloned and the cold-induced promoter and EPS production were tested
Experimental procedure:
1. The mRFP gene was cloned downstream of the cold-induced promoter cspA as described above. The plasmid was introduced into Escherichia coli, inoculated into LB medium, and grown at 37 ° C /25 ° C and 180 rpm for 16h. Absorbance (OD600) and mRFP fluorescence intensity (excitation λ: 584nm/ 10nm, emission λ: 607/10nm) data were measured using a microplate reader (Thermo Fisher Scientific, USA). The relative fluorescence unit (RFU) was obtained by dividing the row mRFP fluorescence intensity by the OD600 value, as shown below.
2.
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{% block detail\_img7 %}https://static.igem.wiki/teams/4987/wiki/notebook/img7.png{% endblock %}
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{% block detail\_title\_a10 %}The effect of the GalU gene on EPS levels was measured at week 11{% endblock %}
{% block detail\_content18 %}Experimental plan: To measure the effect of the GalU gene on EPS levels
Experimental procedure:
1. The seed solution carrying the plasmid was inoculated into LB medium and grown for 16h at 37 ° C and 180 rpm. Using LB to adjust the bacterial OD600=1, 10 mL of the bacterial solution was taken to detect the EPS production using the anthrone sulfuric acid method, and the results are shown below.
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CONTENT

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4987/wiki/cover/engi.jpg{% endblock %}
{% block detail\_content1 %}{% endblock %}
{% block detail\_title\_a1 %}Overview{% endblock %}
{% block detail\_content2 %}Synthetic biology is inherently engineering-oriented. By incorporating engineering principles into the study of life sciences, synthetic biology has established standardized, modular, and systematic research approaches. In our project, we have designed three distinct modules, which are subsequently integrated into a chassis and subjected to efficacy testing. This process follows a cycle of design, construction, testing, and learning, involving the expression of urease through UreABC, utilization of the low-temperature inducible promoter (pCspA), and incorporation of the galU gene. Through multiple iterations, we have successfully constructed this engineered microorganism.
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{% block detail\_title\_a2 %}Cycle 1：Preliminary concept——UreABC {% endblock %}
{% block detail\_content3 %}{% endblock %}
{% block detail\_title\_b1 %}Design{% endblock %}
{% block detail\_content4 %}We utilized the ureA, ureB, and ureC genes derived from Bacillus subtilis to express urease and assess its expression level in CaCO3 formation. The principle and components of this process are illustrated in Figure 1.
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{% block detail\_img1 %}https://static.igem.wiki/teams/4987/wiki/engi/img1.png{% endblock %}
{% block detail\_content5 %}Figure 1 Schematic diagram of UreABC.
{% endblock %}
{% block detail\_title\_b2 %}Build{% endblock %}
{% block detail\_content6 %}UreABC was cloned into the pET23b vector, as shown in Figure 2. Simultaneously, the growth status of the UreABC recombinant strains, the production of CaCO3, and the dependence on nickel ions were tested.
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{% block detail\_img2 %}https://static.igem.wiki/teams/4987/wiki/engi/img2.jpg{% endblock %}
{% block detail\_content7 %}Figure 2 Design of UreABC.
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{% block detail\_title\_b3 %}Test{% endblock %}
{% block detail\_content8 %}The UreABC engineered strains were cultured in large scale, and crude enzyme extracts were collected to determine protein concentration. Finally, the activity of UreABC was measured. As shown in Figure 3a, the urease activity of pT7-UreABC reached 0.4 mg/ml. Meanwhile, the strains were inoculated in urea-CaCl2 medium supplemented with NiCl2 and antibiotics. All samples were pre-washed with hydrochloric acid. The concentration of CaCO3 was then determined using a titration method. As shown in Figure 3b, pT7-UreABC generated more calcium carbonate precipitation. In addition, we studied the dependence of UreABC activity on Ni2+ (Figure 3c). The recombinant strains were cultured in LB medium supplemented with ampicillin and different concentrations of NiCl2. Bacteria were then collected, and urease activity was measured. The results showed that UreABC activity depended on the concentration of Ni2+, but concentrations higher than 5 mM exhibited cell toxicity. Introduction of the urease gene through pT7-UreABC resulted in a significant increase in urease activity. Therefore, more calcium carbonate precipitation was observed. Additionally, it was found that an appropriate concentration of nickel ions could enhance urease activity. However, nickel ions at concentrations higher than 5 mM exhibited toxic effects on bacterial cells.
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{% block detail\_img3 %}https://static.igem.wiki/teams/4987/wiki/engi/img3.png{% endblock %}
{% block detail\_content9 %}Figure 3 Growth status of engineered bacterial strain with UreABC gene, CaCO3 production, and dependency on nickel ions.
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{% block detail\_title\_b4 %}Learn{% endblock %}
{% block detail\_content10 %}Through the above experiments, it was found that the pT7-UreABC engineered strain produced more calcium carbonate precipitation compared to the wild type, and an appropriate concentration of nickel ions could enhance urease activity.However,this experiment was conducted at 37℃, but in reality, the temperature will be lower than 37℃. Consequently, enzyme activity and gene expression levels are likely to decrease. Therefore, it is necessary to design a cold-inducible promoter to enhance gene expression at lower temperatures.
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{% block detail\_title\_a3 %}Cycle 2: Low-temperature expression——Cold-inducible promoter (pCspA){% endblock %}
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{% block detail\_title\_b5 %}Design{% endblock %}
{% block detail\_content12 %}From Pcold E. coli, we discovered a cold-inducible promoter that allows expression at low temperatures. Therefore, we have decided to introduce pCspA and couple it with mRFP downstream of the promoter for detection purposes.
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{% block detail\_title\_b6 %}Build{% endblock %}
{% block detail\_content13 %}The CspA promoter was extracted from Pcold E. coli and inserted upstream of the urease gene, as shown in Figure 4. It was then introduced into E. coli Rosetta. In the pSB1A3 vector, mRFP was cloned downstream of the cold-inducible promoter cspA and transformed into E. coli Rosetta. The transformed cells were inoculated into LB media and cultured. After a certain period, the bacterial liquid concentration and mRFP fluorescence intensity were measured using an microplate reader. The fluorescence intensity expressed per unit of bacterial cell was calculated.
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{% block detail\_img4 %}https://static.igem.wiki/teams/4987/wiki/engi/img4.jpeg{% endblock %}
{% block detail\_content14 %}Figure 4 Design of Cold-inducible promoterp(pCspA)
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{% block detail\_title\_b7 %}Test{% endblock %}
{% block detail\_content15 %}As shown in Figure 5a, the growth of urease at 25 ° C and 37 ° C respectively. We can see that OD600 at 37 ° C (about 1.7) is significantly higher than OD600 at 25 ° C (about 0.9). The absorbance and fluorescence intensity of mRFP were measured using an microplate reader. Finally, the relative fluorescence unit (RFU) was obtained by dividing the fluorescence intensity of row mRFP by OD600. As shown in Figure 5b and5c, the fluorescence intensity of mRFP gene expression coupled at different temperatures showed that the fluorescence of mRFP at 25 degrees (100A.U.) was higher than that of mRFP at 37 degrees (50A.U.). It can be seen that after the introduction of CspA promoter, the expression levels were increased under low temperature conditions.
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{% block detail\_img5 %}https://static.igem.wiki/teams/4987/wiki/engi/img5.png{% endblock %}
{% block detail\_content16 %}Figure 5 The test of Cold-inducible promoter
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{% block detail\_title\_b8 %}Learn{% endblock %}
{% block detail\_content17 %}The research findings suggest that the CspA promoter can enhance the expression of the target gene urease. However, pure calcium carbonate is extremely fragile and easily breakable, so it requires the introduction of specific genes to increase the expression level of extracellular polysaccharides. This is done in order to enhance the adhesion between bricks and overall strength during practical applications.
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{% block detail\_title\_a4 %}Cycle 3: Enhance the adhesion between bricks and overall strength——GalU{% endblock %}
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{% block detail\_title\_b9 %}Design{% endblock %}
{% block detail\_content19 %} Clone GalU into the pET28a vector and transform it into E.coli Rosetta.
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{% block detail\_title\_b10 %}Build{% endblock %}
{% block detail\_content20 %}Inoculate the engineered bacteria into LB medium and induce the expression of UDP glucose pyrophosphorylase (GalU) by adding IPTG chemical inducer. Harvest the bacterial culture and measure the extracellular polysaccharide (EPS) yield using the anthrone-sulfuric acid method and compare it with the EPS yield of the wild-type strain.
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{% block detail\_img6 %}https://static.igem.wiki/teams/4987/wiki/engi/img6.png{% endblock %}
{% block detail\_content21 %}Figure 6 Design of galU
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{% block detail\_title\_b11 %}Test{% endblock %}
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{% block detail\_img7 %}https://static.igem.wiki/teams/4987/wiki/engi/img7.png{% endblock %}
{% block detail\_content23 %}Figure 7 Comparison of EPS production data between the wild-type control bacterial group and the pT7-galU engineered bacterial group.
As shown in Figure 7 above, it can be seen that, compared to the wild-type strain, the EPS yield of the engineered strain induced by IPTG is approximately twice as high.
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{% block detail\_title\_b12 %}Learn{% endblock %}
{% block detail\_content24 %}The results of this study provide clear evidence that the expression of the galU gene effectively enhances EPS production in E. coli. Moreover, the successful application of an EPS coating has significantly improved the resilience and strength of the restored cultural relics, thereby minimizing the potential for secondary damage to these precious artifacts.
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{% block detail\_title\_a5 %}Conclusion{% endblock %}
{% block detail\_content25 %}Our ureaABC has undergone three rounds of iterative improvements, ultimately resulting in the development of an engineered strain that is capable of highly efficient expression and production of calcium carbonate precipitation at low temperatures. Furthermore, this strain exhibits enhanced viscosity and toughness. UreABC holds potential for applications in agriculture, environmental conservation, biotechnology, and medicine.
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CONTENT

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{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block cover\_img %}https://static.igem.wiki/teams/4987/wiki/cover/expe.jpg{% endblock %}
{% block page\_content %}{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

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* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

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* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

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You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

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* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

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