iGEM 2019 — Team KUAS_Korea

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# Team:KUAS Korea/Notebook

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# Notebook

## General notebook

### 2018/12/26

1) set meeting order.

2) ideation

3) distributing roles

4) set monthly plan.

### 2018/1/3

Orientation Day!

we meet up with newcomers and introduced about our team members. Team leader ( Tae Hoon Lee ), Dry Lab leader (Jung Won Choi), Wet lab leader (Han HyunKyu), Human practice leader (Tehyun Eom)

### 2019/1/9

We met with professor In Geol Choi. Professor gaves some lecture about 'What is Synthetic biology' and 'DIY Bio'.

### 2019/1/16

We discussed about Igem ideas. Each have great Ideas such as Zinc finger Motif modification, fine dust remove tactics. Moss wall, and pet excreta smell treatment.

### 2019/1/23

We did Case study session about formal iGEM projects. Moreover, we discussed about our projects and give some feedbacks about them.

Also we set plan for the 2nd recruitment

- make poster

- contact with student center

- online poster upload to student information portal

- online poster upload to our Facebook and Instagram page

### 2019/1/30

Before the recruitment, we decided to open information session for volunteers. We choose date and contetns.>

**Date**

6 / 29

**Contents**

(1) Explain about synthetic biology

(2) Our new HP project ( Homecoming, collaboration)

(3) KUAS TV (youtube channel)

(4) What is iGEM?

### 2019/2/13

We redeemed our homepage. We made QR code and held poster on our homepage.

Also, we met professor and did Ideattion together.

### 2019/3/18

We met our new Recruit member.

### 2019/4/4

we checked our criteria for explanation and set the annual plan.

### 2019/6/30

**Homecoming DAY!**

we met some former igemers and we held some sessions to talk about synthetic biology. Also We can share informations about synthetic biology's future.

### 2019/7/19 ~ 2019/7/24

**DH10B competent cell preparation**

### 2019/7/25

**competent cell TE test(transformation)**

To calculate transformation efficiency, we transformated pB3 vector DNA. Then we give heat shock during 1 min and 30 min recovery.
After that, we spread these DNA to LB ampicillin plate.

### 2019/7/26

**Competent cell TE measurement**

We noticed that there are many colonies on the plate. (The value of TE is 5.23E+3, DH10B+pB3)

**colony select & culture**

We try to get vector from plate, we select two colony randomly, and we inoculate these to 15ml LB broth(liquid). Then conduct shaking incubation. After 5 hours, we frozen at 4℃

### 2019/7/29

**minipreparation**

We did plasmid extraction with cell culture. The density of sample one was 59.560ng/ul, sample 2 was 61.455ng/ul

**electrophoresis**

Only sample 2 shows valid result. ( We checked band on the sample 2.)

### 2019/7/30

**Transformation**

To measure TE value over time, we transformed DH10B with sample 2 pB3 DNA and spread again. We changed heat shock time each 0s/10s/30s/1min.

### 2019/7/31

**colony count**

We checked colonies for all plate. (0s – 434, 10s- 516, 30s – 324, 1min – 283)

colony select & culture

To extract pB3 vector, we choose 2 colony randomly from 60sec plate. Then we Inoculate them to 10ml LB broth(liquid) and conducted shacking incubation for 5 hours. Then we frozen them in 4℃.

### 2019/8/01

**minipreparation**

We extracted plasmid from Cultures grown on 31st. Sample 1 was 46.343ng/ul, sample 2 was 46.512ng/ul

**electrophoresis**

We checked valid band on the both sample successfully.

### 2019/8/6

**FGI(focused group interview) with STEPI**

We met professor and researcher

### 2019/8/7

**transformation**

To get PB vector series (pB1, pB7), transformed each plasmid DNA(5ul) to competent cell and Spread to LB-amp.

### 2019/8/8

**colony select and culture**

Cell was grown in the pB6, pB7. So we choose one colony then inoculation to 15ml LB broth(liquid).
Then we conducted shacking incubation with 5 hours and put them into refrigerator

### 2019/8/9

**minipreparation**

we extracted plasmid from cell broth, which was grown during 24hours. p86 was 90.742ng/㎕ and pB7 was 70.682ng/㎕

**electrophoresis**

We checked valid plasmid band from both samples.

**transformation**

To get plasmid again, we transformed again. Then we changde recovery time 30min to 1hr.

### 2019.8.10

**colony check**

we checked colonies from all plate

### 2019/8/12

**colony selection, culture and minipreparation**

We choose one colony from plate which was checked at 8/10. Then we inoculate them in to 10ml LB broth(liquid) and conducted shacking incubation during 5 hours.

**plasmid extraction**

Then we extracted plasmid from culture.
pB1: 27.087ng/㎕, pB2: 27.201ng/㎕, pB3: 21.234ng/㎕, pB4: 23.218ng/㎕, pB5: 21.298ng/㎕, pB6: 19.266ng/㎕, pB7: 21.398ng/㎕

**enzyme cut & electrophoresis**

To ensure that sma I site is existing in the pB vector’s cloning site, we cut Plasmid with sma I. Then conducted electrophoresis. We found single band for all plasmids. But, pB4’s band was very faint

### 2019/8/14

**DNA(gBlock)primer resuspension**

To calibrate gBlock(luc2, hydroxy)’s concentration, we resuspended each gBlock with 200ul, and 400ul. Also we resuspended primers to become 100pM.
luc2\_pB\_R(277㎕), hydroxy\_pB\_F(346㎕), hydroxy\_pB\_R(262㎕).
After that, we move 5ul for each solution to tubes and diluted with 45ul EB.(10x dilution, total volume 50ul)

**PCR**

Mix gBlock(luc2, hydroxy), primer, Q5 DNA polyemerase, Q5 reaction buffer. Then run the PCR. By electrophoresis, we checked valid band was exposed. (luc2, 747bp, hydroxy.1281bp)

**Vector enzyme cutting**

Use sma I to pB series(pB3~pB7) as restriction enzyme and checked 1 band through electrophoresis.

**clean up the products**

Clean up the pB vector series(pB3~pB7) and check the concentration. ; luc2: 99.371ng/㎕, hydroxy: 60.118ng/㎕, pB3: 16.051ng/㎕, pB4: 5.037ng/㎕, pB5: 7.078ng/㎕, pB6: 32.304ng/㎕, pB7: 11.156ng/㎕

**DNA assembly**

WE used T4 DNA polymerase LIC(ligation independent cloning) method for recombination. Molar ration of Luc2 was 1:5, and hydroxy was 1:3. Then we put enzyme for 2.5 min in room temperature. Then iced to each case including 5 negative control.

**transformation**

After assembly, we transformed DNAs to DH10B competent cell. Then, spread these cell to LB-amp plate.(heat shock 30sec, recovery 1hr)

### 2019/8/15

we checked colony in Luc2-pB7, hydroxy pB3

### 2019/8/20

**Colony PCR**

We conducted colony PCR by pfu DNA polymerase. The result was failed

### 2019/8/21

**colony PCR**

We conducted colony PCR again, then we re-checked result by electrophoresis. Luc2 was 3 of 3, and hydroxy was 7 of 7

**colony culture**

We used 1ml culture as starter which was used in colony PCR. Then we scaled up 10ml and conducted shaking incubation.

### 2019/8/22

**minipreparation**

We used 21th culture for plasmid extract. We extracted two plasmids per gBlock. luc2-pB7\_1: 32.304ng/㎕, luc2-pB7\_2: 31.440ng/㎕, hydroxy-pB3\_1: 28.090ng/㎕, hydroxy-pB3\_2; 33.246ng/㎕

**transformation**

We get 4 different vector and transformed them in to BL21(DE3). Then spread them into LB-amp

### 2019/8/23

**transformation**

The result of 22nd transformation was not clear. So we conducted transformation again. This time, we extended recovery time over 1hr.

### 2019/8/24

we checked colony for all plates.

### 2019/8/26

**overexpression**

we choose one colony for each 7 plates.Then cultured 10ml for each. Then treated with IPTG(without OD measure) Then conducted incubation at 16℃

### 2019/8/27

**Sonication**

Centrifugate overexpressed culture(4℃, 2000g, 20min) and sonicate(80W, 20sec sonicating/40sec restin, repeat 3 times) the culture after resuspend with 600㎕ lysis buffer. After it, keep it in two groups, pellet and supernatant, in 4℃

### 2019/8/28

Solubility test faliure

### 2019/8/29

**Miniprep**

Extract plasmid from the colony which newly cloned 28th, checked with colony PCR and cultured after scaled up

### 2019/9/3

**transformation**

; Transformate the 6 plasmids(luc2-pB3\_4, luc2-pB4\_2, hydroxy-pB3\_3, hydroxy-pB3\_4, hydroxy-pB4\_3, hydroxy-pB4\_6) which extracted at 29th clone and chosen arbitrarily, to Bl21(de3)-star(expression strain). Then spread on LB-AMP agar plate.

**SDS\_PAGE gel preparation**

for expression test (reference: protein method second edition)

### 2019/9/4

**Expression test(by assistant)**

pick 3 colonies per each plasmids(Luc2-pB3, Suc2-pB4, hydroxyl-pB3, hydroxyl-pB4), and conduct IPTG induction(1mM) at 37℃, 3hr. Then heat it at 90℃ using PCR machine to lyse cell, conduct SDS-PAGE;control group-culture which didn’t conduct IPTG induction. As a result, there was a significant difference with control in the band of the desired size.

**SDS-PAGE**

for solubility test

### 2019/9/5

Pick one from each samples of 9/4 and over express them.

### 2019/9/6

**solubility test**

**Sonication**

Centrifuge and remove the supernatant of cultures, mix the pellets with SDS-PAGE buffer. Conduct 1sec sonicating-3sec resting at 80W, repeat it 30 times.

**Centrifugation**

Centrifuge except the total solution. Devide supernatant and pellet and resuspense pellet with buffer.

**SDS-PAGE**

Run samples and ladders(total 44). As a result, proteins that expressed from the DNA assembled at pB4 appeared at both Luc2 and hydroxy in soluble part.

### 2019/9/9

Over express Luc2-pB4, htdroxy-pB4 at 16℃(1mM IPTG), to purificate by the result of 9/7

### 2019/9/10

Centrifugate 9/9 culture and only keep pellet frozen

### 2019/9/16

Prepare LB-AMP plate for new gBlock(nnLuz, cph) cloning.

### 2019/9/17

Prepare LB-CHL plate for characterization

### 2019/9/18

Transformation using RFP coding device parts.

### 2019/9/19

Cannot identify any colony as a result of transformation at 9/18.

### 2019/9/20

**Protein purification failure**

retry from the transformation.

Retry the transformation for characterization. Increase the amount of DNA.

### 2019/9/21

As a result of transformation at 9/20, there’re only one white colony grow up, on 24P.

### 2019/9/22

Conduct additional transformation for characterization.As 24P colony appeared red, picked it and culture for 7 hours, conduct miniprep

### 2019/9/23

transformation: conduct transformation. Luc2-pB4 to BL21(DE3)-star and hydroxyl-pB4 to C43(DE3)

### 2019/9/24

The experiment that conducted on 9/23 all succeed. Picked each colony and made starter.

### 2019/9/25

**Cloning**

by assistant. Cloned new gBlock nnLuz, cph using T4 DNAP LIC method.

### 2019/9/26

**minipreparation**

secure nnLuz, cph DNA, cloning at 9/25

### 2019/9/27

**Transformation**

retransform the 23O, 24P DNA which conducted miniprep to DH10B

### 2019/9/28

Both 23O, 24P which transformed on 9/23 appeared red. Picked each colony and incubate until saturation. Then keep it cold.

Add the starter which made on 9/24 to TB 200ml and set OD600=0.8. Keep it in shaking incubator for 3 day.

### 2019/10/1

saturation density measurement&transformation: Add 23O, 24P (cultured on 9/28) to LB and scaleup. Culture it enough and measure the OD600. Dilute the original culture medium and spread it on LB-CHL plate

### 2019/10/2

**TE**

colony counting

**OD600/cell number relation**

calculate the result by colony counting; About 1687 colony appear when spread the 40㎕ culture that dilute E-4 the culture 23O-DH10B, which OD600=0.418 at 1/5 dilution. It means, there were about 4.2175\*E8 viable cells exist in original culture. In the same vein, there were about 5.4375\*E8 viable cells exist in 24P of OD600=0.596.

### 2019/10/4

**Centrifugation**

centrifuge overexpressed culture (at 9/28) 3500g, 4℃, 20min and remove supernatant, keep it at -20℃

### 2019/10/7

**Transformation**

nnLuz-pB4/cph-pB4\*BL21(DE3)-star/C43(DE3) for expression test, and 23O, 24P transformation for fluorescence measurement

**Standard curve**

measure by dilution. mRFP1 LPETG protein(0.51mg/ml), RLU E-2~E-11.

### 2019/10/8

Successed all transformation conducted on 10/7. Pick colony for expression and culture it in LB broth. Then keep only pellet(4000g, 10min) at -20℃.

### 2019/10/10

Culture C43(DE3) for one day for activity test

Characterization: Culture 23O, 24P single colonies in 10/7 plate for 6 hours. Then measure the OD600 of diluted(1/5) suspension. Set the suspensions to OD600=1 and load 100㎕ to 96well plate, measure RLU 530nm/590nm

### 2019/10/11

Centrifuge C43(DE3) culture on 10/10. Then conduct resuspension it with 4L2/4H6+C43(DE3) pellet cultured on 9/28 and sonicate the suspension. Centrifuge this suspension and 4L2/4H6+BL21(DE3)-star suspension made on 9/20 and take only supernatant. Mix crude extract+NADPH+hispidin and measure luminescent using luminometer.

Contact

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# Team:KUAS Korea/Experiments

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# Experiments

For the development of tattoo, which can identify a cortisol concentration of blood, the goal of our project, the Wet Lab study first focused on visualization methods. So first we focused on securing the genes to build the bioluminescence system. Our system needs 8 genes, 4 genes are the key genes that make up the bioluminescence cycle, and the other 4 genes are those that are involved in the supply of materials and the activation of the cycle. We conducted the experiment first with the aim of acquiring the key elements of the cycle, the luciferase and hispidin 3-hydroxylase(hereinafter H3H) genes and verifying their activity. The process has progressed significantly over time to: the preparation of experimental resources, DNA cloning, and protein level analysis.

[Preparation](#title1)
[DNA work](#title2)
[Protein work](#title3)

## Preparation

### Competent cell preparation

In order to gain genes through cloning, competent cells are needed first. We made competent cells that can change shape by heat shock and used them for all subsequent processes. We will experiment using Escherichia coli, and the strains used are largely three types; DH10B, BL21(DE3)-star, C43(DE3). DH10B is strains to be used for cloning because of their good transformation efficiency, while the other two strains are to be used for gene expression and protein analysis due to their high protein expression. The DH10B strain was tested for its transformation efficiency by transforming pB plasmid vector.

### Vector DNA preparation

Cloning requires a vector to carry genes to the host. We used plasmid vectors because we are going to clone it with E. coli, and we will assemble genes in a way called LIC(Ligation Independent Cloning). Therefore, we decided to use a plasmid called pB vector, which has no MCS and has only one SmaI restriction site(5'-CCCGGG-3'). SmaI makes blunt ended product and these features are suitable for LIC. The pB vector results in protein fusion after cloning, with seven different types of pB vectors depending on the tag sequence. The presence of tags can be used for affinity chromatography in protein purification, and the tags can be removed later, for example, by treating the TEV protease. The pB vector also has the ampicillin resistance gene, which can be used to screen the transgenic strains.

Each pB vector can be introduced in competent cells as a heat shock method, cultured enough, and plasmid can be obtained by minipreparation method. To check whether a restriction site, the most important part of the pB vector is preserved, the extracted plasmid was treated with the SmaI restriction enzyme and then the single band appeared as an electrophoresis.

## DNA work

### PCR

The first gBlock we ordered through IDT is what we call luc2 and hydroxy, each with the fungal luciferase and H3H genes. Two primers(forward, reverse) are designed for each gBlock. Primers are designed to protrude beyond the end of the gBlock, where the protrusion is designed complement to the part of the plasmid vector restriction site for cloning. PCR was performed using primer, gBlock, and pfu polymerase, and the amplified product was checked with electrophoresis.

![](/wiki/images/c/cd/T--Kuas_korea--primer.png)

The electrophoresis result showed the band at the desired length(Luc2: 747bp, hydroxy: 1261bp)

![](/wiki/images/4/45/T--KUAS_Korea--wetlab--fig1.png)

### Vector linearization with registration enzyme

For LIC, you must first cut the plasmid vector with the restricton enzyme. We cut pB vectors of each type with the SmaI restriction enzyme and verified that they are cut with electrophoresis.

![](/wiki/images/3/3f/T--KUAS_Korea--wetlab--fig2.png)

The electrophoresis result confirmed one band in all.

### DNA cleanup

When assembling DNA, the ratio and purity of two DNAs are important. To make samples without impurities as much as possible, purify the restriction enzyme-treated plasmid and gBlock amplified with PCR.

### Assembly

T4 DNA polymerase was used for DNA assembly. When PCR products, linearized vectors, and T4 DNA polymerase are mixed without dNTP, only 3'→5' exonuclease activity of the pol. is activated, making the end of each component sticky and can assembled each other. Performing this method, the time to treat T4 DNA pol. is important and it is useful because carried out successfully, self-assembly can be reduced. Assembled DNA will be transformed into host cells without the treatment of ligase, and ligation depends on the DNA repair technique and cloning process of host cells. We used the LIC method to assemble the luc2 and hydroxy genes into the pB vector.

### Transformation

The recombinant DNA was then transformed into the DH10B strain and then spread on a medium with ampicillin added. Since DH10B does not have its own ampicillin resistant gene, subsequent colony is almost likely to be recombinant strains.

### Colony PCR

Since bacterial cells with only vectors-without gBlock assembled-can survive against antibiotics, arbitrary colonies should be selected to confirm the presence of insertion. Colony PCR is a method of breaking cells after growing colonies and amplifying the desired sequence of pieces with PCR. If the primer which is the same thing as gBlock PCR is used at this time, PCR products will be produced only when the desired gene is inserted. By electrophoresis, you can screen out the desired recombinant strains.

![](/wiki/images/9/9a/T--KUAS_Korea--wetlab--fig3.png)

Only the desired colonies can be screened by checking whether the band exists on the predicted size.

### Minipreparation

By cultivating selected colonies and extracting plasmids, we can obtain recombinant plasmids.

### Other genes

In addition to Luc2 and hydroxy, we are continuously carrying out cloning genes such as nnluz, nnh3h, and cph. This text will mainly explain the luc2 and hydroxy genes but cloning of the rest is also essential to complete our bioluminescence cycle. As of October 2019, cloning of the nnluz, nnh3h genes has also been completed. The two genes encode luciferase and H3H, respectively, derived from a fungus called Neonothopanus nambi(‘nn’ means N.nambi).

## Protein work

### Expression test

Once a gene has been acquired, it needs to be checked if the gene actually expresses the protein with the expected activity. The steps of activity verification are divided into three categories: expression test, solubility test, and activity test in chronological order. Among them, an expression test is a way to determine if the desired gene is expressing protein. It is necessary to perform SDS-PAGE analysis for the cells which are over-expressed the desired genes with the control group, a normal expressing group. The amount of all protein in the cell is checked by electrophoresis. If you check for unusual bands compared to controls and they are the same size as the ones you want to encode, you may say that the genes present proteins.

### Solubility test

Just because proteins are expressed, it doesn’t mean that they are always active. For having a normal function, they should exist in an active form. For example, there are many cases of over-expression failure by IPTG processing, since the protein does not form a normal structure and may become tangled together to form a aggregates. These insoluble particles are not of the desired type because they do not have normal activity or function. Therefore, it is necessary to verify that the proteins exist in an active form. When analyzing all proteins in a cell, this can be available by centrifuging the total homogenate to separate with soluble and insoluble parts, and then SDS-PAGE them together. If over-expressed bands are identified in the lane of the soluble part, it suggests that the protein was expressed in its normal(soluble) form.

### Activity test

Since we have confirmed that proteins exist in soluble form, we have decided to examine each gene's activity using the luc2-pB4 and hydroxy-pB4 plasmid recombinant strains. The ideal test for gene activation is to purify proteins and perform quantitatively, but you can quickly conduct an active verification test before purifying them. If the active verification fails, the time and effort wasted is large, so if the activity is confirmed in simple way, then proceed with purification and start quantitative analysis. This simple test means just using of unpurified cell fractures(crude extract). However, we have confirmed that protein exists in a soluble state, so we decided to crush cells and centrifugate them to verify their activity using only the supernatant.

So how do we identify the activity of the proteins? For luc2 gene, we should focus on the feature, luminescence. We need to determine if luminescence is observed by adding the substrate of luciferase into the supernatant. The measurement of luminous intensity is possible by using a luminometer. But the problem was the active identification of the hydroxy gene. The only known function of this gene is that it acts as a catalyst for the synthesis of luciferin. Determining whether it produce fungal luciferin, 3-hydroxyhispidin, would be a way of verifying the activity of the hydroxy gene. But we couldn't use this method because this process requires high-level chemical techniques such as NMR. So, we decided to use the alternative method of identifying the two genes at once. The hydroxy gene hydroxylates hispidin under the presence of NADPH. And the resulting substance, 3-hydroxyhyspidin, becomes the substrate of fungal luciferase, that is, luciferin. Thus, it is known that a combination of hispidin, NADPH, H3H, and luciferase can be expected to produce luminescence through two steps of biochemical reactions. Although this solution was difficult to troubleshoot when failed to check activity, we conducted an active verification experiment with this idea because it was worth a try in our situation.

There are 4 major substances involved in this test; hispidin, NADPH, H3H crude extract, and luciferase crude extract. We cannot expect luminescence if even only one of any of these factors are omitted. Therefore, at least 4 control groups are required by excluding one of each element. Moreover, the luminometer should be aware of the background noise for the mixture we measure, since it tells us the relative value(RLU; Relative Light Unit) of the current output by the measured luminescence. Therefore, the luminous intensity of 6 objects is measured. We transformed each gene into 2 strains, BL21(DE3)-star and C43(DE3). Of these, only the culture of BL21(DE3)-star was tested for solubility, but we decided to conduct an experiment with both cases. The table below shows the composition of each target. Omphalotus japonicus is a species of luminous mushroom that acts as a positive control group to verify that hispidin works normally as a precursor to luminescence.

![](/wiki/images/c/cf/T--KUAS_Korea--wetlab--fig4.png)

The results of the experiment showed luminescence only in the experimental group that added all components. In order to ascertain specifically the variation in luminous flux over time, we conducted an additional experiment to observe the variation in luminous flux for 12 hours. Now we will enter the stage of obtaining pure luciferase and H3H through protein purification and identifying the specific characteristics of enzymes through quantitative analysis.

### Other genes

Since then, we have continued to conduct cloning and verifying protein activity on other genes. Currently, the nnluz and nnh3h genes are being tested for expression.

## Protocols

### **Competent Cell Preparation (for Heat Shock Method)**Materials Calcium chloride dihydrate sol.(1M), LB media, properly selected marker(antibiotics), centrifuge, Sorvall tube, micropipette Method 1. Spread the microbial strains on the LB-agar plate and incubate them at the appropriate temperature. 2. Pick a suitable cluster from 2 and perform a small shaking incubation in the LB-marker media. 3. Apply the culture of 2 to the LB-agar-marker plate and incubate at the appropriate temperature. 4. Pick a suitable colony from 3 and perform a shaking incubation at the LB-marker media. 5. Check the OD600 value frequently. When OD600=0.4~0.5 is reached, stop the incubation. 6. Put the culture fluid in a sterilized and frozen tube and centrifugate 20min at 4°C, 4000g. 7. Remove the supernatant solution, add a suitable amount of calcium chloride to the pallet, and mix it gently. 8. Wait for at least 30 minutes in the condition of 7, and then perform centrifugation again as provided in 6. 9. Remove the supernatant and pour the calcium chloride into the pallet in an appropriate amount (0.1 M calcium chloride 2 ml per 50 ml of original culture) and mix it gently. 10. Use immediately for transformation or mix glycerol and store at -70°C. **Transformation**Materials competent cell, DNA, Eppendorf tube, LB media, micropipette Method 1. Put a suitable amount of competent cell in a tube. 2. Add DNA (less than 50 ng per 10μl of competent cell) to 1 and ice it for 30 minutes. 3. Heat the tube in a 42°C water bath for about 1 minute. Do not shake at this time. 4. Add 800μl of LB media to the tube and culture with shaking incubation at the appropriate temperature for at least 1 hour. 5. Spread a culture of 4 to the LB-agar-marker plate (if present) and incubate it at an appropriate temperature. **Agarose Gel Electrophoresis for DNA**Materials agarose, TAE buffer, DNA loading buffer, mold, DNA, ladder (marker DNA fragment), electrophoresis kit, power supply, micropipette Method 1. Mix agarose in a TAE buffer solution at the desired percentage, heat it, and dissolve it. 2. Pour 1 into a mold and stick the comb in and harden. 3. Put the hardened gel in the kit and fill it with buffer. 5. Mix sample DNA in the loading buffer (about 0.8x). 6. Load ladder and 5 into each well. 7. Apply a suitable voltage (100~150V) for 10~20 minutes. 8. Examine the gel for UV rays and observe the results. **Minipreparation (Using MG MED Kit)**Materials culture, buffers (S1, S2, S3, AW, PW, EB), centrifuge, column, Eppendorf tube, micropipette Method 1. Centrifugate the microbial culture at 4000g for 20min. 2. Remove the supernatant completely and add S1 buffer (250μl per 5 ml of culture) and mix it. 3. Add 250μl of S2 buffer to 2, then mix very gently and quickly. 4. Add 350μl of S3 buffer to 4 and mix them quickly. Centrifugate the solution (13500g, 10min) . 5. Put the supernatant in the column and place it in the Eppendorf tube and centrifugate it (13500g, 1min). 6. Discard the filtrate, add 300μl of the AW buffer to the column, and perform centrifugation (13500g, 1min). 7. Discard the filtrate, add 500μl of the PW buffer to the column, and perform centrifugation (13500g, 1min). 8. Throw away the filtrate and perform centrifugation again (13500g, 1min). 9. Replace the Eppendorf tube, add EB to the column as much as desired (50~100μl), wait for one minute and centrifugate it (13500g, 1min). 10. Discard the column and take a filtrate. 11. Check the results with electrophoresis. **PCR**Materials DNA template, primer(F/R), DNA pol., DNA pol. Buffer, pure water, PCR tube, thermocycler, micropipette Method 1. Mix ingredients in a PCR tube in appropriate proportion. DNA polymerase is the last to be mixed. \*for pfu pol.; DW: 10x buffer: primer F: primer R: 10mM dNTP : template : pfu = 39 : 5 : 1 : 1 : 1 : 1 : 2 (total 50) \*for Q5 pol.; DW: 5x buffer: Primer F: Primer R: 10mM dNTP : template : Q5 = 29.5 : 10 : 2.5 : 2.5 : 4 : 1 : 0.5 (total 50) 2. After placing the tube on the thermocycler, repeat the cycle (denaturation-binding-annealing) as desired and react to the temperature conditions according to the primer. 3. Check the result with electrophoresis. \*Colony PCR is the same method except that the ‘liquid medium’ is used instead of the ‘template’. **PCR Product Cleanup (Using MG MED Kit)**Materials sample DNA, buffers(PB, NW, EB), Eppendorf tube, column Method 1. Mix a PB buffer equal to five times the amount of DNA. 2. Put the column in the Eppendorf tube and put 1 in the column. 3. Perform centrifugation (13500g, 1min) and remove the filtrate. 4. Add the NW buffer of 700μl to 3, and perform centrifugation (13500g, 1min). 5. Remove the filtrate of 4 and centrifugate it one more time (13500g, 1min). 6. Replace the Eppendorf tube, add EB to the column as much as desired (50 to 100μl), wait for one minute and centrifugate (13500g, 1min). 7. Discard the column and take a filtrate. **Vector Restriction Enzyme Cut**Materials vector, restriction enzyme, enzyme buffer Method 1. Mix vector and enzyme buffer. 2. Add the restriction enzyme to 1 and wait at room temperature for 10 minutes. 3. Check the results with electrophoresis. **LIC**Materials T4 DNA pol. and buffer, insert DNA, vector, pure water, competent cell Method 1. Mix T4 DNA pol. buffer, insert DNA, vector, and pure water in proportion and keep them cold. 2. Add T4 DNA pol. to 1 and then react 2.5min at room temperature and quickly put it into ice. 3. Perform transformation by mixing the solution of 2 and the component cell, and then incubate it in a suitable medium plate. **Overexpression (IPTG induction)**Materials media, IPTG(1M), micropipette Method 1. Cultivate the desired microorganisms and measure the OD600 value frequently. 2. When OD600=0.8 is reached, stop incubation and add IPTG (0.1 mM - 1 mM). 3. Culture by shaking incubation at 4°C for 12 hours. **SDS-PAGE**Materials protein sample, PAGE gel, loading buffer, running buffer, staining/washing buffer, PAGE kit, power supply, micropipette Method 1. Assemble the gel to the kit and pour the running buffer. 2. Mix the protein sample with the loading buffer. 3. Load 2 into 1 gel well and run for 50 minutes. 4. Take out the gel from kit and observe the results after staining (1 hour) and washing

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# Team:KUAS Korea/Results

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# Results

[Preparation](#title1)
[DNA work](#title2)
[Protein work](#title3)

## Preparation

### Competent cell preparation

For DH10B, the number of colonies was calculated and the transformation efficiency of the DH10B strain was around 5.23E+3.

![](/wiki/images/4/4a/T--KUAS_Korea--wetlab--fig5.png)

### Vector DNA preparation

The extraction concentration of each vector is as follows; pB1: 27.087ng/㎕, pB2: 27.201ng/㎕, pB3: 21.234ng/㎕, pB4: 23.218ng/㎕, pB5: 21.298ng/㎕, pB6: 19.266ng/㎕, pB7: 21.398ng/㎕. Electrophoresis results show a single band on all vectors.

![](/wiki/images/5/57/T--KUAS_Korea--wetlab--fig6.png)

## DNA work

### Transformation

Clones for several pB vectors were obtained. The picture below shows the results of successful cloning of pB3 and pB7 vectors. Although there is only one picture, we repeated this process several times, eventually adopting a cloned strain using pB3 and pB4 vectors.

![](/wiki/images/a/a5/T--KUAS_Korea--wetlab--fig7.png)

### Minipreparation

In the overall cloning process, about two failures were experienced, resulting in the acquisition of luc2-pB3 and luc2-pB4, hydroxy-pB3 and hydroxy-pB4 plasmid.

![](/wiki/images/2/2f/T--KUAS_Korea--wetlab--fig8.png)

## Protein work

### Expression test

![](/wiki/images/c/c3/T--KUAS_Korea--wetlab--fig9.png)

As a result of SDS-PAGE, the bands were identified in both the luc2 and hydroxy genes’ recombinants, so the desired protein was expressed in the host. The different bands have different heights depending on the type of vector, because different tags are linked to the protein. The tag of the pB3 vector has a size of 3,128Da and the tag of pB4 vector is 43,623Da.

### Solubility test

![](/wiki/images/8/8a/T--KUAS_Korea--wetlab--fig10.png)

![](/wiki/images/3/36/T--KUAS_Korea--wetlab--fig11.png)

Test’s result shows that only genes assembled into the pB4 plasmid showed protein in the soluble part. Therefore, we decided that subsequent identification of activity would be carried out using only this plasmid.

### Activity test

The results of observing the luminous flux of the experimental group with the control group for 10 minutes are as follows for each strain.

![](/wiki/images/c/c7/T--KUAS_Korea--wetlab--fig12.png)

The luminous flux of O.japonicus with the addition of hispidin was also measured, and the results are as follows.

![](/wiki/images/5/5b/T--KUAS_Korea--wetlab--fig13.png)

This confirmed that hispidin works as a fungal luminescent element. We can therefore see the activity of the fungal luminous enzyme in the test tube. However, compared with the amount of light emitted in mushrooms, it seems that in vitro emits are quite low levels. It can be reasonable to think that the activation factor for luminescence may additionally act in vivo, or the results may vary depending on the proportion of necessary components. In other words, this result show the possibility that our system can produce strong enough light through optimization tasks.

The results of the measurement of luminous flux for an additional 12 hours(5-minute interval) are as follows.

![](/wiki/images/8/86/T--KUAS_Korea--wetlab--fig14.png)

From the above results, the luminous flux graphs for each strain are shown below, which subtracts the mean of background noise represented by wild type, C43(DE3).

![](/wiki/images/c/cd/T--KUAS_Korea--wetlab--fig15.png)

At the beginning of measurement, it can be seen the luminous flux gradually increases and then decreases slowly after reaching its peak. In addition, the early experiments of 10 minutes-measurement were not sure, but a small amount of luminescence was still observed in the C43(DE3) strain. Although C43(DE3) is uncertain because it has not been tested for solubility, it can be expected that most enzymes have been converted into insoluble proteins in the process of over-expression.

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