iGEM 2018 — Team Gifu

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# Protocol

---

### Medium

LB medium(100 mL)

|  |  |
| --- | --- |
| Tryptone | 10g |
| Yeast Extract | 0.5g |
| NaCl | 1.0g |||  |  |
| --- | --- |
| H2O | 100 ml |
| agar (in case of agar medium) | 2.0g |

SOC medium(100 mL)

|  |  |
| --- | --- |
| Tryptone | 2.0g |
| Yeast Extract | 0.5g |
| 1M NaCl | 1.0 mL |
| 1M KCl | 0.25 mL |
| H2O | (up to 100 mL) |

YPD medium(100 mL)

|  |  |
| --- | --- |
| Yeast Extract | 1.0g |
| Glucose | 2.0g |
| Peptone | 2.0g |
| H2O | 100 mL |

### Restriction digests

#### Materials

Ice and a bucket/a container
DNA to be digested
CutSmartTM buffer
Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
Incubater

#### Procedure (an example)

1.Add 43 ul of DNA to be digested into a 1.5 ml microcentrifuge tube.
2.Add 5.0 ul of 10x CutSmartTM buffer.
3.Add 1.0 ul of EcoRI.
4.Add 1.0 ul of SpeI.
5.There should be a total volume of 50 ul. Mix well and spin down briefly.
6.Incubate the restriction digest at 37℃ for 30 min.
7.Run a portion of the digest on a gel (6 ul) and check that part length is accurate.

### Ligation

1. Add digested fragment A.
2. Add digested fragment B.
3. Add ligation mixture.
4. Ligation 16℃ for 30 min.
*Note*: Make the amount of fragment A and B equimolar. The volume of ligation mixture is equivalent to the total volume of the mixed solution of fragment A and B. We used a constant temperature water bath.

### Transformation

1.Dispense 25 ul of competent cells to each 1.5 ml tubes.
2.Add 1 ul of DNA to each tube.
3.Close tubes and incubate the cells for 30 min on ice.
4.Heat shock the cells at 42℃ for 60 seconds.
5.Incubate the cells on ice for 2 minutes.
6.Add 225 ul of SOC medium to each tube.
7.Incubate cells at 37℃ for 30 minutes.
8.Spread 50 μL of cells onto each plate containing appropriate antibiotics.
9.Culture the cells.

### DNA densitometry(NanoDrop)

1.Activate the device.
2.Choose “Nucleic Acid” at main menu.
3.Wash the upper and lower optical surfaces of the microspectrophotometer sample retention system with distilled water. Wipe off both optical surfaces with a Kimwipe gently.
4.Choose a sample type. There are DNA-50(double-strand), DNA-33(single-strand), RNA.
5.Pour 1 μL distilled water to test a blank and clean both optical surfaces with a Kimwipe
6.Pour 1 μL of the sample to measure the concentration.

### Colony PCR

#### Materials

##### Prepare reaction mixture(45 μL)

|  |  |
| --- | --- |
| Nuclease free water | 27.5 µL |
| 10×PCR buffer | 5 μL |
| 2 mM dNTP | 5 μL |
| 5 pmol/µL Fw primer | 2.5 μL |
| 5 pmol/µL Rv primer | 2.5 μL |
| 0.5 U/μL Taq polymerase | 2.5 μL |

#### Procedure

1. Pick a colony on a plate and suspend it in 100 μL of LB medium(liquid)
2. Add 5 μL of diluted cell culture to reaction mixture.(Total volume is 50 μL)
3. Do PCR amplification.
4. Measure the length of the DNA by Electrophresis.

### Electrophoresis

#### Materials

##### Prepare 100 mL of agarose solution

|  |  |
| --- | --- |
| Agarose | 2g |
| 50×TAE buffer | 2 mL |
| Deionized water | 98 mL |

#### Procedure

1. Heat agarose solution in a microwave oven to dissolve agarose completely.
2. Cool the solution to an appropriate temperature.
3. Pour the solution into a mold with a comb.
4. Remove bubbles in the solution.
5. After the gel harden, remove the comb carefully.
6. Transfer the mold into a phoresis tank.
7. Immerse the mold in 1×TAE buffer.
8. Mix 5 µL of DNA solution and 1 µL of 6× loading buffer.
9. Pour the mixture into the well.
10. Electrophorese at 100 V.
11. Stop the electrophoresis when the band of dye move up to 3/4 of the gel.
12. Pick out the gel and then stain it with ethidium bromide (0.5 µg/mL) for 20 minutes.
13. Observe DNA bands with UV transilluminator.

### Miniprep

#### Materials

##### Prepare following solutions

SolⅠ: 50 mM glucose/25 mM Tris-HCl/10 mM EDTA
SolⅡ: 0.2N NaOH/1%SDS
SolⅢ: 3M acetic acid/5M potassium

#### Procedure

1. Dispense 1000 μL of culture to a 1.5 ml tube.
2. Centrifuge(14,000rpm, room temperature,1 minutes) and remove the top layer by decantation.
3. Add 50 μL of SolⅠand mix it by vortex.
4. Add 100 μL of SolⅡ and mix it very slowly and put it gently on ice for 1 minute.
5. Add 75 μL of SolⅢ and mix it slowly and put it gently on ice for 5 minutes.
6. Centrifuge(14,000rpm, 4℃, 5 minutes) and move the top layer to a new tube.
7. Add 225 μL of chloroform/phenol(1:1) and mix it .
8. Centrifuge(14,000rpm, room temperature, 10 minutes) and move the top layer to a new tube.
9. Add 225 μL of chloroform and mix it.
10. Centrifuge(14,000rpm, room temperature, 5 minutes) and move the top layer to a new tube.
11. Add 225 μL of propan-2-ol and mix it and put it gently for 5 minutes.
12. Centrifuge(14,000rpm, room temperature, 10 minutes) and remove the top layer.
13. Add 500 μL of 70% ethanol and turn upside-down to clean the inside of the tube.
14. Centrifuge(14,000rpm, room temperature, 5 minutes) and remove the top layer completely and dry it by vacuum drying.
15. Melt the precipitation by adding 20 μL of 20 μg/mL RNase A/TE buffer.
16. Incubate it for 30 minutes in 37℃.

### Gel Extraction with FastGene™ Gel/PCR Extraction Kit

#### Sample preparation

1.Cut down a DNA fragment from an agarose gel. Remove surplus agarose to make the gel fragment as small as possible.
*Note*: Recommended concentration of agarose is under 2.5%.
2.Transfer the gel fragment (up to 300 mg of gel) to a centrifugal tube.
3.Add 500 µL of GP1 to a sample and voltex the tube.
4.Incubate the sample at 55℃ for 10~15 minutes (until the gel fragment completely dissolves). Invert the tube every 2~3 minutes while incubating.

#### Sample loading

1.Insert FastGene™GP column into a collection tube.
2.Dispense (up to 800 µL of) sample solution prepared previously into FastGene™GP columns　and then centrifuge it(13,000rpm, 30 seconds).
3.Throw filtrate away and then return the column to the collection tube.
4.Caution: If the volume of the sample solution is over 800 µL, repeat the step of DNA bonding.

#### Membrane washing

1.Add 600 µL of GP2 to the column and then centrifuge it(13,000rpm, 30 seconds).
2.Throw filtrate away and then return the column to the collection tube.
3.Caution: If you use TAE gel, proceed to the next step. If you use TBE gel, repeat this step to remove boric acid completely.

#### Membrane drying

1.Centrifuge a column matrix(13,000rpm, 2 minutes) to desiccate it.

#### DNA Elution

1.Insert a FastGene™GP column into a new centrifugal tube.
2.Add 20~50 µL of elution buffer GP3 to the center of column matrix.
3.Keep the same state for 2 minutes at room temperature to ensure GP3 is absorbed by the column matrix,.
4.Centrifuge it(13,000rpm, 2 minutes) and then elute refined DNA.

### PCR Purification with FastGene™ Gel/PCR Extraction Kit

#### Sample preparation

1.Mix GP1 and PCR products in the ratio of 5 to 1 in a centrifuge tube(e.g. 200 µL of GP1 per 40 µL of PCR products).
Caution: If the volume of the sample is under 40 µL, add GP1 or water for PCR products to adjust the volume of the sample to 40 µL.

#### Sample loading

1.Insert FastGene™GP column into a collection tube.
2.Dispense (up to 800 µL of) sample solution prepared previously into FastGene™GP columns　and then centrifuge it(13,000rpm, 30 seconds).
3.Throw filtrate away and then return the column to the collection tube.
4.Caution: If the volume of the sample solution is over 800 µL, repeat the step of DNA bonding.

#### Membrane washing

1.Add 600 µL of GP2 to the column and then centrifuge it(13,000rpm, 30 seconds).
2.Throw filtrate away and then return the column to the collection tube.
3.Caution: If you use TAE gel, proceed to the next step. If you use TBE gel, repeat this step to remove boric acid completely.

#### Membrane drying

1.Centrifuge a column matrix(13,000rpm, 2 minutes) to desiccate it.

#### DNA Elution

1.Insert a FastGene™GP column into a new centrifugal tube.
2.Add 20~50 µL of elution buffer GP3 to the center of column matrix.
3.Keep the same state for 2 minutes at room temperature to ensure GP3 is absorbed by the column matrix,.
4.Centrifuge it(13,000rpm, 2 minutes) and then elute refined DNA.

### HisLink Spin Protein Purification

#### Materials to Be Supplied by the User

・Nuclease-Free Water
・rotor
・wide-bore pipette tips
・5M NaCl solution
・tabletop centrifuge
・1.5 ml microcentrifuge tubes

#### Preparation of FastBreak™ Reagent/DNase I solution

1.Add 80 μL of Nuclease-Free Water to the vial of DNaseⅠ.
2.Mix completely to dissolve the powder.
3.Remove the DNase solution from the vial and add it to 1 mL of Nuclease-Free Water.
Mix well.
4.Mix 5.8 μL of the FastBreak™ Reagent and 64.2 μL of the DNase I solution.

#### Procedure of centrifugation

1.Pipet 700 μl of bacterial culture into a 1.5 ml microcentrifuge tube. Add 70 μl of the FastBreak™ Reagent/DNase I solution.
2.Resuspend the resin and allow it to settle. Once the resin has settled, use a wide-bore pipette tip to transfer 75 μl of the HisLink™ Resin from the settled resin bed to the 1.5 ml microcentrifuge tube. To successfully transfer resin, place the wide-bore pipette tip deep into the resin and pipet slowly to assure that a consistent amount of resin is drawn into the pipette. Allow the resin to resettle between each pipetting.
*Note*: We recommend optimizing the amount of HisLink™ Resin used for low- (<1 mg/ml) or high- (>1 mg/sample) expressing proteins. For low-expressing proteins, less resin should be used; similarly, for high-expressing proteins, more resin per sample can be used.
3.Incubate the sample and resin for 30 minutes, mixing frequently on a rotating platform or shaker to optimize binding.
4.Place a Spin Column onto a Collection Tube (or a new 1.5 ml microcentrifuge tube). Use a wide-bore pipette tip to transfer the lysate and resin from the original 1.5 ml microcentrifuge tube in Step 3 to the spin column. If resin remains in the 1.5 ml microcentrifuge tube, add HisLink™ Binding/Wash Buffer to the tube, then transfer the buffer and remaining resin to the spin column.
5.Centrifuge the spin column with the collection tube for 5 seconds or until the liquid clears the spin column.
6.To save the flowthrough, remove the spin column from the collection tube and transfer the flowthrough from the collection tube to a new 1.5 ml microcentrifuge tube. Otherwise, discard the flowthrough.
7.Place the spin column back onto the collection tube. Add 500 μl of HisLink™ Binding/Wash Buffer to the spin column, then cap the spin column. Centrifuge for 5 seconds or until the Binding/Wash Buffer clears the spin column. Discard the flowthrough. Repeat for a total of two washes.
8.Take the spin column off the collection tube and wipe the base of the spin column with a clean absorbent paper towel to remove any excess HisLink™ Binding/Wash Buffer.
9.Place the spin column onto a new 1.5 ml microcentrifuge tube. Add 200 μl of HisLink™ Elution Buffer. Cap the spin column and tap or flick it several times to resuspend the resin. Wait 3 minutes.
10.Centrifuge the spin column and microcentrifuge tube at 14,000rpm for 1 minute to collect the eluted protein.

### SDS-PAGE

#### Protein extraction and Sample preparation

1.Add 50 µL of cell suspension that was cultured overnight to LB medium, incubate at 37℃ for two hours.
2.Measure the turbidity of *E. coli* culture at a wavelength of 660 nm. Dilute the cell suspension with LB liquid and make the optical density(660 nm) equivalent to 0.5. Add IPTG to the cell suspension.
3.Cultivate it for 3 hours and then store it at low temperature.
4.Dispense 1 mL of the suspension into a 1.5 ml tube. Centrifuge it(13,000rpm, 4°C, 15 minutes) and then remove the supernatant .Repeat this step twice.
5.Add 300 µL of PBS to the precipitation and then mix it.
6.Sonicate the cell suspension for 15 sec and cool it for 60 sec on ice. Repeat this step 4times.
7.Centrifuge(13,000rpm, 4°C, 20 minutes)and transfer the supernatant into another tube.
8.Add 100 μL of PBS to the precipitation. Vortex the tube.
9.Dispense 8 µL of the supernatant(=cell extract) into a new tube and 8 µL of the precipitation suspension into another tube.
10.Add 2 µL of 5×loading dye to the tubes and then denature it at 90℃.
11.Centrifuge and prepare the sample.
12.Apply 10 µL of the sample to SDS gel.

#### Making a SDS-PAGE gel and Electrophpresis

1 Mix and shake reagents quickly to prepare the separation gel.
2 Construct a plate for electrophoresis.
3 Pour separation gel into a gap of the plate (until about 2 cm below a comb).
Note: Wipe a plate for electrophoresis with 70% ethanol.
Hold a plate for electrophoresis not to spill the gel.
4 Pour a proper quantity of Milli Q water into a gap of the plate and then incubate for an hour at room tempareture.
5 Mix and shake quickly reagents for stacking gel (except APS and TEMED).
6 Slant the gel plate and absorb multistoried Milli Q water.
7 Add APS and TEMED to the mixture. (step5)
8 Fill the gap of the plate with stacking gel and then insert the comb into the gap of the plate.
Note: Be careful not to generate bubbles in gel.
9 Take out the plate and gel together after stacking gel coagulates.
10 Put the plate and gel into a migration tank with the plate toward outside.
11 Pour 300 µL of electrophoresis buffer into a phoresis tank. Immerse the gel completely.
12 Apply 10 µL of the sample and 5 µL of a marker.
13 Electrophorese at 40 mA in the stacking gel until a pigment comes at the separation gel.
After that, electrophorese at 60 mA in the separation gel.
14 Stop electrophoresis and then collect the gel carefylly.
Note: Use tweezers.
15 Remove the buffer for electrophoresis and dye the separation gel with CBB.
16 Wash the gel plate and the electrophoretic tank with neutral detergent and rinse it steadily.

#### Staining with CBB

1 Put the gel into fixing solution.
2 Leave the gel on the shaker until a band is dyed yellow.
3 Remove the fixing solution and then put the gel into CBB dyeing liquid.
4 Wrap it and then heat it until it is almost boiling with a microwave oven.
5 Remove the wrap carefully and then let vapor out slowly.
6 Remove the CBB dyeing liquid and pour deionized water into a container carrying the gel. Put Kim wipe into the container.
7 Infiltrate deionized water into the gel for several tens of minutes. Transfer waste liquid into a tank.
Take a picture under UV light and then dry the gel and store it.

### Transformation of yeast

#### For transformation of yeast, iGEM Gifu used *S. cerevisiae* Direct Transformation Kit from Wako.

1 Prepare 2 mL of YPD medium and inoculate yeast colonies.
2 Adjust OD600 to 0.01.
3 Transfer 1 mL of the medium to 1.5 mL of centrifuge tube and incubate it with shaking for approximately 20 hours.
4 Prepare 1.5 mL centrifuge tube and add 20 μL of Sc Transformation Reagent, 1 μg of plasmid DNA and 2 μL of Carrier DNA.
5 Add water to make the mixture 25 μL. Mix by vortexing.
6 Add 25 μL of the suspension and vortex it.
7 Incubate it for 2 hours at 42℃
8 Transfer 150 μL of water to the mixture and perform plating.
9 Incubate the plate for 2-3 days and perform colony PCR to confirm the transformation.


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# Team:Gifu/Experiments

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[JUDGING FORM](https://igem.org/2018_Judging_Form?team=Gifu)

# Plasmid design

---

The design of DNA fragment (Seq.1) we used this year is shown below. In PURE system, T7 RNA polymerase is contained. Basically, we need to use T7 promoter designated by the inventors for transcription. SD sequence and Red fluorescent protein lie between BBa\_K1859015 and BBa\_K1859016. As terminator of this alignment, T7 terminator (BBa\_K731721) was selected. In case of myTXTL, it uses σ70 promoter. When you use T7 promoter, you need to add T7 RNA polymerase. The recommended concentration of the polymerase is 2U/μL. SD sequence (RBS) and RBS are sandwiched by ribosomes. As T7 promoter, we did not use the sequence which has been registered in iGEM catalog. Instead of this, we used the new sequence of T7 promoter. RFP coding also contain TEV protease recognition site.

\*Caution: BBa\_K1332002 contains histidine tag. This time we excluded histidine tag. The function of the coding of this sequence is exactly the same as [BBa\_E1010](http://parts.igem.org/Part%3ABBa_E1010) except for stop codon.

![](https://static.igem.org/mediawiki/2018/2/2a/T--Gifu--new5.png)
**Seq.1 T7p-Ribozyme-SD-RFP-Ribozyme-T7t**

As proof of improvement of previous composite part of iGEM, [BBa\_K1491033](http://parts.igem.org/Part%3ABBa_K1491033) was provided by iGEM HQ. Typical alignment of T7 promoter is used in this part. However, in PURE system, the enhancer of the stem-loop structure need to be located in the downstream of T7 promoter to make an expression of protein intense. Thus we also design the part (Seq.2) below to show the improvement and this may contribute to the development of synthetic biology and iGEM competition. In Fig.1 we showed the detail of T7 promoter we used this year. Translation enhancer upstream of the SD sequence of mRNA, enhance the biosynthesis of protein. The enhancer may contribute to a direct interaction with ribosome protein S1. Fig.1 shows the detail of the enhancer.

\*Caution: BBa\_K1332002 contains histidine tag. This time we excluded histidine tag. The function of the coding of this sequence is exactly the same as [BBa\_E1010](http://parts.igem.org/Part%3ABBa_E1010). In Seq.2 it has s stop codon.

![](https://static.igem.org/mediawiki/2018/f/f7/T--Gifu--improve3.png)
**Seq.2 T7p--SD-RFP-T7t**

![](https://static.igem.org/mediawiki/2018/0/09/T--Gifu--promoter.png)
![](https://static.igem.org/mediawiki/2018/6/6e/T--Gifu--BASE.png)
**Fig.1 Explanation of enhancer of T7 promoter**

# Experiments

---

For activation of cell-free kit, both circular DNA and linear DNA is workable. We used linear DNA this time since we could not construct the plasmids. To amplify DNA fragment, PCR amplification or subcloning in *E.coli* is necessary. For the production, 0.5~3 ng/1 kb・μL of DNA for PURE system, 5 nM (Final concentration) of DNA for myTXTL was given to the reaction tube. Incubation time was typically 2 to 4 hours for PURE system, 15 hours for myTXTL. Observation of RFP under UV and SDS-PAGE was performed to confirm the expression. Flowchart of the cell-free expression is shown in Fig.2. You can perform PCR and cell-free expression in the same day. All DNA fragments were synthesized by IDT as gBlock fragment. The samples were sequenced and the alignments are all correct.

![](https://static.igem.org/mediawiki/2018/d/d4/T--Gifu--Flow.png)
**Fig.2 Flowchart of cell-free expression**

# Cell free system

---

We used two types of cell-free system. The difference is shown in Table.1.
![](https://static.igem.org/mediawiki/2018/e/e3/T--Gifu--difference_of_cell-free.png)
**Table.1 Cell-free system**

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# Team:Gifu/Results

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TEAM

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PROJECT

[Description](https://2018.igem.org/Team%3AGifu/Description)
[Experiments](https://2018.igem.org/Team%3AGifu/Experiments)
[Protocol](https://2018.igem.org/Team%3AGifu/Protocol)
[InterLab](https://2018.igem.org/Team%3AGifu/InterLab)
[Results](https://2018.igem.org/Team%3AGifu/Results)
[Improve](https://2018.igem.org/Team%3AGifu/Improve)
[Attributions](https://2018.igem.org/Team%3AGifu/Attributions)
[Calendar](https://2018.igem.org/Team%3AGifu/Calendar)
[Reference](https://2018.igem.org/Team%3AGifu/Reference)

[PARTS](https://2018.igem.org/Team%3AGifu/Parts)
[SAFETY](https://2018.igem.org/Team%3AGifu/Safety)

HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3AGifu/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3AGifu/Public_Engagement)

[JUDGING FORM](https://igem.org/2018_Judging_Form?team=Gifu)

# Results

---

# PCR

The result of PCR is shown in Fig.1~Fig.3. All PCR was performed by primers in table 1. All PCR were succeeded. We used PrimeSTAR Max premix for amplification.

![](https://static.igem.org/mediawiki/2018/4/45/T--Gifu--_BBa_K1491033.JPG)
**Fig.1 PCR of BBa\_K1491033**

![](https://static.igem.org/mediawiki/2018/4/43/T--Gifu--Seq1_PCR.jpg)
**Fig.2 PCR of Seq.1**

![](https://static.igem.org/mediawiki/2018/f/f9/T--Gifu--Seq2_PCR.jpg)
**Fig.3 PCR of Seq.2**

![](https://static.igem.org/mediawiki/2018/4/45/T--Gifu--Primers.png)
**Table.1 Primers**

# Cell-free expression

Here we show the result of expression. (Fig. 4~7) Seq.2 (monomer RFP, mRFP) was expressed well in PURE system and slightly expressed in myTXTL. Seq.1 (polymer RFP, pRFP) has no fluorescence. In myTXTL, T7 RNA polymerase (Takara) was supplemented before the use. For Seq.2, we added 50U. For Seq.1, we prepared two conditions, 50 U and 25 U.

![](https://static.igem.org/mediawiki/2018/c/c4/T--Gifu--BUVPURE.jpg)
 **Fig.4 After expression in PURE system (No UV)**

P: Positive (DHFR gene), N: Negative (Water), tem.: Template DNA

mRFP tem. and mRFP PCR was slightly turned into red.

![](https://static.igem.org/mediawiki/2018/f/f4/T--Gifu--BUV_myTXTL.jpg)
 **Fig.5 After expression in myTXTL (No UV)**

P: Positive (eGFP), N: Negative (Water)

Positive (eGFP) shows green color.

![](https://static.igem.org/mediawiki/2018/7/7d/T--Gifu--UV_PURE.jpg)
 **Fig.6 Tubes of PURE system under UV**

P: Positive (DHFR gene), N: Negative (Water), tem.: Template DNA

mRFP shows the clear fluorescence. mRFP tem. and mRFP PCR shows the same result. mRFP shown in the figure is mRFP tem.

![](https://static.igem.org/mediawiki/2018/f/f7/T--Gifu--UV_myTXTL.jpg)
 **Fig.7 Tubes of myTXTL under UV**

P: Positive (eGFP), N: Negative (Water)

Positive (eGFP) turned into green.

# SDSPAGE

We performed SDSPAGE to confirm whether pRFP was expressed or not. The result is shown below.

![](https://static.igem.org/mediawiki/2018/7/76/T--Gifu--SDSPAGE.jpg)
![](https://static.igem.org/mediawiki/2018/6/6f/T--Gifu--SDSPAGE-LANE.png)
**Fig.8 SDSPAGE**

As a result of SDS-PAGE, both myTXTL and PURE system shows the intense expression of the protein. In PURE system, we could see the monomer RFP. With the eye, a slight expression of mRFP in myTXTL was confirmed but not in SDSPAGE. In both cell-free system, the long-chain protein and mRFP was not confirmed with Seq.1.

# Discussion

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3-dimensional structure of the enhancer, stem-loop structure may inhibit the function of ribozymes. Ribozymes need to maintain certain structures to catalyze the reaction. Chemically, it is possible that the enhancer changes the ribozymes' conformation. We need to make the same plasmid without the enhancer so that we can confirm whether the stem-loop structure effects on the ribozymes. We should extract circular RNA from the tube confirm if there is circular RNA.

Furthermore, in myTXTL, even we confirmed the large expression of eGFP, we could not see enough expression of RFP. This may be because our T7 RNA polymerase does not fit in myTXTL crude extract. We should change it into T7 RNA polymerase from Thermo at a conc. of 2 U/μL. This is functional and confirmed by the company.

This time we did not use TEV protease site. Before performing the cut, we should consider whether no indispensable components for transcription and translation do not have the site. In addition, TEV protease can work in low temperature. To make monomer RFP, incubation temperature can be at low temperature. With this tip, the folding rate of the expressed protein would be much slower and the cutting rate may be effective.

# Future work

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Our final goal of the project is mass-production of cell-free system. Cell-free system is free system (Open system), so we can add any components from outside. It means "If amino acids may be running out, we can add them". If the system can be developed to industrial level, our idea is really interesting.

This year we focus on making monomer RFP from circular RNA. Circular RNA is a definitely amazing technique for cell-free system to cover its low ability of production of protein. We inserted TEV protease upstream the coding region. But when it comes to an operon, for example *E.coli* operon, they have a functional protein from one long mRNA. Technically, all protein of such mRNA has stop codons. Between each protein, they have spacer alignment. With the spacer region, even Ribosomes recognize the stop codons, it keeps binding to mRNA. This mechanism can be applied to circular RNA.
![](https://static.igem.org/mediawiki/2018/b/b7/T--Gifu--operon.png)

In addition to this, we tried to use PIE method to make circular RNA. However, the efficiency is estimated at 2.5% of all transcripted RNA. In cell-free system, plasmids can not be replicated and the efficiency of constructing the circular RNA is possibly quite small. Thus next year we would like to use *in vitro* circularization with RNA ligase. *in vitro* circularization has really high efficiency and we can use the RNA to quantify the speed of expression.

![](https://static.igem.org/mediawiki/2018/2/24/T--Gifu--in_vitro.png)

In conclusion, our future work is...

![](https://static.igem.org/mediawiki/2018/5/5a/T--Gifu--Future_work.png)

Focusing on the specific spacer, we would like to make it possible to have eternal production of protein, literally, iVEPOP in the near future.

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