iGEM 2021 — Team ICJFLS

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# Team:ICJFLS/Notebook

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[Human Practices](https://2021.igem.org/Team%3AICJFLS/Human_Practices)
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# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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# Team:ICJFLS/Engineering

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PROJECT

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[JUDGING FORM](https://igem.org/2021_Judging_Form?team=ICJFLS)

# We created some new parts.

In this virus epidemic season, we are lucky to have the opportunity to do some experiments in the laboratory. Although there is a limitation on the number of people in the lab for preventing infection of virus, we cloned some genes involved in the amylose synthesis. For creating parts, they were cloned into the plasmid pSB1C3, hoping they are useful contribution for future iGEMer.

## 1.BBa\_K3867001

This part is ADG1 which serves as the small subunit of ADP glucose pyrophosphorylase (AGPase). The reaction catalyzed by AGPase is as flows:

![](https://static.igem.org/mediawiki/2021/6/6b/T--ICJFLS--Results-1.png)

AGPase catalyzes Glucose-6-phosphate (Glu-6-P) converted to adenosine diphosphate glucose (ADPG). It is a key enzyme in amylose synthesis and catalyzes the first, rate limiting step in amylose biosynthesis. The AGPase gene contains 2 subunits, ADG1 and APL1. ADG1 encodes the small subunit which is the catalytic isoform responsible for AGPase activity. And APL1 encodes the large subunit which is the large subunit playing a regulatory role in the catalytic process.

![](https://static.igem.org/mediawiki/2021/1/12/T--ICJFLS--Engineering-1.png)

Fig.1 The sequence information of ADG1.

For construction the new part, ADG1 (1563bp) was inserted into the vector pSB1C3. PCR method was used to identify the new part, showed in Fig.2.

![](https://static.igem.org/mediawiki/2021/1/1b/T--ICJFLS--Engineering-2.png)

Fig.2 The identification result of ADG1 gene cloning. M: Marker; 1: plasmid of pSB1C3-ADG1; 2: PCR result of ADG1 gene.

When the ADG1 gene was constructed to the expression vector pETDuet-1, ADG1 protein was expressed. His tag was used to purify ADG1 protein. The identification result was showed in Fig.3.

![](https://static.igem.org/mediawiki/2021/7/70/T--ICJFLS--Engineering-3.png)

Fig.3 The expression of ADG1 cloned in pETDuet-1-ADG1 in E. coli, M. Marker; 1. Purified ADG1; 2. The whole protein of E. coli with IPTG induction; 3. The whole protein of E. coli without IPTG induction.

## 2.BBa\_K3867005

For construction the new part BBa\_K3867005, the APL1 gene (1569bp) serving as the large subunit of AGPase, was inserted to the vector pSB1C3 by restriction enzyme (EcoRI and Pstl). Figure 4 shows the sequence information of APL1.

![](https://static.igem.org/mediawiki/2021/f/f2/T--ICJFLS--Engineering-4.png)

Fig.4 The sequence information of APL1.

The Fig.5 showed the identification result for construction of pSB1C3-APL1.

![](https://static.igem.org/mediawiki/2021/5/59/T--ICJFLS--Engineering-5.png)

Fig.5 The identification result of APL1 gene cloning. M: Marker; 1: plasmid of pSB1C3-APL1; 2: PCR result of APL1 gene.

After the APL1 gene was constructed into the expression vector pETDuet-1-APL1, APL1 protein was expressed. His tag was used to purify APL1 protein. The identification result was showed in Fig.6.

![](https://static.igem.org/mediawiki/2021/7/74/T--ICJFLS--Engineering-6.png)

Fig.6 The expression of APL1 cloned in pETDuet-1-APL1 in E. coli, M. Marker; 1. The whole protein of E. coli without IPTG induction; 2. Purified APL1; 3. The whole protein of E. coli with IPTG induction.

## 3.BBa\_K3867007

The part BBa\_K3867007 is about Granule-bound starch synthase 1 (GBSS1). The reaction catalyzed by GBSS1 is as follows:

![](https://static.igem.org/mediawiki/2021/b/bf/T--ICJFLS--Results-4.png)

GBSS1(1883bp) is another key enzyme for the amylose synthesis. It belongs to UDP-Glycosyltransferase superfamily protein. GBSS1 is an enzyme that catalyzes the transfer of glucose from to glucose-containing polysaccharides with α1,4-linkages, which is the main gene controlling amylose synthesis.

In order to construct the new part, GBSS1 (1833bp) gene was inserted into the vector pSB1C3 with restriction enzymes EcoRI and PstI.

![](https://static.igem.org/mediawiki/2021/4/41/T--ICJFLS--Engineering-7.png)

Fig.7 The sequence information of GBSS1.

![](https://static.igem.org/mediawiki/2021/e/ef/T--ICJFLS--Engineering-8.png)

Fig.8 The identification result of GBSS1 gene cloning. M: Marker; 1: plasmid of pSB1C3-GBSS1; 2: PCR result of GBSS1 gene.

After the GBSS1 gene was constructed into the expression vector pETDuet-1-GBSS1, GBSS1 protein was expressed. His tag was used to purify GBSS1 protein. The identification result was showed in Fig.9.

![](https://static.igem.org/mediawiki/2021/c/ca/T--ICJFLS--Engineering-9.png)

Fig.9 The expression of GBSS1 cloned in pETDuet-1-GBSS1 in E. coli, M. Marker; 1. The whole protein of E. coli without IPTG induction; 2. The whole protein of E. coli with IPTG induction; 3. Purified APL1.

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# Team:ICJFLS/Experiments

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# Key experiments protocols

## Plasmid Isolation Protocol

1. The bacteria that contained target plasmid were cultivated at 37°C overnight.

2. Take out 1,500uL solution of cultivated bacteria to a 2mL micro-centrifuge tube. The tube was centrifuged at 12,000rpm for 1 min at room temperature, and then the supernatant was discarded. In this tube, repeating these steps three times to increase the concentration of the bacteria.

3. Add 250uL Solution I buffer to suspend the precipitation. Complete suspension is vital for obtaining good plasmid yield by vortex or pipetting up and down.

4. Add 250uL Solution II buffer, mix gently upside down 4-6 times to make a cleared lysate. A 2-3 min incubation at room temperature may be necessary. However, this step should not be more than 5 min, and avoid vigorous mixing as doing so will shear chromosomal DNA and lower plasmid purity.

5. Add 350uL Solution III buffer, mix gently and fully upside down 6-8 times, centrifuge the tubes at 12,000rpm for 10 min at room temperature.

6. Put a HiBind DNA Mini Column into a 2mL Collection Tube.

7. The supernatant in step 4 is transferred to the Mini Column, and centrifuged at 12,000rpm for 1 min, the filtrate is discarded.

8. Put the Column back into the Collection Tube, add 500uL HBC Buffer, centrifuge at 12,000rpm for 1 min, the filtrate is discarded.

9. Put the Column back into the Collection Tube, add 700uL Washing buffer, centrifuge at 12,000rpm for 1 min, the filtrate is discarded. Repeat the washing step once.

10. Put the Column back into the Collection Tube and centrifuge at 12,000rpm for 2 min.

11. Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2 min at room temperature. Then add 50uL ddH2O to the center of the Column matrix, and leave it at room temperature for 1 min. Centrifuge at12,000 rpm for 1 min. Discard the HiBind DNA Mini Column.

12. Measure concentration of extracted plasmid DNA by Nano drop 1000, and store plasmid DNA at -20°C.

## Plasmid DNA Restriction Endonuclease Digestion

DNA for downstream applications is usually digested with restriction endonucleases. Type II restriction enzymes are the most widely used in molecular biology application.

The reaction volume are carried out in 20µL.

1. Add components to a clean tube in the order as follow:

1 µL DNA (1 µg/µL)

2 µL 10x buffer

1 µL each restriction enzyme (EcoR1 and Pst 1)

15 µL ddH2O

2. Mix gently and fully, and incubate the tube at certain temperature (37°C) for 1 h.

3. Stop the reaction by heat inactivation (65°C for 15 min).

4. The digested DNA is ready for use in research applications.

## DNA Extraction from Agarose Gel

Buffer Preparation. Before starting, add 24mL 100% ethanol (26mL 95% ethanol) to the 6mL DNA Wash Buffer concentrate. Add 96mL 100% ethanol (104mL 95% ethanol) to the 24mL DNA Wash Buffer concentrate.DNA Wash Buffer included with D4001S and D4001T is supplied ready to use and does not require the addition of ethanol prior to use.

1. The digested DNA was run in 1% agarose gel electrophoresis.

2. Excise the DNA fragment from the agarose gel using a razor blade, scalpel or other device and transfer it in to a 1.5 mL micro-centrifuge tube.

3. Add 3 volumes of ADB to each volume of agarose excised from the gel (e.g. for 100µL (mg) of agarose gel slice adding 300µL of ADB).

4. Incubate it at 56°C for 5-10 min until the gel slice is completely dissolved. Vortex the tube every 2-3 min.

5. Transfer the melted agarose solution to a Zymo-Spin™ Column in Collection Tube.

6. Centrifuge for 1 min at 12,000 rpm. The filtratewas discarded.

7. Add 500µL of DNA Wash Buffer to the column and centrifuge for 1 min. The filtrate was discarded. Repeat the wash step once.

8. Put the Column back into the Collection Tube and centrifuge at 12,000rpm for 2 min.

9. Put the Column into a new 1.5mL micro-centrifuge tube and leave it for 2 min at room temperature.

10. Add 20µL ddH2O directly to center of the column matrix, centrifuge for 1 min to elute DNA. Ultra-pure DNA is now ready for use.

## DNA Ligation Reaction

The plasmid and insert DNAs (APL1, ADG1 and GBSS1, respectively) are digested with endonucleases prior to beginning a ligation. Following gel-purification of digested plasmid and insert DNAs, DNA concentrations are measured by a spectrophotometer to determine the concentration of the purified plasmid and insert.

1. Add components to a clean tube in 20µL volume in the order as follow:

9 µL ddH2O

3 µL Promoter DNA

5 µL Plasma DNA

1 µL 10x buffer

1 µL T4 DNA Ligase

2. Mix them gently and fully, and incubate the tube at 22°C for 20 min. The ligation product is ready to use.

## The Competent Cell Preparation

1. Streak the E. coli stock on a LB-agar plate. Incubate the plate at 37°C overnight.

2. Pick a single well-isolated colony and inoculate it into 3mL of LB broth solution. Incubate it at 37°C overnight (more than 16 h) with shaking at 220 rpm.

3. Transfer 250uL of the saturated overnight solution to a sterile 50mL polypropylene tube containing 25mL of LB medium. Incubate the E. coli at 37°C with shaking at 220 rpm until OD600 reaching 0.6. This usually takes 2.5 h. Check the OD every 30 min after 1 h to avoid overgrowth.

4. When the medium reaches an OD600 of 0.6, chill the tube on the ice for 30 min and then centrifuge at 8000rpm for 1min at 4°C, discard the supernatant.

5. Resuspend the E. coli in 2.5mL of ice-cold TSS solution with gentle swirling. Incubate on ice for 10 min. Aliquot in 50uL per tube. Now the competent cells are ready to be transformed.

Note: Transformation and Storage Solution (TSS) contains LB medium 85% 8.5mL, PEG (wt/vol, Mw 8000) 10% 1.0g, DMSO (vol/vol) 5% 500μL, and MgCl2(pH 6.5) 50mM 500μL.

## Transforming the competent cells

1. Thaw TSS cells on ice.

2. Add 5uLDNA in TSS cells, pipette gently to mix (1μl of prepped plasmid is used as control).

3. Incubate it on ice for 30 min with occasional mixing.

4. Heat shock at 42°C for 90 sec.

5. After heat shock, put it on ice for 2 min, add 0.8mL LB medium into the cells.

6.Shake and incubate at 37°C for 60 min at 120 rpm.

7.Take out 100uL medium and spread them on the appropriate agar plates with certain antibiotic.

8.Incubate the plates at 37°C overnight.

## PCR protocol

In the PCR protocol, reaction components are assembled as described below. The final volume should be 20µL.

In the PCR protocol, reaction components are assembled as described below. The final volume should be 20µL.

1. Thaw all reagents on ice.

2. Assemble reaction mix into 20µL volume in a thin walled 0.2mL PCR tubes as follows:

8uL ddH2O

1 µL DNA Template

0.5uL Primer F

0.5uL Prmer R

10uL 2x Taq mixture

3. Prepare negative control reaction without template DNA.Prepare positive control reaction with template of known size and appropriate primers.

4. Gently mix by tapping tube. Briefly centrifuge to settle tube contents.

5. Put the tube into a PCR instrument.

6. Set up the following PCR program: initial denaturing at 94°C for 4 min, followed by 30 cycles of 94°C for 30 sec, 55°C for 30 sec, and 72°C for 1 min, plus a final extension at 72°C for 10 min.

7. Check the PCR product by agarose gel electrophoresis.

## Purification of His/S-tagged protein

All the procedures were performed at 4°C or on ice unless otherwise specified.

1. 10mL BL21 cells transformed with a pETDuet-APL1-ADG1 or pET28a-GBSS1 were cultured at 37°C until an OD600 of approx. 0.8.

2. Add IPTG to the culture (final 0.67mM IPTG)

3. Cultivate at 30°C for 2h.

4. Collect cells in 5mL tube, centrifuge at 5000rpm at 15 min.

5. Suspend cells in 5mL of TBS, transfer the content to 1.5mL tube.

6. Collect cells at 10000rpm at 1 min. The supernatant was discarded.

7. Add 500uL of ice-cold lysis buffer to the cell pellet.

8. Sonicate cell (4°C).

9. Clarify lysate by centrifugation at to 10000rpm at 10 min.

10. After centrifugation, save 20uL of supernatant for SDS-PAGE sample.

11. Transfer supernatant to the 1.5mL tube containing pre-balanced resin bead gel.

12. Shake gently (4°C) for 30 min.

13. Centrifuge the tube 1200rpm for 30 sec. The supernatant was discarded.

14. Wash resin bead gel with washing buffer, Centrifuge the tube 1200rpm for 30 sec. The supernatant was discarded. Repeat it once.

15. Elute the protein 3 time with 20uL of elution buffer. It is ready to prepare the SDS-PAGE sample.

## SDS Gel Electrophoresis

1. Make up 30 mL of running gel by adding components to a clean glass in the order as follow:

12.3 mL H2O

7.5 Ml 1.5 M Tris-HCl, pH 8.8

0.15 mL 20% (w/v) SDS

9.9 mL Acrylamide/Bis-acrylamide(30%/0.8% w/v)

0.15 mL 10% (w/v) ammonium persulfate (APS)

0.02 mL TEMED

2. Mix the ingredients mentioned above.

3. Pour the solution quickly into a gel casting form, and leave about 2cm below the bottom of the comb for the stacking gel. Then layer the top of the gel with water very carefully. Wait for about 30min for the gel to polymerize completely.

4. While waiting, mix the reagents for the stacking gel (4% Acrylamide) by adding components to a clean glass in the order as follows:

3.075 mL H2O

1.25 mL 0.5 M Tris-HCl, pH 6.8

0.025 mL 20% (w/v) SDS

0.67 mL Acrylamide/Bis-acrylamide (30%/0.8% w/v)

0.025 mL 10% (w/v) ammonium persulfate (APS)

0.005 ml TEMED

5. Mix the ingredients mentioned above.

6. Remove the water on the top of running gel, and pour the stacking gel on top of the running gel. Insert the combs by trying not to produce bubbles stuck underneath and allow another 30 min - 1h for complete polymerization.

7. Preparesamples: mix the protein 4:1 with the loading buffer. Heat the sample by Boiling for 5-10 min.

8. Run gel: clamp the gel and fill both buffer chambers with gel running buffer. Pipet the sample and molecular weight standard marker into the gel. Attach the power leads and run the gel until the blue dye reaches the bottom.

9. Visualize the proteins using Coomassie Brilliant Blue.

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# Team:ICJFLS/Results

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[Human Practices](https://2021.igem.org/Team%3AICJFLS/Human_Practices)
[Integrated Human Practices](https://2021.igem.org/Team%3AICJFLS/Human_Practices#integrated)
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[JUDGING FORM](https://igem.org/2021_Judging_Form?team=ICJFLS)

# 1.Gene cloning from Arabidopsis thaliana

Starch consists of amylopectin and amylose. Amylopectin is a branched glucan polymer with α-1,6 glucosidic bonds that connect linear chains, which is synthesized by the catalytic action of several enzymes, such as ADP glucose pyrophosphorylase (AGPase), starch synthases (SSs), starch branching enzymes (BEs), and starch debranching enzymes (DBEs). Differently, amylose is essentially linear and composed of 1,4-linked α-D-glucan chains; AGPase and granule-bound starch synthase (GBSS) are involved in amylose production.

To realize the production of amylose synthesis in E. coli, we synthesized 2 genes related to the synthesis of amylose, which were ADP glucose pyrophosphorylase (AGPase) containing 2 subunits ADG1 and APL1, and granule-bound starch synthase 1 (GBSS1) based on the sequence from Arabidopsis thaliana.

## 1.1 ADP glucose pyrophosphorylase (AGPase)

![](https://static.igem.org/mediawiki/2021/6/6b/T--ICJFLS--Results-1.png)

AGPase catalyzes Glucose-6-phosphate (Glu-6-P) converted to adenosine diphosphate glucose (ADPG), which is a key enzyme in amylose synthesis and catalyzes the first, rate limiting step in amylose biosynthesis. The ADP glucose pyrophosphorylase gene contains 2 subunits, ADG1 and APL1. ADG1encodes the small subunit and APL1 encodes the large subunit of ADP-glucose pyrophosphorylase. The large subunit plays a regulatory role whereas the small subunit (ADG1) is the catalytic isoform responsible for ADP-glucose pyrophosphorylase activity.

In order to insert ADG1(1563bp) and APL1(1569bp) genes to the bi-expressing vector pETDuet-1, the ADG1 and APL1 genes were synthesized by adding restriction enzyme sites sequences at the 5’ and 3’ ends, which were EcoRI and PstI on flanking sequence of ADG1 gene, NdeI and Xhol on flanking sequence of APL1 gene, respectively.

First, we constructed the plasmid pETDuet-1-ADG1. The ADG1gene and vector pETDuet-1were digested by EcoRI and PstI. Gel extraction of digestion production of the ADG1gene and vector pETDuet-1 were performed, and were ligated together to construct the plasmid pETDuet-1-ADG1. Figure 1 shows the identification result for the plasmid pETDuet-1-ADG1 construction.

![](https://static.igem.org/mediawiki/2021/9/93/T--ICJFLS--Results-2.png)

Fig.1. The identification result of plasmid pETDuet-1-ADG1construction.

M. Marker; 1. Plasmid pETDuet-1-ADG1 digested by EcoRI and PstI; 2. Plasmid pETDuet-1-ADG1.

Second, the APL1 gene was inserted to the plasmid pETDuet-1-ADG1 by restriction enzyme (NdeI and Xhol) digestion method, similar to those mentioned above. Figure 2 shows the identification result of the plasmid pETDuet-1-ADG-APL1 construction.

![](https://static.igem.org/mediawiki/2021/3/3d/T--ICJFLS--Results-3.png)

Fig.2. The identification result of plasmid pETDuet-1-ADG1-APL1construction. M. Marker; 1.

Plasmid pETDuet-1-ADG1-APL1; 2. Plasmid pETDuet-1-ADG1-APL1 digested by NdeI and Xhol.

## 1.2 Granule-bound starch synthase 1 (GBSS1)

![](https://static.igem.org/mediawiki/2021/b/bf/T--ICJFLS--Results-4.png)

Another enzyme for the amylose synthesis is Granule-bound starch synthase 1 (GBSS1), which belongs to UDP-Glycosyl transferase super family protein.

GBSS1 is an enzyme that catalyzes the transfer of glucose to glucose-containing polysaccharides with α1,4-linkage, which is the main gene controlling amylose synthesis.

In order to insert GBSS1(1833bp) gene to the vector pET-28a (+), the GBSS1 gene was synthesized by adding restriction enzyme sites sequences at the 5’ and 3’ ends, which were EcoRI and Xhol on flanking sequence of GBSS1 gene, respectively.

The GBSS1 gene was inserted to the vector pET-28a (+) by restriction enzyme digestion method. Figure 3 shows the identification result for the plasmid pET-28a (+)-GBSS1 construction.

![](https://static.igem.org/mediawiki/2021/2/2e/T--ICJFLS--Results-5.png)

Fig.3. The identification result of plasmid pET-28a (+)-GBSS1 construction.

M. Marker; 1. Plasmid pET-28a (+)-GBSS1; 2. Plasmid pET-28a (+)-GBSS1 digested by EcoRI and Xhol.

# 2.The expression of ADG1, APL1 and GBSS1 in E. coli

In order to synthesize amylose in E. coli, the two plasmids of pETDuet-1-ADG1-APL1 and pET-28a (+)-GBSS1 were co-transformed into E. coli (BL21) cells. This is a key step to realize the production of disposable and biodegradable straw made from amylose.

## 2.1 The expression of ADP glucose pyrophosphorylase (AGPase)

The ADG1 and APL1 were cloned into the EcoRI/PstI and NdeI/Xhol sites of pETDuet-1. Overnight cultures of E. coli (BL21) cells transformed with plasmid pETDuet-1-ADG1-APL1 were cultured in fresh LB medium containing ampicillin. Soluble cytoplasmic proteins were prepared from BL21 cells with and without IPTG induction, respectively. The purification of recombinant protein ADG1 with a 6xHis was performed by His-tag, and APL1 with fused S-tag in its C-terminus by S-tag according to the instruction. The SDS-PAGE result shows the ADG1 and APL1 expression in E. coli in figure 4 and 5, respectively.

![](https://static.igem.org/mediawiki/2021/2/2d/T--ICJFLS--Results-6.png)

Fig.4. The expression of ADG1 in transformed E. coli.

M. Marker; 1. Purified ADG1 protein (57kD) with IPTG induction; 2. Whole proteins of E. coli with IPTG induction; 3. Whole proteins of E. coli without IPTG induction.

![](https://static.igem.org/mediawiki/2021/8/88/T--ICJFLS--Results-7.png)

Fig.5. The expression of APL1 in transformed E. coli.

M. Marker; 1. Whole proteins of E. coli without IPTG induction; 2. Purified APL1 protein (57.5kD) with IPTG induction; 3. Whole proteins of E. coli with IPTG induction.

## 2.2 The expression of Granule-bound starch synthase 1 (GBSS1)

The GBSS1 was cloned into the EcoRI/Xhol sites of pET-28a (+). Overnight cultures of E. coli (BL21) cells transformed with plasmid pET-28a (+)-GBSS1 were cultured in fresh LB medium containing kanamycin. Soluble cytoplasmic proteins were prepared from transformed BL21 cells containing pET-28a (+)-GBSS1 with and without IPTG induction, respectively. The purification of GBSS1 protein was performed by His-tag according to the instruction. The SDS-PAGE result shows the GBSS1 expression in E. coli in figure 6.

![](https://static.igem.org/mediawiki/2021/5/5f/T--ICJFLS--Results-8.png)

Fig.6. The expression of GBSS1 in transformed E. coli.

M. Marker; 1. Whole proteins of E. coli without IPTG induction; 2. Whole proteins of E. coli with IPTG induction; 3. Purified GBSS1 protein (67kD) with IPTG induction.

# 3.The optimization of culture media conditions of E. coli

After the identification of the recombinant genes construction, we hoped to culture the E. coli to optimize some conditions for high amylose production yield. We searched some references, and figure out that the iodine binding e assay in a 96-well plate format for measuring amylose at both OD620 nm and OD510 nm. The initial testing to produce a standard curve, utilizing amylose as a standard, was scaled to a 96-well plate format. The dual wavelength method exhibited a very high coefficient of determination (R2= 0.993) with plate to plate repeatability on slope and intercept values. The level of accuracy and repeatability of the standard curves allows for quantification of amylose in samples.

We found that the temperature, pH value, fermentation time, inoculum size, and the carbon sources are the important conditions. So, we tried to culture the E. coli for amylose production with different conditions. And the results are showed in the following figures (Fig. 7 - Fig. 11).

![](https://static.igem.org/mediawiki/2021/6/6b/T--ICJFLS--Results-9.png)

Fig.7. The effect of temperature on amylose production.

![](https://static.igem.org/mediawiki/2021/6/6c/T--ICJFLS--Results-10.png)

Fig.8. The effect of pH value on amylose production.

![](https://static.igem.org/mediawiki/2021/c/ca/T--ICJFLS--Results-11.png)

Fig.9. The effect of fermentation time on amylose production.

![](https://static.igem.org/mediawiki/2021/6/63/T--ICJFLS--Results-12.png)

Fig.10. The effect of inoculum size on amylose production.

![](https://static.igem.org/mediawiki/2021/1/1d/T--ICJFLS--Results-13.png)

Fig.11. The effect of carbon sources on amylose production.

Glu, Glucose; Man, Mannitol; Suc, Sucrose; Fru, Fructose; GluA, glucosamine; 2-DOG, 2-deoxyglucose; 3-OMG, 3-O-methylglucose.

The optimization experiment results indicated that 37°C, pH7.6, fermentation 22h, 14% inoculum size, and glucose serving as the carbon source are the best culture conditions for high amylose production yield in E. coli. And we know that for the amylose production in E. coli, it still need further experiments to realize disposable and biodegradable straw made from amylose produced by E. coli.