iGEM 2023 — Team subcat-china

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**2023/4/15:
Consulting information pertaining to Current Genetic Engineering Research**

Our
team attended an online video conference, consulting knowledge of the field of current genetic engineering
research with Prof.
Wang. We obtained a general understanding of how some techniques of synthetic biology works, and a
comprehensive overview of the general procedure of genetic engineering. Also, we were deeply impressed by
the great influence and future potential synthetic biology has.

**2023/5/27: Online
conference with Mr.** **Wen****for consulting designing and assembly of
parts**

Our
team consulted Dr. Wen for getting a general understanding and inspirational ideas pertaining to designing
and assembly of parts. After the conference, we also knew about some of the techniques of connecting DNA
sequence and the interesting metaphor that synthetic biology is akin to building blocks. We were ready to
start our own fantastic and brand-new journey into synthetic biology.

**2****023/6/15: Online conference for identifying our
research project topic**

We
held an online conference regarding our team research topic for hours, discussing about the resources and
research viewed online pertaining to our topic. We together started our meaningful and valuable topic
which is to enable our construct engineered yeast to self-secrete amylase
and saccharifying enzyme. The problem we desire to address is that the expensive cost of exogenous enzymes
added during alcoholic fermentation. At the same time, we are also trying to introduce sweet potato
residue as a raw material of alcoholic fermentation.

**2****023/6/****27****:** **all materials are
ready with the help of SubCat and WANG XING.**

**2023/7/2****：Team
assembly and Project Kick-Off: Establishing the EcoNzyme Alchemist Team Identity**

Our
team embarked on four significant activities. First, we engaged in ice-breaking exercises, fostering
introductions and facilitating team bonding through interactive games. Next, we held preliminary
elections, allowing prospective team leaders to present their speeches and campaign for the role, which
served as an important step before finalizing our team leadership. Following this, we collectively
undertook a comprehensive review of our project to ensure a common understanding of our goals. To
conclude, we together defined our team identity – "EcoNzyme Alchemist", with an inspiring slogan,
distinctive visual identity (VI), and self-designed
team uniform.

![](https://static.igem.wiki/teams/4845/wiki/notebook/1-1.png)

**2023/7/3-7/4****: Preparation of Luria-Bertani (LB) Solid
Medium, Bacterial Culturing, and Plasmid Extraction**

Our
team successfully prepared the LB and YPD culture media, followed by the inoculation of bacterial strains
CCTCC M44055, GA, temA, and Cas-9. Then, we extracted plasmids from strains X-2, XII-5; XI-2, X-3, as well
as CCTCC M94055, GA, TemA, and pHCas9, and accurately measured their concentrations.

![](https://static.igem.wiki/teams/4845/wiki/notebook/2-1.png)
![](https://static.igem.wiki/teams/4845/wiki/notebook/2-2.png)

**2023/7/5****: Exploring Molecular Biology: A Hands-On
Journey through PCR and Gel Electrophoresis**

Our
team delved into several fundamental molecular biology techniques today. We studied the principles of
Polymerase Chain Reaction (PCR) and carried out the process ourselves. Subsequently, we prepared agarose
gel and conducted gel electrophoresis. It was a day of both theoretical learning and hands-on
experience.

![](https://static.igem.wiki/teams/4845/wiki/notebook/3.png)

**2023/7/6-7/8****: Gel Documentation, Gel Extraction, and Over
PCR: A Day of Precision and Progress**

Our
team began by using a gel documentation system to analyze the results of the gel electrophoresis.
Following this, we extracted the desired DNA fragments from the gel for further use.

Finally,
we embarked on Overlapping PCR (Over PCR), a technique that allows for the fusion of DNA fragments at
their overlapping regions. We prepared specific PCR mixtures for four different plasmids. Notably, we
successfully fused the GAP promoter, GA gene fragment and CYC1 terminator in one set of reactions, and the
TEF1 promoter, GA gene fragment and ADH1 terminator in another. This step was crucial for the assembly of
our desired genetic constructs.

![](https://static.igem.wiki/teams/4845/wiki/notebook/4.png)

**2023/7/9****: Competent Cell Preparation, DNA Manipulation,
and Gel Preparation**

We
prepared competent yeast cells for transformation, performed restriction digestion and ligation to
assemble our desired plasmid, and prepared an agarose gel for electrophoresis. These steps are crucial in
our journey towards successful genetic manipulation.

![](https://static.igem.wiki/teams/4845/wiki/notebook/5.png)

**2023/7/10****: Preparation and Transformation of Competent
Cells**

We
made significant strides in our project by successfully transforming yeast cells using the lithium acetate
method. We began by preparing our competent yeast cells and transformation reagents, then proceeded to
introduce our plasmids into the yeast cells. After the transformation, we allowed the cells to recover in
a YPD liquid medium. The transformed cells were then spread onto selective agar plates to promote the
growth of colonies. In parallel, we prepared a soluble starch agar medium for future experiments. This day
marked a pivotal milestone in our project as we successfully incorporated a new
genetic material into our yeast cells.

**2023/7/11****:****One Day
off**

**2023/7/12-13****: Functional Testing and Solution
Preparation**

We
focused on functional testing of our recombinant yeast cells (GA-temA). We extracted proteins from the
cells and analyzed them using SDS-PAGE, which confirmed the successful expression of our target protein.
Concurrently, we prepared starch solutions at various pH levels for upcoming enzymatic activity tests.
These days were a
blend of analytical and preparatory tasks, each crucial for assessing the functionality of our genetic
constructs and preparing for subsequent steps in our project.

![](https://static.igem.wiki/teams/4845/wiki/notebook/6.png)

**2023/7/14****: Revisiting SDS-PAGE and Initiating****Single****Clonal Verification**

We
revisited the SDS-PAGE procedure due to initial results that required further refinement. Our goal was to
achieve more precise protein detection. Simultaneously, we initiated the process of single clone
verification to confirm the successful gene transformation in yeast. We also prepared an iodine solution,
which will be used to confirm successful starch degradation, indicative
of the functional expression of our target gene, GA-temA. Despite the need for repetition, this day was a
testament to the perseverance and progress inherent in scientific discovery, reminding us that the path to
breakthroughs often involves trial and error.

![](https://static.igem.wiki/teams/4845/wiki/notebook/7.png)

**2023/7/15****：****Gene Expression Confirmation and Enzyme
Activity Testing**

We
made use of the iodine solution we had prepared before.
This was used to conduct iodine staining, which confirmed the functional expression of our target gene,
GA-temA. We also analyzed the overnight destained SDS-page Protein
gel,
using a gel documentation system, confirming the successful protein expression. Additionally, we initiated
the activity testing of our recombinant enzymes, GA and temA, using the Folin-Wu colorimetric method. This
day marked significant progress in our project, as we were able to verify the successful expression and
functionality of our target gene.

![](https://static.igem.wiki/teams/4845/wiki/notebook/8.png)

**2023/7/16****：****Wet lab and Dry lab assembly to discussion
about Wiki edition and presentation video**

Our
team assembled together to distribute work of editing Wiki content,
and we also discussed about the framework of presenting our project. To present our project more pithily,
we also consulted our supervisor,

and
did an efficient mock presentation together to get more familiar with the presentation.

![](https://static.igem.wiki/teams/4845/wiki/notebook/9.png)

**2023/7/17****：****Mock Presentation and Communicating with
audiences for advice**

After
tidying up our content and modifying our presentation slides, our team presented again in front of some
invited audiences with professional knowledge of Synthetic biology. In the latter part of the
presentation, we received lots of sincere suggestions from the audiences regarding to our slides design
and project introduction, we were really grateful and determined to refine on it further.

![](https://static.igem.wiki/teams/4845/wiki/notebook/10-1.png)
![](https://static.igem.wiki/teams/4845/wiki/notebook/10-2.png)

**2023/7/25 Online
meeting for reviewing project and experiment**

Our
team held a meeting together online, inviting our supervisor together to review the whole project and
experiment process together through daily notes we took during the assembly period and the general outline
of experiments. Also, we had a Q-and-A section with our supervisor, consulting our confusion and asking
for some advice.

**2023/7/27 Online
meeting regarding work distribution and progress discussion**

Our
team held an online meeting discussing progress we have made and our future plans for preparing for Wiki
and presentation, it was really glad to meet everyone all online again and we did share a meaningful time
together distributing work to make our later collaboration more organized and tighter. Furthermore,
everyone reported their work progress to keep pace with the project progress.

**2023/****8****/****26~28 Yeast
fermentation experiment for alcohol production test**

After discussion
with our instructor, we decided to conduct the alcohol production test to see if our yeast-1974 could
successfully produce alcohol. This work lasted 3 days with great help from our instructor Bella and the
data was collected for further calculation with the guidance of the kit manual.

**2023/****8/29 ~  Wiki
documentation & construction, Videos editing & submission, et al**

Per our work allocation, each one was diligently focused on
completing tasks to prepare the final jamboree session and of courses, school classes and
homework.

![](https://static.igem.wiki/teams/4845/wiki/notebook/11.png)

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Overview

Engineering Design (DBTL) Cycles

Reference

Overview

We
successfully constructed all four plasmids required to meet our aim eventually. To introduce, these four
plasmids(X-2-2GA, XII-5-2GA, X-3-2temA，XI-2-2temA) are grouped into two separate rounds of engineering
according to their key genes (GA, temA) expressed. To be more specific, GA is the gene expressing glucoamylase, and temA is the
gene expressing amylase.X-2-2GA and XII-5-2GA share the same gene sequence with length of 8802bp, but
different plasmid backbones (X-2 and XII-5).X-3-2temA and XI-2-2temA share the same gene sequence with
length of 9433bp (total plasmid length), but different plasmid backbones (X-3 and XI-2). What is more, 2GA
or 2temA indicate the number of the key genes inserted in the plasmid, we inserted two key genes into each
plasmid to promote starch decomposing efficiency by expressing more amylases or glucoamylases.
Furthermore, the reason why we constructed four plasmids that two of them share the same gene sequence but
difference plasmid backbones is that two same plasmid backbones are not able to be transformed into a
single cell together since they share the same replicon(ori) (Novick;
Austin and Nordstrom; Bouet, Nordstrom and Lane; Diaz, Rech and Bouet; Helinski; Johnson and Nolan;
Meacock and Cohen).

**Table 1: clear information
expressed in the form of chart to ensure comprehension**

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
| **Plasmid Name** | **Key genes** | **Length of plasmid** | **Enzyme expressed** | **Round** |
| **X-2-(2)GA** | GA\*2 | 8802bp | Glucoamylase | 1 |
| **XII-5-(2)GA** | GA\*2 | 8802bp | Glucoamylase | 1 |
| **X-3-(2)temA** | temA\*2 | 9433bp | Amylase | 2 |
| **XI-2-(2)temA** | temA\*2 | 9433bp | Amylase | 2 |
| **Success?** | √ | √ | √ | √ |

The
whole project design does follow the methodology of synthetic biology (Figure 1), and our experiment
involved the engineering design cycle（DBTL） comprehensively.

![](https://static.igem.wiki/teams/4845/wiki/engineering-success/1.png)

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LB
and YEPD Culture Media Preparation and E.coli Inoculation

Material

|  |  |
| --- | --- |
| **R****eagent** | **V****olume/mass** |
| Tryptone | 30g |
| Yeast Extract | 15g |
| NaCl | 10g |
| Glucose | 20g |
| Agar | 6g |
| ddH2O | / |
| Carbenicillin | 1g |
| Chloramphenicol | 250mg |
| CCTCC M94055 (E.coli) | 5ul |
| GA (E.coli) | 5ul |
| TemA (E.coli) | 5ul |
| pHCas9 (E.coli) | 5ul |

|  |  |
| --- | --- |
| **Apparatus** | **Amount** |
| **C****onstant temperature shaker** | 1 |
| **Electronic balance** | 2 |
| **C****lean Bench** | 1 |

Procedure

1. Mix 10g tryptone, 5g yeast extract, 10g NaCl and
3g agar (to solidify, if the LB media is liquid, then ignore agar) together to prepare LB culture
medium.

2. Add ddH2Oure to 1L and do sterilization under 121℃
for 20 min.

3. Mix 20g tryptone, 10g yeast extract, 20g glucose
and 3g agar (to solidify, if the YEPD culture media is liquid, then ignore agar) together to prepare YEPD
culture medium.

4. Add ddH2O to the mixture to 1L and do sterilization under
121℃ for 20min.

5. Weigh 1g Carbenicillin, and mix it with 10ml
ddH2O, do filtration
sterilization, store the agent of 1ml each for Ampicillin of 100mg/ml.

6. Weigh 250mg Chloramphenicol, and mix it with 10ml ddH2O, do filtration sterilization, store the
agent of 1ml each for Chloramphenicol of 25mg/ml.

7. Take 5ul cryopreserved E.coli CCTCC M94055, GA, TemA
and pHCas9 respectively, inoculate them into test tube with 3ml liquid LB culture media.

8. Add 5ul antibiotic into each test tube with
inoculated E.coli (Carbenicillin for GA and TemA, Chloramphenicol for CCTCC M94055, pHCas9). Culture the
E.coli in constant temperature shaker under 37℃, 220rpm for 15h (12-18h interval is
acceptable).

9. Add 50ul/ml mixture of E.coli and antibiotic to prepared
plate with solid LB culture media in the clean bench, label them with E.coli name + antibiotic.

10.  Placed the prepared E.coli in the
bacterial incubator under 37℃

Plastid
Extraction (CCTCC
M94055, GA, TemA and
pHCas9 plasmids)

Material

|  |  |
| --- | --- |
| **Reagent** | **V****olume/mass** |
| CCTCC M94055-CAM | 2ml |
| GA-Amp | 2ml |
| TemA-Amp | 2ml |
| pHCas9-CAM | 2ml |
| Buffer S1 | 1ml |
| Buffer S2 | 1ml |
| Buffer S3 | 1.4ml |
| Washing Buffer (ethanol-containing) | 4ml |
| Eluent | 200ul |

|  |  |
| --- | --- |
| Apparatus | Amount |
| Centrifuge | **2** |
| 4℃fridge | **4** |
| Water Bath Cauldron | **1** |
| spin column | **4** |
| Centrifuge Tube | **8** |

Procedure (For each
group)

1. Take 2ml bacterial liquid (for example, GA-Amp)
prepared, centrifuge it under 12000rpm for 1min, discard the supernatant liquid (for collecting
bacteria).

2. Add 250 μl Buffer S1 taken from 4℃ fridge (for
suspending the bacteria).

3. Add 250 μl Buffer S2, turn over the centrifuge
tube mildly and sufficiently for 4-6 times till the solution is translucent (for sufficient lysate of
bacteria), this step should be within 5 min.

4. Add 350μl Buffer S3, turn over the centrifuge tube mildly and
sufficiently for 6-8 times, centrifuge it under 9000rpm for 1min.

5. Transfer the supernatant liquid to spin column,
place the spin column into 2ml centrifuge tube, centrifuge it under 9000 rpm for 1min, discard the
filtrate.

6. Take out the spin column, add 500μl Washing
buffer, centrifuge under 9000 rpm for 1 min, discard the filtrate.

7. Repeat the Step 6.

8. Place the spin column back in the 2ml centrifuge
tube, centrifuge it under 12000 rpm for 1min.

9. Take out and place the spin column into 1.5ml
centrifuge, wait 1-2 min for volatilization of ethanol, add 50μl 60-65℃ Eluent solution to the center of the spin
column.

10. Place the spin column in 60-65℃ water bath for 1-2 minutes, take it out and
centrifuge it under 12000rpm for 1min.

PCR

Material

For
X-2-GA plasmid:

PCR system:

#2mix
contains dNTPs,
Taq DNA polymerase, buffer, cofactor (mgcl2)

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **N****ame**  **X-2-TEF1(429bp)** | **V****olume**  **(****μ****l)** | **N****ame**  **GA(1580bp)** | **Volume(****μ****l)** | **N****ame**  **X-2-ADH1t(216bp)** | **V****olume****(ul)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **X-2-TEF1-F1** | 2 | ADH1t-GA-R1 | 2 | GA-ADH1t-F1 | 2 |
| **GA-TEF1p-R1** | 2 | TEF1p-GA-F1 | 2 | X-2-ADH1t-R1 | 2 |
| **pH Cas9** | 1.5 | GA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

The
primer sequences for PCR（X-2-GA-2）are as follows :

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| GAPp-R1 | ATatactcgagTCATTATCAATACTGCCATTTCAAAG |
| GA-GAPp-F1 | caatctgatcatTTTGTTTGTTTATGTGTGTTTATTC |
| GAPp-GA-R1 | CATAAACAAACAAAatgatcagattgaccgttttc |
| CYC1t-GA-F1 | cataactaattacatgattacaataattcgatcaacttg |
| X-2-CYC1t-F1 | atatagagctcgcaaattaaagccttcgagcgtcccaaaac |
| GA-CYC1t-R1 | gttgatcgaattattgtaatcatgtaattagttatgtcac |

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| X-2-TEF1p-F1 | GATGGgtttaaaccatagcttcaaaatgtttctactc |
| GA-TEF1p-R1 | cggtcaatctgatcatcttagattagattgctatgc |
| ADH1t-GA-R1 | aatcataagaaattcgcttacaataattcgatcaacttg |
| TEF1p-GA-F1 | gcaatctaatctaagatgatcagattgaccgttttc |
| X-2-ADH1t-R1 | tttgcgagctcccggtagaggtgtggtcaataag |
| GA-ADH1t-F1 | tgatcgaattattgtaagcgaatttcttatgatttatg |

**F****or
XII-5-****GA**

PCR
system:

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **N****ame**  **XII-5-GAP(690bp)** | **V****olume (****μ****l)** | **N****ame**  **GA(1579bp)** | **V****olume(****μ****l)** | **N****ame**  **XII-5-CYC1t(278bp)** | **V****olume(****μ****l)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **GAPp-R1** | 2 | GAPp-GA-R1 | 2 | XII-5-CYC1t-F1 | 2 |
| **GAPp-F1** | 2 | CYC1t-GA-F1 | 2 | GA-CYC1t-R1 | 2 |
| **CCTCC M94055** | 1.5 | GA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Name**  **XII-5-TEF1p** | **Volume**  **(****μ****l)** | **N****ame**  **XII-5-GA (1580bp)** | **Volume**  **(****μ****l)** | **N****ame**  **XII-5-ADH1t (216bp)** | **Volume**  **(****μ****l)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **XII-5-TEF1p-F1** | 2 | ADH1t-GA-R1 | 2 | GA-ADH1t-F1 | 2 |
| **GA-TEF1p-R1** | 2 | TEF1p-GA-F1 | 2 | XII-5-ADH1t-R1 | 2 |
| **pH Cas9** | 1.5 | GA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

The
primer sequences for PCR（XII-5-GA-2）are as follows :

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| GAPp-R1 | ATatactcgagTCATTATCAATACTGCCATTTCAAAG |
| GA-GAPp-F1 | caatctgatcatTTTGTTTGTTTATGTGTGTTTATTC |
| GAPp-GA-R1 | CATAAACAAACAAAatgatcagattgaccgttttc |
| CYC1t-GA-F1 | cataactaattacatgattacaataattcgatcaacttg |
| XII-5-CYC1t-F1 | ATATAactagtgcaaattaaagccttcgagcgtccc |
| GA-CYC1t-R1 | gttgatcgaattattgtaatcatgtaattagttatgtcac |

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| XII-5-TEF1p-F1 | ACCGTgtttaaaccatagcttcaaaatgtttctactcc |
| GA-TEF1p-R1 | cggtcaatctgatcatcttagattagattgctatgc |
| ADH1t-GA-R1 | aatcataagaaattcgcttacaataattcgatcaacttg |
| TEF1p-GA-F1 | gcaatctaatctaagatgatcagattgaccgttttc |
| GA-ADH1t-F1 | tgatcgaattattgtaagcgaatttcttatgatttatg |
| XII-5-ADH1t-R1 | atttgcactagtccggtagaggtgtggtcaataagag |

**F****or
XI-2-****t****emA**

**PCR****system****：**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **N****ame**  **XI-2-GAP278bp** | **V****olume**  **(****μ****l)** | **V****olume**  **XI-2-temA-1888bp** | **Volume**  **(****μ****l)** | **N****ame**  **XI-2-CYC1t-273bp** | **V****olume**  **(****μ****l)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **XI-2-GAPp-F1** | 2 | D-GAPp-temA-F1 | 2 | D-temA-CYC1t-F1 | 2 |
| **D-TemA-GAPp-R1** | 2 | D-CYC1t-temA-R1 | 2 | XI-2-CYC1t-R1 | 2 |
| **CCTCC M94055** | 1.5 | TemA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **N****ame**  **XI-2-TEF1p-423bp** | **A****mount(****μ****l)** | **N****ame**  **XI-2-temA-1886bp** | **A****mount(****μ****l)** | **N****ame**  **XI-2-ADH1t-214bp** | **Amount(****μ****l)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **D-temA-TEF1p-F1** | 2 | D-ADH1t-temA-F1 | 2 | XI-2-SacI-R1 | 2 |
| **XI-2-Mss1-R1** | 2 | D-TEF1p-temA-R1 | 2 | D-temA-ADH1t-R1 | 2 |
| **pH Cas9** | 1.5 | temA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

The
primer sequences for PCR（XI-2-temA-2）are as follows :

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| XI-2-GAPp-F1 | TataggcgcgccTCATTATCAATACTGCCATTTC |
| D-temA-GAPp-R1 | aaaggcgtcatTTTGTTTGTTTATGTGTG |
| D-GAPp-temA-F1 | CAAACAAAatgacgcctttcgtcctc |
| D-CYC1t-temA-R1 | taattacatgactatctccatgtgtcgac |
| D-temA-CYC1t-F1 | acacatggagatagtcatgtaattagttatgtc |
| XI-2-CYC1t-R1 |  |

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| D-temA-TEF1p-F1 | aaggcgtcatcttagattagattgctatg |
| XI-2-Mss1-R1 | TACCCgtttaaaccatagcttcaaaatgtttctactc |
| D-ADH1T-temA-F1 | aaattcgcctatctccatgtgtcgac |
| D-TEF1p-temA-R1 | taatctaagatgacgcctttcgtcctc |
| X-2-Sac1-R1 | tttgcgagctcccggtagaggtgtggtcaataag |
| D-temA-ADH1t-R1 | acacatggagataggcgaatttcttatgatttatg |

**Fo****r X-3-temA**

**P****CR
system****:**

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Name**  **X-3--GAP278bp** | **Amoun****t(****μ****l)** | **Name**  **X-3-temA-1888bp** | **Amount(****μ****l)** | **N****ame**  **X-3-CYC1t-273bp** | **A****mount(****μ****l)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **XI-2-GAPp-F1** | 2 | D-GAPp-temA-F1 | 2 | D-temA-CYC1t-F1 | 2 |
| **D-TemA-GAPp-R1** | 2 | D-CYC1t-temA-R1 | 2 | XI-2-CYC1t-R1 | 2 |
| **CCTCC M94055** | 1.5 | TemA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

|  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- |
| **Name**  **X-3-TEF1p-423bp** | **A****mount(****μ****l)** | **N****ame**  **X-3-temA-1886bp** | **A****mount(****μ****l)** | **N****ame**  **X-3-ADH1t-214bp** | **A****mount(****μ****l)** |
| **2mix** | 25 | 2mix | 25 | 2mix | 25 |
| **D-temA-TEF1p-F1** | 2 | D-ADH1t-temA-F1 | 2 | XII-5-Bcu1-R1 | 2 |
| **X-3-Mss1-R1** | 2 | D-TEF1p-temA-R1 | 2 | D-temA-ADH1t-R1 | 2 |
| **pH Cas9** | 1.5 | temA | 1.5 | CCTCC M94055 | 1.5 |
| **ddH2O** | 19.5 | ddH2O | 19.5 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 | Total | 50 |

The
primer sequences for PCR（X-3-temA-2）are as follows :

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| XI-2-GAPp-F1 | TataggcgcgccTCATTATCAATACTGCCATTTC |
| D-temA-GAPp-R1 | aaaggcgtcatTTTGTTTGTTTATGTGTG |
| D-GAPp-temA-F1 | CAAACAAAatgacgcctttcgtcctc |
| D-CYC1t-temA-R1 | taattacatgactatctccatgtgtcgac |
| D-temA-CYC1t-F1 | acacatggagatagtcatgtaattagttatgtc |
| XI-2-CYC1t-R1 |  |

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| D-temA-TEF1p-F1 | aaggcgtcatcttagattagattgctatg |
| X-3-Mss1-R1 | AGTGGgtttaaaccatagcttcaaaatgtttc |
| D-ADH1T-temA-F1 | aaattcgcctatctccatgtgtcgac |
| D-TEF1p-temA-R1 | taatctaagatgacgcctttcgtcctc |
| XII-5-Bcu1-R1 | atttgcactagtccggtagaggtgtggtcaataagag |
| D-temA-ADH1t-R1 | aaattcgcctatctccatgtgtcgac |

|  |  |
| --- | --- |
| **App****a****ratus** | **A****mount** |
| **P****CR** | **1** |

**Pr****ocedure in the form of PCR
program setting chart**

|  |  |  |
| --- | --- | --- |
| Step | Temperature(℃) | Time |
| 1 | **9****5** | **5****min** |
| 2 | **9****5** | **3****0sec** |
| 3 | **5****5** | **3****0sec** |
| 4 | **7****2** | **2****min** |
| 5 | **7****2** | **1****0min** |
| 6 | **4** | **Forever** |

**Repeat
Step2-4 for 30 times**

Agarose Gel Electrophoresis

Material

|  |  |
| --- | --- |
| **R****eagent** | **V****olume/mass** |
| **T****AE buffer** | / |
| **A****garose** | 1g |
| **Gel Red (nucleic acid dye)** | 5ul |
| **D****NA marker** | 10ul |

|  |  |
| --- | --- |
| **A****pparatus** | **A****mount** |
| **E****lectrophoresis apparatus** | 1 |
| **M****icrowave** | 1 |
| **Erlenmeyer Flask** | 1 |
| **G****el tray** | 1 |
| **B****lue light transilluminator** | 2 |

Procedure:

1. Add
100ml TAE buffer into the Erlenmeyer Flask, weigh 1g agarose and place it into the Erlenmeyer
Flask.

2. Heat
the Erlenmeyer Flask in the microwave till the components melt completely. Take out and shake it
well.

3. Add
5ul
<
 nucleic
acid Gel Red to the agarose gel.

4. Assemble
the gel tray and comb. Pour the liquid gel mildly onto the gel tray, remove the bubble, insert a comb on a
gel, wait for solidification of gel.

5. Take
out the comb, transfer
the solidified gel to the electrophoresis tank, add TAE buffer till the gel is immersed completely.

6. Add
samples into each sampling hole respectively and record the order, add 10ul DNA
Marker after adding samples.

7. Power
on with 180 V for 15min.

8. Take
out and place the gel under the blue lightTransilluminator for
observation.

Gel Extraction

Material

|  |  |
| --- | --- |
| **R****eagent** | **V****olume/mass** |
| **B****uffer B3** | 250ul |
| **W****ashing solution** | 1ml |
| **E****lution buffer** | 500ul |

|  |  |
| --- | --- |
| **Ap****paratus** | **A****mount** |
| **c****entrifuge** | 1 |
| **Vortex oscillator** | 2 |
| **1****.5ml centrifuge tube** | 1 |
| **A****bsorption column** | 1 |

Procedure

1. Cut
out the target section from gel according to its position shown under blue light transilluminator.

2. Transfer
the product to 1.5ml centrifuge tube, add 250μl buffer B3, vortex it on the vortex
oscillator.

3. Transfer the mixture to the absorption column,
centrifuge under 8000 rpm for 30 seconds, discard the filtrate.

4. Add 500μl Washing solution, centrifuge under 9000 rpm for
1min, discard the filtrate.

5. Repeat Step 4

6. Centrifuge under 9000 rpm for 1min.

7. Add 20μl  Elution buffer into absorption film,
place it under room temperature for 1-2 min, centrifuge under 9000 rpm for 1min, obtain the DNA.

8. Measure the concentration of DNA, store it under
the temperature of -20 ℃.

Overlap Extension PCR (Over PCR)

Materials

For
X-2-GA plasmid

PCR
system:

|  |  |  |  |
| --- | --- | --- | --- |
| **Name**  **X-2-GAP-GA-CYC1t-2485bp** | **A****mount(****μ****l)** | **N****ame**  **X-2-TEF1-GA-ADH1-2160bp** | **A****mount(****μ****l)** |
| **2mix** | 25 | 2mix | 25 |
| **GAPp-R1** | 2 | X-2-TEF1p-F1 | 2 |
| **X-2-CYC1t-F1** | 2 | X-2-ADH1t-R1 | 2 |
| **X-2-GAP** | 1 | X-2-TEF1 | 1 |
| **GA** | 1 | X-2-GA | 1 |
| **X-2-CYC1t** | 1 | X-2-ADH1 | 1 |
| **ddH2O** | 18 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 |

The
primer sequences for PCR（X-2）are as follows :

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| GAPp-R1 | ATatactcgagTCATTATCAATACTGCCATTTCAAAG |
| X-2-CYC1t-F1 | atatagagctcgcaaattaaagccttcgagcgtcccaaaac |
| X-2-TEF1p-F1 | GATGGgtttaaaccatagcttcaaaatgtttctactc |
| GA-TEF1p-R1 | cggtcaatctgatcatcttagattagattgctatgc |
|  |  |

**F****or XII-5-GA plasmid**

**P****CR system:**

|  |  |  |  |
| --- | --- | --- | --- |
| **Name**  **X****II****-****5****-GAP-GA-CYC1t-2485bp** | **Amount****(****μ****l)** | **N****ame**  **X****II****-****5****-TEF1-GA-ADH1-2160bp** | **A****mount(****μ****l)** |
| **2****mix** | 25 | 2mix | 25 |
| **GAPp-R1** | 2 | XII-5-TEF1p-F1 | 2 |
| **XII-5-CYC1t-R1** | 2 | XII-5-ADH1t-R1 | 2 |
| **XII-5-GAP** | 1 | XII-5-TEF1 | 1 |
| **XII-5****-****GA** | 1 | XII-5-GA | 1 |
| **XII-5-CYC1t** | 1 | XII-5-ADH1 | 1 |
| **ddH2O** | 18 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 |

The primer sequences for PCR（XII-5）are as follows
:

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| GAPp-R1 | ATatactcgagTCATTATCAATACTGCCATTTCAAAG |
| XII-5-CYC1t-R1 | atttgcactagtccggtagaggtgtggtcaataagag |
| X-2-Sac1-R1 | tttgcgagctcccggtagaggtgtggtcaataag |
| D-temA-ADH1t-R1 | acacatggagataggcgaatttcttatgatttatg |
|  |  |

**For****X****I-2-****temA**

**PCR****sys****tem:**

|  |  |  |  |
| --- | --- | --- | --- |
| **N****ame**  **X****I****-****2****-GAP-GA-CYC1t-****2804 bp** | **A****mount(****μ****l)** | **Name**  **X****I****-****2****-TEF1-GA-ADH1-2160bp** | **Amount****(****μ****l)** |
| **2****mix** | 25 | 2mix | 25 |
| **X-2-CYC1t-F1** | 2 | X-2-Sac1-R1 | 2 |
| **XI-2-GAPp-F1** | 2 | D-temA-ADH1t-R1 | 2 |
| **X****I****-****2****-GAP** | 1 | XI-2-TEF1 | 1 |
| **X****I****-****2-****GA** | 1 | XI-2-GA | 1 |
| **X****I****-****2****-CYC1t** | 1 | XI-2-ADH1 | 1 |
| **ddH2O** | 18 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 |

The primer sequences for PCR（XI-2）are as follows
:

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| X-2-CYC1t-F1 | atatagagctcgcaaattaaagccttcgagcgtcccaaaac |
| XI-2-GAPp-F1 | TataggcgcgccTCATTATCAATACTGCCATTTC |
| X-2-Sac1-R1 | tttgcgagctcccggtagaggtgtggtcaataag |
| D-temA-ADH1t-R1 | acacatggagataggcgaatttcttatgatttatg |
|  |  |

**For****X-****3-****temA**

**P****CR system:**

|  |  |  |  |
| --- | --- | --- | --- |
| **Name**  **X-****3****-GAP-GA-CYC1t-****2804 bp** | **A****mount(****μ****l)** | **N****ame**  **X-****3****--TEF1-GA-ADH1-2160bp** | **A****mount(****μ****l)** |
| **2****mix** | 25 | 2mix | 25 |
| **XI-2-GAPp-F1** | 2 | XII-5-Bcu1-R1 | 2 |
| **XII-5-CYC1t-R1** | 2 | X-3-Mss1-R1 | 2 |
| **X-****3****--GAP** | 1 | X-3--TEF1 | 1 |
| **X-****3****-****-****GA** | 1 | X-3--GA | 1 |
| **X-****3****--CYC1t** | 1 | X-3-ADH1 | 1 |
| **ddH2O** | 18 | ddH2O | 19.5 |
| **Total** | 50 | Total | 50 |

The
primer sequences for PCR（X-3）are as follows :

|  |  |
| --- | --- |
| **Primer** | sequences**(5'to3')** |
| XI-2-GAPp-F1 | TataggcgcgccTCATTATCAATACTGCCATTTC |
| XII-5-Bcu1-R1 | atttgcactagtccggtagaggtgtggtcaataagag |
| X-3-Mss1-R1 | AGTGGgtttaaaccatagcttcaaaatgtttc |
| XII-5-CYC1t-R1 | atttgcactagtccggtagaggtgtggtcaataagag |
|  |  |

|  |  |
| --- | --- |
| Apparatus | Amount |
| PCR | **1** |

**P****rocedure in the form of PCR program
chart**

|  |  |  |
| --- | --- | --- |
| Step | Temperature（℃） | Time |
| 1 | **9****5** | **5****min** |
| 2 | **9****5** | **3****0sec** |
| 3 | **5****5** | **3****0sec** |
| 4 | **7****2** | **2****min** |
| 5 | **7****2** | **1****0min** |
| 6 | **4** | **Forever** |

**Repeat
step 2-4 for 30 times**

Restriction Endonuclease Digestion and Ligation

For
X-2
restriction endonuclease digestion system:

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **Amount(****μ****l)** | **N****ame** | **Volume(****μ****l)** |
| **X-2 plasmid skeleton** | 30 | X-2-GAP-GA-CYC1t | 30 |
| **Cut****Smart Buffer** | 5 | Cut Smart Buffer | 5 |
| **Xho1** | 1 | Xho1 | 1 |
| **Sac1** | 1 | Sac1 | 1 |
| **ddH2O** | 13 | ddH2O | 13 |
| **Total** | 50 | Total | 50 |

For
XII-5
restriction endonuclease digestion system:

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **V****olume(****μ****l)** | **N****ame** | **V****olume(****μ****l)** |
| **XII-5 plasmid skeleton** | 30 | XII-5-GAP-GA-CYC1t | 30 |
| **Cut****Smart Buffer** | 5 | Cut Smart Buffer | 5 |
| **Xho1** | 1 | Xho1 | 1 |
| **Bcu****1** | 1 | Bcu1 | 1 |
| **ddH2O** | 13 | ddH2O | 13 |
| **Total** | 50 | Total | 50 |

For
XI-2
restriction endonuclease digestion system:

|  |  |  |  |
| --- | --- | --- | --- |
| **N****ame** | **A****mount(****μ****l)** | **N****ame** | **Amount(****μ****l)** |
| **XI****-2** **plasmid skeleton** | 30 | XI-2-GAP-GA-CYC1t | 30 |
| **Cut****Smart Buffer** | 5 | Cut Smart Buffer | 5 |
| **Sac****1** | 1 | Sac1 | 1 |
| **Sgs1** | 1 | Sgs1 | 1 |
| **ddH2O** | 13 | ddH2O | 13 |
| **Total** | 50 | Total | 50 |

For
X-3 restriction endonuclease digestion system:

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **V****olume(****μ****l)** | **N****ame** | **Volume(****μ****l)** |
| **X****-3** **plasmid skeleton** | 30 | XI-2-GAP-GA-CYC1t | 30 |
| **Cut****Smart Buffer** | 5 | Cut Smart Buffer | 5 |
| **B****cu1** | 1 | Bcu1 | 1 |
| **Sgs1** | 1 | Sgs1 | 1 |
| **ddH2O** | 13 | ddH2O | 13 |
| **Total** | 50 | Total | 50 |

Ligation
PCR system:

|  |  |
| --- | --- |
| **Name** | **V****olume(****μ****l)** |
| **P****las****mid skeleton (eg. X-2)** | 4 |
| **Inserted gene (eg. X-****2****-GAP-GA-CYC1t)** | 16 |
| **T4 ligase** | 1 |
| **T4 ligase buffer** | 5 |
| **ddH2O** | 25 |
| **Total** | 50 |

|  |  |
| --- | --- |
| **Appa****ratus** | **A****mount** |
| **P****CR** | 1 |

 Procedure

1. Prepare
restriction endonuclease digestion PCR system

2. Do
restriction endonuclease digestion under 37℃ PCR
for 30min.

3. Take
out and reclaim the reagents, prepare the Ligation system according to the material chart.

4. Do
Ligation under 16℃ PCR
for 1.5 hours.

Competent cell preparation and Yeast Cell
Transformation

Material

|  |  |
| --- | --- |
| **R****eagent** | **V****olume/mass** |
| **PEG****3350 (500g/L)** | 240ul |
| **L****iAc (1M)** | 36ul |
| **S****almon sperm ssDNA(2ml/L)** | 50ul |
| **d****dH2O** | / |
| **Peptone** | 10g |
| **B****ee****f extract** | 5g |
| **N****aCl** | 5g |
| **Soluble Starch** | 2g |
| **A****gar** | 15g |
| **1****974 yeast cultured** | 200ul |
| **Y****EPD culture media** | 1ml |
| **D****NA (GA+ temA) after Over PCR+ ddH2O** | 34ul |
| **Sterile water** | 25ml |

|  |  |
| --- | --- |
| **A****pparatus** | **Amount** |
| **N****anodrop** | 1 |
| **C****entrifuge** | 2 |
| **1.5ml Centrifuge Tube** | 1 |
| **B****ox with ice** | 1 |
| **W****ater bath** | 1 |
| **C****onstant temperature shaker** | 1 |
| **C****onstant temperature incubator** | 1 |
| **V****ortex oscillator** | 2 |
| **E****lectronic Balance** | 1 |

Procedure:

1. Dilute
yeast liquid (over-night cultured) to 1/10 concentration, test OD600 of diluted liquid.

2. Transfer
the diluted yeast liquid to 250 ml Erlenmeyer flask with 25ml 2\*YEPD culture medium.

3. Culture
the yeast under the temperature of 30℃, 240r/min
in constant temperature shaker till its OD600 is 0.3-0.8.

4. 3000
rpm Centrifuge the yeast liquid for 5min for collecting yeast.

5. Add
25ml sterile water to suspend the yeast, 3000rpm centrifuge under the temperature of 20℃ for
5min.

6. Repeat
the Step 5 twice.

7. Add
1 ml sterile water to suspend the yeast, transfer the yeast liquid to 1.5ml centrifuge tube, 12000rpm
centrifuge for 30s, discard the supernatant liquid, collect the competent yeast cell.

8. Prepare
components for LiAc transformation liquid reagent according to the chart.

|  |  |
| --- | --- |
| **R****eagent** | **F****ormula** |
| **P****EG 3350 (500g/L)** | 25g PEG 3350 + 50ml ddH2O |
| **L****iAc (1M)** | 4.94g LiAc + 50ml ddH2O |
| **S****almon sperm DNA (2mg/L)** | 0.02g ssDNA+10ml ddH2O. Place the 2mg/L ssDNA in 100℃ water bath for 5min, take it out and put it on the ice rapidly |

9. AddHO
240ul
 PEG
3350 (500g/L), 36ulLiAc
(1M), 50ul*ssDNA
and 34 ul* DNA
(GA, temA) to centrifuge tube with competent cells in order.

10. Water
bath the mixture under the temperature of 42℃ for
15min, 12000 centrifuge for 30 seconds, discard the supernatant liquid, add 1ml YEPD liquid culture media,
suspend yeast, seal the
opening.

11. Place
it in the 240rpm constant temperature shaker under the temperature of 30℃
for
1 hour. 3000rpm Centrifuge for 4min, discard 600ul supernatant
liquid.

12. Add
5g beef extract, 10g Peptone, 5g NaCl, 2g soluble starch and 15g agar, add ddH2O till
the solution volume is up to 1 L, sterilize under 115℃ for
20min. (Preparation of soluble-starch solid culture media)

13. Evenly
distribute the yeast on the plate with soluble-starch solid culture media, place the sealed plate up-side
down in the incubator for two days under 30℃.

Ultrasonic Cell Disruptor

Material

|  |  |
| --- | --- |
| **R****eagent** | **V****olume/mass** |
| **Yeast****G****A-temA** | / |
| **Y****east 1947** | / |
| **P****rotein extract solution** | 15ml |

|  |  |
| --- | --- |
| **A****pparatus** | **A****mount** |
| **5****0ml centrifuge tube** | 2 |
| **Ultrasonic Disruptor** | 1 |

Procedure:

1. Transfer
the yeast GA-temA and yeast 1974 to 50ml centrifuge tube, 8000rpm centrifuge for 5min, discard the
supernatant liquid, add nutrition materials on the precipitate

2. 8000
rpm centrifuge for 5min, collect all the nutrition materials.

3. Add
15ml Protein extract solution (100mmol/L NaCl, 10mmol/L EDTA, pH8.0), use pipette to blow to suspend the
precipitate.

4. Put
the centrifuge tube on the test-tube stand, put the metal head of ultrasonic disruptor into the centrifuge
tube, adjust the position of the tube, turn on the ultrasonic disruptor.

5. Ultrasonically
disrupt the yeast with 80W Power for 2 seconds and 2 seconds of interval, 5 cycles once, the total
disruption lasts for 20min.

6. 8000rpm
centrifuge for 5min.

7. Take
out centrifuge tube, carefully transfer the supernatant liquid to a new centrifuge tube, store under
4℃.

SDS-PAGE electrophoresis

**Material**

|  |  |
| --- | --- |
| Reagent | Volume/mass |
| Yeast 1974 | **/** |
| Recombinant yeast | **/** |
| YPD-G418 liquid culture media | **5****0ml** |
| Protein buffer | **/** |

|  |  |
| --- | --- |
| Apparatus | Amount |
| Centrifuge | **2** |
| Water bath | **1** |
| SDS PAGE | **1** |
| Coomassie Blue Staining Kit | **1** |

**P****rocedure:**

1. Inoculate yeast 1974 and recombinant saccharomyces
cerevisiae to 50ml YPD-G418 liquid culture media, culture them under 30℃, 240rpm in constant temperature incubator for 72
hours.

2. Add protein buffer into the supernatant liquid
after centrifuge, 100℃ water bath for 5min for denaturation.

3. Do
SDS-PAGE electrophoresis for recombinant GA-temA
yeast and original saccharomyces cerevisiae 1974 under 130V for 30min for concentration, turn the volt to
90V and continue separation for 60min.

4. Dye
the
SDS-PAGE gel through Coomassie
Blue Staining Kit.

Iodine Staining, Transparent Circle Observation and Enzymatic
Activity Test

Material

|  |  |
| --- | --- |
| **R****eagent** | **V****olume/mass** |
| **Recombinant yeast GA-temA** | / |
| **Y****east 1974** | / |
| **Iodine** | 0.13g |
| **I****odide potassium** | 0.35g |
| **d****dH2O** | 250 ml |
| **C****itric Acid** | 101g |
| **S****odium citric** | 941g |
| **S****tarch** | 0.2g |

|  |  |
| --- | --- |
| **A****pparatus** | **A****mount** |
| **Constant temperature shaker** | 1 |
| **N****an****odrop** | 1 |

Procedure:

1. Mix
101g citric acid and 100ml ddH2O for
preparation of citric acid solution A

2. Mix
941g sodium citric acid and 100ml ddH2O for
preparation of sodium citric solution B

3. Weigh
27.5g solution A and 72.5g solution B, add 0.2g starch, adjust the pH value to 3, 4, 5, 6 and 7
respectively (soluble starch solution with different pH values).

4. Dissolve
0.13g iodine and 0.35g iodide potassium in 50ml ddH2O.

5. Add
small amount of iodine solution to the soluble-starch solid culture medium with yeast 1974 and recombinant
yeast GA-temA,
observe the transparent circle.

6. Add
original yeast 1974 and recombinant yeast GA-temA to soluble-starch solution with pH values of 3, 4, 5, 6
and 7 respectively.

7. Inoculate
yeast 1974 and recombinant yeast GA-temA to 3ml YPD test, culture it under 30℃,
240rpm in the constant temperature shaker overnight.

8. Take
the culture liquid and dilute it to 1/10 concentration, test the OD600 value through nanodrop.

9. Inoculate
200ul yeast
liquid to 50ml YEPD liquid culture media, culture under 30 ℃,
240rpm in the constant temperature shaker for 72 hours.

10.  Take
proper amount of yeast liquid, 12000rpm centrifuge for 10min for enzymatic activity test.

Alcohol content
determination

1.Yeast
100ul 1974 and 1974-GA-temA were added to 100ml YPD, and then 100 Amp was added, cultured at 37 °C for
24h, 36h, 72h.

2.
The sample 12000Xg was
centrifuged for 10 min, and the supernatant solution was taken to measure the alcohol content of 100ul
1974 and 1974-GA-temA with the alcohol Megazyme kit. [( Ethanol Assay Kit )](http://megazyme.jinpanbio.com/958.html)

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Overview

Results of our work

Overview

Our
project tends to enable Saccharomyces cerevisiae to
self-secrete alpha-amylase and saccharifying enzyme by
inserting four integrated plasmids X-2-GA, XII-5-GA, XI-2-temA and X-3-temA constructed by our group. We
picked and extracted two genes expressing to produce alpha-amylase and saccharifying enzyme from bacteria
cultured, and we then integrated these genes with plasmid backbones through restriction-ligation method.
After obtaining the constructed plasmids, we transformed them into saccharomyces cerevisiae,
enabling it to achieve self-secreting enzymes mentioned above. Therefore, we achieve reducing the cost of
addition of exogenous enzymes during alcoholic fermentation.

 ![](https://static.igem.wiki/teams/4845/wiki/result/1.png) |

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