iGEM 2022 — Team cafa-china

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{% block title %}Notebook{% endblock %}
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# N O T E B O O K

## Notebook

#### 20220718

#### The Synthetic gene synthesized gene is transferred to DH5a

Import the plasmid carrying the fragment of interest into DH5α for preservation

##### Experimental operation:

1. Plasmid dry powder (transport at room temperature, store at -20 degrees, 90 days shelf life, please be sure to use after converting and extracting plasmid)

2. Centrifuge the plasmid tube, 13000g for 3min, then add 80uL of sterile water to the tube, shake well, dissolve the plasmid dry powder;

3. Add 10μl of plasmid, chemically transform into DH5α strain competent state, named: pUC57-SulAp\_eGFP, pUC57-hrpR, pUC57-hrpS\_PhrpL, pUC57-SulAp\_relE, and coated on resistant plates;

4. Incubate the plate forward for 1h, and then invert at 37 °C for 14h;

（If there are too many colonies, dilute the plasmid before transformation. If there are no colonies, add 10 μl of plasmid for transformation. In addition, do not directly transform to competent cells, but first transform cloned competent cells, re-extract plasmids and then transform into expression competent cells）

---

#### 20220719

#### 1. SulAp\_eGFP、hrpR、hrpS\_PhrpL Fragment sequencing

Pick transformed single colonies (host bacterial DH5a) from yesterday's plates (pUC57-SulAp\_eGFP, pUC57-hrpR, pUC57-hrpS\_PhrpL), design primer amplification SulAp\_eGFP, hrpR, hrpS\_PhrpL fragments, agarose gel electrophoresis to verify fragment size and send for sequencing, run the program above.

##### Primer list：

![](https://static.igem.wiki/teams/4226/wiki/en01.png)

##### PCR outcome：

##### PCR outcome：

![](https://static.igem.wiki/teams/4226/wiki/en02.png)

#### 2. Save bacteria and shake bacteria

- Inoculate the Successfully transformed colonies of pUC57-SulAp\_eGFP, pUC57-hrpR, pUC57-hrpS\_PhrpL into 4 ml of LB medium (with antibiotics) at 220 rpm overnight;

- Pick a single colony of pSB1C3 (43) from the plate and inoculate into 4 ml of LB medium (with antibiotics) at 220 rpm overnight;

---

#### 20220720

#### 3. Plasmid extraction and amplification of the fragment of interest

##### Experimental operation：

1. Plasmid extraction: Extract the plasmids of pUC57-SulAp\_eGFP, pUC57-hrpR, pUC57-hrpS\_PhrpL, pSB1C3(43) according to the instructions;

2. PCR to obtain the gene and skeleton of interest

##### Gene of interest: from pUC57-SulAp\_eGFP

![](https://static.igem.wiki/teams/4226/wiki/en03.png)

##### Skeleton: From pSB1C3（43）

![](https://static.igem.wiki/teams/4226/wiki/en04.png)

##### Configure the PCR system: (refer to the instructions for using high-fidelity polymerases):

![](https://static.igem.wiki/teams/4226/wiki/en05.png)

##### Procedure (refer to instructions for use of high-fidelity polymerases):

![](https://static.igem.wiki/teams/4226/wiki/en07.png)

##### PCR products run glue

##### Agarose Gel DNA Recovery (Enhanced DNA Recovery Kit)

##### At this point, the target genes and skeleton are obtained

---

#### 20220721

#### 4. Plasmid construction

- Connect eGFP fragments to the pSB1C3 skeleton; Attach the SulAp\_eGFP fragment to the pSB1C3 skeleton;

- The homology arm has been added when the primers are designed in the front to facilitate subsequent Gibson connections:

- DNA fragments and Backbone are mixed in a 3:1 ratio, using Gibson connections.

##### Reaction system:

![](https://static.igem.wiki/teams/4226/wiki/en08.png)

Note: Linearized vectors with generally 10-50 ng/10ul reaction system, and the molar ratio of the insert fragment to the vector is 2-3:1 is the best.

Use a PCR instrument at 50°C, incubate for 1-2h or at 16°C overnight

#### 5. Chemical Conversion:

##### - After being connected by Gibson:

• Take 50 μL of E. coli competent cells and place on ice to slowly thaw.

• Add the assembly product, mix well with a flick and leave in an ice bath for 20 min.

• After 42 °C thermal excitation for 60 s, quickly ice bath for 2 min, the process does not shake the EP tube.

• Add 300 μL of LB liquid medium without antibiotics and resuscitate the culture in a 37 °C shaker for 60 min.

• For plasmid transformation (the amount of plasmid DNA added to the transformation >5 ng is sufficient), take 100 μL of the recovered bacterial solution, coat on a solid plate containing Canas, and invert at 37 °C for about 16 h.

(BL21 transformation colonies are relatively dense, so do not centrifuge before coating, but blow and aspirate the bacterial solution at the bottom)

---

#### 20220722

#### 6. Verification of plasmid construction

##### Colony PCR

![](https://static.igem.wiki/teams/4226/wiki/en09.png)

Agarose gel electrophoresis validation

• Send for sequencing

• Compare sequencing results with target sequences

##### The construction of the pSB1C3-j23119\_eGFP and pSB1C3-SulAp\_eGFP plasmids has been completed

##### - PUC57-SulAp-relE plasmid is converted to DH5a

• Flat coating

• PCR verification

• Pick-up

• plasmids exaction

---

#### 20220723

#### 7. SulAp\_eGFP Characterization

##### Experimental operation:

1. Seed solution preparation: pSB1C3（43 BL21）、pSB1C3-j23119\_eGFP（BL21）、pSB1C3-SulAp\_eGFP（JM109）、pSB1C3-SulAp\_eGFP（BL21）

2. Pick the transformed single colonies from the plate and transfer to 4 ml of liquid LB (C+), 37 °C, 220 rpm overnight incubation, use tin foil to block out the light;

3. Expand culture and protein expression: Add 10 ml lb (C+) to a 250 ml Erlenmeyer flask, take 100 ul of overnight culture liquid and add LB culture medium to OD595 = 0.4-0.5 (about 3-4 h) at 37 °C, 220 rpm, use tin foil to block out the light;

4. UV induction at different times, 37 °C, 200 rmp culture overnight or for 6-8 h, use tin foil to block out the light;

5. Collect bacterial bodies: microplate reader measures OD600, fluorescence value (excitation light 485 nm, absorbed light 535 nm, gain value 50)

![](https://static.igem.wiki/teams/4226/wiki/en10.png)

- The results of the fluorescence value were positively correlated with the UV induction time

- Repeat the experiment 3 times

---

#### 20220724

#### 8. SulAp-hrpR-eGFP plasmid construction, unsuccessful

- Attach the Sulp-relE fragment to the pSB1C3 skeleton:

- Amplify SulAp-relE fragments from pUC57-SulAp-relE

- Amplifyied pSB1C3 fragments as skeletons

---

#### 20220725

#### 9. SulAp-hrpR-eGFP plasmid construction, unsuccessful

2. pSB-SulAp-relE plasmid construction

- - Gibson procedure connects SulpAp-relE fragments and pSB1C3 backbones, competently transforms and cultures

---

#### 20220726

#### 10. pSB-SulAp-relE plasmid construction:

Colony PCR validation of SulAp-relE fragments

![](https://static.igem.wiki/teams/4226/wiki/en11.png)

Bacterial P verification: relE1, 3 colonies successful, sent for sequencing (1 ,3)

pSB1C3-SulAp\_eGFP fragment length is incorrect (5,6)

hrpR has no stripes and construct unsuccessfully (7, 8, 9)

---

#### 20220727

#### 1.RelE1,3-extracting plasmid, PCR, gel electrophoresis detection

#### 2.Test pSB1C3-SulAp\_eGFP fragments converted on July 22

![](https://static.igem.wiki/teams/4226/wiki/en12.png)

##### relE, strip correct, etc. sequencing results;

##### pSB1C3-SulAp\_eGFP et al. (7.21 and 7.27) conversion results;

---

#### 20220728

#### 1.pSB1C3-relE sequencing was successfully construct

Sequencing Results: Success

pSB1C3-relE, PadR-DC\_let4X (extract plasmid tomorrow)，pick up colony, ready to construct SulAp-relE-3WJ-Bro

#### 2.Test pSB1C3-SulAp\_eGFP fragments converted on July 27

![](https://static.igem.wiki/teams/4226/wiki/en13.png)

No. 21 ligation product, colony 3 with the correct band, sent for sequencing

No. 27 ligation product, colony 4 with the correct band, sent for sequencing

![](https://static.igem.wiki/teams/4226/wiki/en14.png)

![](https://static.igem.wiki/teams/4226/wiki/en15.png)

---

#### 20220729

#### 1.pss1 PCR products sequencing

pSB1C3-SulAp\_eGFP,Colony 27 convertes to colony 2

![](https://static.igem.wiki/teams/4226/wiki/en16.png)

#### Construct plasmid Sulp-relE-3WJ-Bro

Gibosn ligation, chemical conversion

---

#### 20220730

#### pSB1C3-SulAp\_eGFP sequencing was successful

Sequencing Results: Success

pSB1C3-SulAp\_eGFP,pick up colony, extract plasmid and prepare to construct pSB1C3-SulAp-hrpR-eGFP

#### 2.SulAp-relE-3WJ-Bro verification

##### 1. PCR results of SulAp-relE-3WJ-Bro colony:

![](https://static.igem.wiki/teams/4226/wiki/en17.png)
![](https://static.igem.wiki/teams/4226/wiki/en18.png)

The first No. 4 + the second No. 3, No. 5 band approach, sent for sequencing (note that the 3F+R and 5F1+R1 primers are different)

#### 3.Activity of Sulp after UV induction

Experimental operation:

1. Seed solution preparation: take non-recombinant BL21、pSB1C3-SulAp\_eGFP (BL21);

2. Pick a single colony from the plate and transfer to 4ml liquid LB (C+), 37 °C, 220 rpm culture,use tin foil to block out the light;

3. Induction of expression: Add 10 ml lb (C+) to a 50 ml centrifuge tube, take 100 ul of overnight culture solution and add LB culture medium to OD595 = 0.4-0.5 (~3-4 h) at 37 °C, 220 rpm, use tin foil to block out the light;

4. UV induction at different times, 37 °C, 200 rmp culture, 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, use microplate reader to measure of OD600 and fluorescence values respectively (excitation light 485 nm, absorbed light 535 nm, gain value 50)

![](https://static.igem.wiki/teams/4226/wiki/en19.png)

##### Experimental data is available and the results are as follows:

0730 SulAp Startup Effect (Notes)

![](https://static.igem.wiki/teams/4226/wiki/en20.png)

---

#### 20220731

#### 1.Construct pSB1C3-SulAp-hrpR\_eGFP (unsuccessful)

1. Extract the successfully constructed pSB1C3-SulAp\_eGFP plasmid

2. Amplify hrpR fragments and pSB1C3-SulAp\_eGFP skeleton

#### 2.construct pSB1C3-T4 lysis-lacZ

1. Amplify fragments and skeletons

![](https://static.igem.wiki/teams/4226/wiki/en21.png)

2. Gel recovery, Gibson ligation + transformation

---

#### 20220801

#### 1.pSB1C3-SulAp-eGFP validation

1. The sequencing is correct, the fragment size is correct, and the unseen of skeleton P is not because of plasmid

2. Build the pSB1C3-SulAp-hrpR\_eGFP, the skeleton is still not amplified, and it needs to be redesigned

![](https://static.igem.wiki/teams/4226/wiki/en22.png)

#### 2.pSB1C3-T4 lysis-lacZ authentication

1. The lacZ clip is not connected

![](https://static.igem.wiki/teams/4226/wiki/en23.png)

---

#### 20220802

#### 1.SulAp-relE-3WJ-Bro sequencing was successful

##### Plasmid profile completion:

#### 2.Plasmid construction

1. pSB1C3-SulAp-hrpR\_eGFP

![](https://static.igem.wiki/teams/4226/wiki/en24.png)

2. pSB1C3-T4 lysis-lacZ

![](https://static.igem.wiki/teams/4226/wiki/en25.png)

Strip correctly, cut glue recycled, Gibson connected, transformed

---

#### 20220803

#### 1.Plasmid ligation test

pSB1C3-SulAp-hrpR\_eGFP has suspected bands:

![](https://static.igem.wiki/teams/4226/wiki/en26.png)

pSB1C3-T4 lysis-lacZ，Colony 4 may be correct:

![](https://static.igem.wiki/teams/4226/wiki/en27.png)

Colonies are too dense, dilute inoculation after transformation ，try again

#### 2.Test the effect of relE-3WJ-Bro

Experimental Materials:

##### SulAp-relE-3WJ-Bro

Experimental operation:

1. Seed solution preparation: Take SulAp-relE (DH5a), SulAp-relE-3WJ-Bro (DH5a)；

2. Pick a single colony from the plate and transfer to 4ml liquid LB (C+), 37 °C, 220 rpm culture, use tin foil to block out the light；

3. Induction of expression: Add 10 ml lb (C+) to a 50 ml centrifuge tube, take 100ul of overnight culture broth and add LB culture medium to OD595 = 0.4-0.5 (approximately 3-4 h) at 220 rpm, use tin foil to block out the light;

4. UV induction at different times, 37 °C, 200 rmp culture, 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, use microplate reader to measure of OD600 and fluorescence values respectively

##### Invalid experimental data:

![](https://static.igem.wiki/teams/4226/wiki/en28.png)

---

#### 20220804

#### 1.Plasmid ligation test

pSB1C3-SulAp-hrpR\_eGFP：

![](https://static.igem.wiki/teams/4226/wiki/en29.png)

##### Send for sequencing: Colonies 1, 2, 3, 6

pSB1C3-T4 lysis-lacZ：

![](https://static.igem.wiki/teams/4226/wiki/en30.png)

---

#### 20220806

#### 1.The pSB1C3-SulAp-hrpR\_eGFP was successfully construct

1. Sequencing result: Success

#### 2.Colony inoculation culture

1. pSB1C3-SulAp-hrpR\_eGFP (colony 3, Extract plasmid, construct Amp30E)

2. pSB1C3-T4 lysis-lacZ (colony 8, cultured and then extracted plasmid PCR test, banded and then sequenced)

3. pSB-SulAp-relE-3WJ-Bro (Extract plasmid, PCR verified)

4. pSS5（DH10B）

5. pSB1C3（DH10B）

---

#### 20220807

#### 1/Experimental preparation

1. Extract plasmid

2. Prepare different concentrations of arabinose

---

#### 20220808

#### 1.pSB1C3-SulAp-Amp30E\_eGFP construct

1. Expansion of skeletons and fragments

No strips

#### 2.pSB1C3-T4 lysis-lacZ construction

![](https://static.igem.wiki/teams/4226/wiki/en31.png)

Recycle fragments, connect and convert Gibson

---

#### 20220809

#### 1.pSB1C3-SulAp-Amp30E\_eGFP construction

![](https://static.igem.wiki/teams/4226/wiki/en32.png)

Recycle fragments, connect and convert Gibson

#### 2.pSB1C3-T4 lysis-lacZ authentication

![](https://static.igem.wiki/teams/4226/wiki/en33.png)

send Colonies 3 and 8 for sequencing

#### 3.pSS5new\_pSB1C3\_pBAD\_T4 lysis device test

- transfer pSS5 plasmid to the DH10B competent state

- Transfer pSB1C3 plasmid to the DH10B competent state

##### - Different concentrations of arabinose 0, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250mM (10μL)

1. seed solution preparation: take pSS5, pSB1C3, pick up a single colony from the plate, transfer to 4 ml of liquid LB(C+), 37 °C, 220 rpm overnight culture;

2. Expand culture and protein expression: add 20 ml of LB (C+) to a 50 ml centrifuge tube, take 100 ul of overnight culture broth and add LB culture medium to OD595 = 0.4-0.5 (~3-4 h) at 37 °C, 220 rpm;

3. Different concentrations of arabinose induction, 37 °C, 200 rmp culture.

4. Bacterial measurement: induction 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, respectively, microplate reader measurement OD600, record data.

---

#### 20220810

#### 1.pSB1C3-SulAp-Amp30E\_eGFP authentication

![](https://static.igem.wiki/teams/4226/wiki/en66.png)

Colonies 1 and 2, send for sequencing

---

#### 20220811

#### 1.pSB1C3-T4 lysis-lacZ sequencing comparison

1. Sterilization verification

---

#### 20220812

#### 1.pSB1C3-T4 lysis-lacZ construction

#### 2.pSB1C3-SulAp-Amp30E\_eGFP construction

---

#### 20220813

#### 1.pSS5new\_pSB1C3\_pBAD\_T4 lysis device repeat the experiment

---

#### 20220814

#### 1.pSB1C3-T4 lysis-lacZ authentication

![](https://static.igem.wiki/teams/4226/wiki/en67.png)

Colony No. 8 (posterior), Band No. 1 is weak Send sequencing, experimental verification

#### 2.pSB1C3-SulAp-Amp30E\_eGFP verify

![](https://static.igem.wiki/teams/4226/wiki/en35.png)

---

#### 20220815

#### 1.pSB1C3-SulAp-Amp30E\_eGFP test

1. PCR, strip correct, send for sequencing tomorrow

#### 2.pSB1C3\_pBAD\_T4 lysis-lacZ test

1. PCR, strip correct, send for sequencing tomorrow

2. Colors appear

![](https://static.igem.wiki/teams/4226/wiki/en36.png)

---

#### 20220817

#### 1.pSB1C3-SulAp-Amp30E\_eGFP the test was successful

#### 2.pBAD\_T4 lysis-lacZ the test was successful

#### 3.Amp30E amplification system effect verification

Experimental operation:

1. Seed solution preparation: take non-recombinant BL21, pSB1C3-SulAp\_eGFP (BL21); pSB1C3-Amp30E\_eGFP (BL21), pick up a single colony from a plate and transfer to 4 ml of liquid LB(C+), 37 °C, 220 rpm culture, use tin foil to block out the light;

2. Induction of expression: Add 10 ml lb (C+) to a 50 ml centrifuge tube, take 100 ul of overnight culture liquid and add LB culture medium to OD595 = 0.4-0.5 (about 3-4 h) at 37 °C, 220 rpm, use tin foil to block out the light;

3. UV induction at different times, 37 °C, 200 rmp culture, 0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, respectively use microplate reader measurement of OD600 and fluorescence values (excitation light 485 nm, absorbed light 535 nm, gain value 50)

![](https://static.igem.wiki/teams/4226/wiki/en37.png)
![](https://static.igem.wiki/teams/4226/wiki/en38.png)

##### Experimental data need to be duplicated

---

#### 20220819

#### 1.relE-3WJ-Bro construct

#### 2.Amp30E-relE-3WJ-Bro construct

Recycle the connection

![](https://static.igem.wiki/teams/4226/wiki/en39.png)

#### 20220820

#### 1.Test the scale-up system

1. The steps are the same, the experiment is successful, and the data is available (just visualize):

0821-Amp30E Effect Test - Fluorescence ÷OD

#### 2.relE-3WJ-Bro inspection

![](https://static.igem.wiki/teams/4226/wiki/en40.png)

Colonies 5/6/10/17 are available and will be amplified tomorrow for testing

#### 3.Amp30E-relE-3WJ-Bro inspection

![](https://static.igem.wiki/teams/4226/wiki/en41.png)

Colonies 2/4/17 are available and will be amplified tomorrow for testing

---

#### 20220821

##### DH10B、T4 lysis（DH10B）

Arabinose induction lysis device (data available)

##### DH10B、T4lysis-lacZ（DH10B)

Arabinose induces lacZ color reaction

##### pSB1C3（DH10B）

##### pSB1C3-relE（DH10B）

Existing (colonies 1 and 9 are ok)

##### pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）

---

#### 20220823

#### 1.Amp30E-relE-3WJ-Bro（DH5a）Conversion results

![](https://static.igem.wiki/teams/4226/wiki/en42.png)

Select colonies 10 and 11 (expect 4200 bp)

#### 2.Amp30E-relE-3WJ-Bro（DH10B）Conversion results

![](https://static.igem.wiki/teams/4226/wiki/en43.png)

##### Select colonies 1 and 3 (expected 4200 bp)

#### 3.The T4-lacZ build is complete and the blue effect is displayed

1. Transfer pBAD\_T4 lysis-lacZ plasmid into DH10B competent state;

2. Prepare 8 Erlenmeyer flasks (250 ml) containing M9 liquid medium (50 ml) and sterilize;

3. Add 100ul lacZ bacterial solution to the medium (+25 μl chloramphenicol), overnight culture at 37 °C, 220 rpm;

4. Different conditions induce expression, and samples in Erlenmeyer flasks are used to observe the blue phenomenon.

5. Put into a shaker for culture and observe the phenomenon

![](https://static.igem.wiki/teams/4226/wiki/en44.png)

---

#### 20220824

#### 1.Amp30E-relE-3WJ-Bro fragment amplification for testing

Fail

#### 2.Amp30E-relE (DH5a) colony PCR

![](https://static.igem.wiki/teams/4226/wiki/en45.png)

Take colonies 2 and 9 for culture, tomorrow PCR, and if successful, extract the plasmid

#### 3.pSB1C3（DH10B）、pSB1C3-relE（DH10B）、pSB1C3-Amp30E-relE（DH10B）Induce expression

(Using pSB1C3-relE9 colony, Amp30E-relE-3WJ-Bro1 colony)

![](https://static.igem.wiki/teams/4226/wiki/en46.png)

![](https://static.igem.wiki/teams/4226/wiki/en47.png)

The data needs to be duplicated

---

#### 20220825

#### 1.pSB1C3-Amp30E-relE Clips are sent for testing

![](https://static.igem.wiki/teams/4226/wiki/en48.png)

---

![](https://static.igem.wiki/teams/4226/wiki/en49.png)

#### 2.pSB1C3（DH5a）、pSB1C3-relE（DH5a）、pSB1C3-Amp30E-relE（DH5a）Induce expression

Repeat yesterday's experiment to continue testing, experimental results are not satisfactory, continue to test with DH10B

---

#### 20220826

#### 1. pSB1C3-Amp30E-relE Sequencing results

The relE fragment is lost, design the primer , and test whether there is a relE fragment on the ligation product tomorrow

Transfer Sulp-relE-3WJ-Bro to DH10B ， use PCR to screen colonies tomorrow

---

#### 20220827

#### 1.RelE toxin protein and tag system testing

![](https://static.igem.wiki/teams/4226/wiki/en50.png)

##### pSB1C3-SulAp-Amp30E-relE has a band that conforms to the length of relE (with Colony 2)

#### 2.pSB1C3-SulAp-relE-3WJ-Bro（DH10B）colony PCR

Obtain pSB1C3-SulAp-relE (DH10B) available (colonies 1 and 9 are ok)

##### pSB1C3（DH10B）

##### pSB1C3-SulAp-relE-3WJ-Bro（DH10B）

##### pSB1C3-SulAp-Amp30E-relE-3WJ-Bro（DH10B）

---

#### 20220828

#### 1.pSB1C3（DH10B）、pSB1C3-relE-3WJ-Bro（DH10B）、pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）Test

Repeat the experiment, the results need to be debugged:

##### pSB1C3-SulAp-relE-3WJ-Bro（DH10B）Previously constructed Colony 9 is not available

##### pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）Colony No. 1 is available and data are available for testing

---

#### 20220829

#### 1.pSB1C3-SulAp-relE-3WJ-Bro (DH10B) Second Build Test (Successful)

1. Colonies all have target bands

![](https://static.igem.wiki/teams/4226/wiki/en51.png)

2. Take the PCR reaction product above and detect whether there are relE and 3WJ-Bro fragments respectively

The relE fragment is correct:

![](https://static.igem.wiki/teams/4226/wiki/en52.png)

3WJ-Bro fragment correct:

![](https://static.igem.wiki/teams/4226/wiki/en53.png)

#### 2.pSB1C3-Amp30E-relE-3WJ-Bro（DH10B）【succeed】

1. Detect the presence of releE, 3WJ-Bro, and Amp30E fragments

Colony 1 (data available): left reelE, right 3WJ-Bro

![](https://static.igem.wiki/teams/4226/wiki/en54.png)

Colony 3: Left reelE, right 3WJ-Bro

![](https://static.igem.wiki/teams/4226/wiki/en55.png)

Amp30E fragment: Both 1 and 3 colonies have the correct bands

![](https://static.igem.wiki/teams/4226/wiki/en56.png)

##### Colony 1 was tested, and the experimental data matched the trend, Preserve bacteria

#### 3.pSB1C3（BL21）、pSB1C3-relE-3WJ-Bro（BL21）、pSB1C3-Amp30E-relE-3WJ-Bro（BL21）Test

pSB1C3（BL21）possess

pSB1C3-relE-3WJ-Bro（BL21）Both a and b have bands, start with colony bpSB1C3-Amp30E-relE-3WJ-Bro（BL21）In transformations of 1 and 3 , colony 1 is used first

---

#### 20220830

#### 1.pSB1C3-Amp30E-relE-3WJ-Bro (BL21) Colony PCR Verification (Incorrect)

![](https://static.igem.wiki/teams/4226/wiki/en57.png)

#### 2.pSB1C3-Amp30E-relE (DH10B) colony PCR Verificationvalidation (no relE fragments)

![](https://static.igem.wiki/teams/4226/wiki/en58.png)

1. With Amp30E strip (2366bp)

![](https://static.igem.wiki/teams/4226/wiki/en59.png)

2. No relE stripe (383bp)

![](https://static.igem.wiki/teams/4226/wiki/en60.png)

---

#### 20220831

#### 1.RelE experimental data

0830-Amp30E-relE-Absorbance

![](https://static.igem.wiki/teams/4226/wiki/en61.png)

#### 2.pSB1C3-SulAp-relE-3WJ-Bro (DH10B) build and test colonies 9 and 11 for the second time

##### Both relE and 3WJ-Bro bands were correct, perform follow-up experiments were performed with colony 11, which was successfully tested yesterday

![](https://static.igem.wiki/teams/4226/wiki/en62.png)

---

#### 20220901

#### Test the mSarlet-I-3WJ-Bro label

---

#### 20220902

#### 1.pSB2C3-Amp30E-relE-3WJ-Bro（BL21）

1. With Amp30E strip (2366bp)

![](https://static.igem.wiki/teams/4226/wiki/en63.png)

2. With 3WJ-Bro strip (aroundabout 900bp)

![](https://static.igem.wiki/teams/4226/wiki/en64.png)

3. RelE band (aroundabout 300bp)

![](https://static.igem.wiki/teams/4226/wiki/en65.png)

---

#### 20220906

#### 1. Arabinose-induced T4 lysis device

Get replicated experiment data

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#### 20220909

#### 1.Arabinose-induced T4 lysis device

Get replicated experiment data

---

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{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
{% block page\_content %}

# E N G I N E E R I N G

## Engineering Success

#### Summary

"Kill switch" is defined as an artificial system that causes cell death under certain conditions. In other iGEM team projects, kill switch is usually used to ensure the biological safety of the project. In this project, our team designed death programs to program and control life and present a series of "engineering bacterial death simulators" in the form of installation art, in which bacteria will present their life performances.
In Plan A, we designed a UV-inducible switch to simulate the impact of excessive UV radiation from the ozone layer hole caused by complex human activities and atmospheric chemical process. The death of engineered bacteria warns people that if environmental damage is not prevented in time, our lives will also be negatively affected. We hope to warn everyone that we need to curb the deterioration of air pollution, ozone hole and other environmental problems through our efforts.
In Plan B, our T4 lysis device and beta-galactosidase synthesis pathway showed the effect of blue color reaction, symbolizing the tears of Horus' eyes in art. The engineered bacteria died and the solution turned blue. The mixture of solutions slowly dripped from the container, as if the tears of giant's eyes fell from the sky. Macroscopically, it is a visual spectacle, while microcosmically, it is the doomsday aesthetics presented by innumerable cell deaths to reflect thoughts on bioethics.

#### Plan A: Death from UV pollution

In our design, we used UV-inducible switch to turn on the expression of lethal gene. After full exposure of sunlight, bacteria can synthesize toxins and die. We have referenced the design of UT-Tokyo iGEM team in 2011 and SZ-SHD team in 2020, and decided to adopt the UV-inducible switch, which is sulA promoter (BBa\_K518010) proposed by iGEM11\_UT-Tokyo (2011). And the lethal gene we chose is the relE toxin (BBa\_K185047), an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons [1]. The expression of the relE gene has been shown to severely inhibit translation and prevent colony formation.

##### 1. sulAp promoter testing

In plan A, we expected the engineered bacteria to turn on relE gene expression and die under UV induction. In order to achieve this goal, we first studied the activity of sulAp with and without UV irradiation with the help of eGFP gene. We constructed the circuit containing sulAp and eGFP with the vector pSB1C3 (Gene circuit 1), and then transformed it into BL21.

![](https://static.igem.wiki/teams/4226/wiki/g1.jpg)

- Gene circuit 1: pSB1C3-sulAp-eGFP

We cultured the recombinant bacteria in the dark to optimum cell growth (OD600) and irradiated them under ultraviolet C (UVC, 254nm) with an intensity of 15mWcm-2 for different periods of time. Then, each sample was cultured in incubating-shaker in darkness for 8 hours during which the fluorescence per OD was calculated.
Fluorescence can be observed after UVC induction. Where there were 2-3 times increments in fluorescence than normal, and there was a positive correlation between the time of UVC induction and fluorescence intensity, as displayed in figure 1.
The experimental results prove that the sulAp promoter successfully triggered the expression of eGFP under UV induction.

![](https://static.igem.wiki/teams/4226/wiki/g2.jpg)

Figure 1: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Green fluorescence spectrum: 485/510nm. (eGFP -UV-: non-recombinant BL21 under no UV irradiation; eGFP -UV+: non-recombinant BL21 under UV irradiation for 1 min; 0 min: recombinant BL21 without UV irradiation; 0.5 min: irradiated under UV for 0.5 min; 1 min: irradiated under UV for 1 min and so on in a similar fashion).

##### 2. Expression of relE toxin gene

We then replaced the eGFP gene with relE toxin gene (Gene circuit 2) to test whether sulA promoter could induce the expression of relE.

![](https://static.igem.wiki/teams/4226/wiki/g3.jpg)

Gene circuit 2: pSB1C3-sulAp-relE

Although the expression of relE gene results in bacterial growth slower than the control group, the effect of relE was not obvious as we expected, which is shown in figure 2. We still hope to obtain a better effect on inhibiting bacterial growth.

![](https://static.igem.wiki/teams/4226/wiki/g4.jpg)

Figure 2: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-11h: data collection time after UVC induction.

##### 3. Use of Amp30E Amplification Device

We analyzed the results, and speculated that it might be caused by the low expression of relE gene. Therefore, we made improvements to the existing circuits based on literature research.
We applied a typical cellular sensor that can be abstracted as a three-stage processor comprising a sensing module that recognizes and transduces external signals into intracellular transcriptional signals, a computing module that modulates the transduced sensor signals, and an output actuating module that executes physiological responses.
We used the promoter of sulA as sensor module. The amplifier module (Amp30E Amplification Device) includes hrpR (BBa\_K4226001), hrpS (BBa\_K4226002) and PhrpL (BBa\_K4226003) are engineered multi-layered transcriptional amplifiers that can be able to sequentially boost output expression level of target genes [2].The eGFP was used as an output module to test the effect of amplifier module.
In this part, we improved the parts: HrpR Gene (BBa\_K1014001), HrpS Gene (BBa\_K1014000) and Promoter hrpL (BBa\_K1014002) uploaded by iGEM13\_HIT-Harbin.
Gene circuits 3 was designed to test the effect of Amp30E using reporter gene eGFP:

![](https://static.igem.wiki/teams/4226/wiki/g5.jpg)

- Gene circuit 3: pSB1C3-sulAp-Amp30E-eGFP- The results showed that the Amp30E Amplification Device significantly increased the fluorescence expression of eGFP. This result greatly demonstrates the excellent capability of Amp30E Amplification Device as shown in Figure 3:

    ![](https://static.igem.wiki/teams/4226/wiki/g6.jpg)

    Figure 3: The result of eGFP expression (Fluorescence per OD) after irradiated under UVC (254nm). Competent host cell: BL21. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction.

    ##### 4. Amp30E increased the expression of relE

    On this basis, we replaced the eGFP gene with relE toxin gene to test whether Amp30E system can increase the expression of relE gene. Gene circuit 4 was constructed as follows:

    ![](https://static.igem.wiki/teams/4226/wiki/g7.jpg)

    Gene circuit 4: pSB1C3-sulAp-Amp30E-relE

    As we expected, the OD600 of the recombinant bacteria decreased after UVC induction (pSB1C3-sulAp-relE), and the Amp30E Amplification Device significantly increased the inhibitory effect of relE on bacterial growth (pSB1C3-sulAp-Amp30E-relE). So far, we have achieved better results through reconstructing the gene circuits.

    ![](https://static.igem.wiki/teams/4226/wiki/g8.jpg)

    Figure 4: The OD600 of recombinant bacteria after irradiated under UVC (254nm). Competent host cell: DH10B. 0-8h: data collection time after UVC induction.

    ##### 5. Verification of increased expression of relE

    In order to further confirm the phenomenon of increased expression of relE at transcriptional level, we introduced RNA and protein reporting systems including 3WJ-Bro (BBa\_K4226000) and mScarlet-I (BBa\_K3977002).
    The relE toxin gene and the mScarlet-I gene were fused to construct a fusion gene, in order to detect the amount of relE protein synthesis. We removed the terminator of relE and fused it to the mScarlet-I gene. And the 3WJ-Bro was used to ligate fluorescent aptamers to examine the relE RNA synthesis at transcriptional level (Gene circuit 5).

    ![](https://static.igem.wiki/teams/4226/wiki/g9.jpg)

    Gene circuit 5: pSB1C3-sulAp-Amp30E-relE-mScarlet-I-3WJ-Bro

    ###### RNA synthesis of relE:

    After bacterial culture and UV induction, we measured OD600 and fluorescence values under 485/510nm. RNA synthesis of relE was almost undetectable in circuits without Amp30E. The curve of experimental group was significantly higher than that of control group and the amount of relE RNA synthesis gradually increased with incubation time (Figure 5). The results indicate that the Amp30E Amplification Device significantly increased the RNA synthesis of relE gene as expected.

    ![](https://static.igem.wiki/teams/4226/wiki/g10.jpg)

    Figure 5: 3WJ-Bro was ligated with fluorescent aptamers in order to examine the level of relE RNA synthesis. Competent host cell: DH10B. Green fluorescence spectrum: 485/510nm. 0-8h: data collection time after UVC induction.

    ###### Protein synthesis of relE:

    Then, we measured OD600 and fluorescence values under 579/616nm to examine the protein synthesis of relE. The curve of experimental group was also higher than that of control group (Figure 6), indicating the mScarlet-I correctly displayed the increasing tendency of relE protein synthesis under the effect of Amp30E Amplification Device:

    ![](https://static.igem.wiki/teams/4226/wiki/g11.jpg)

    Figure 6: The mScarlet-I gene was fused with relE gene to detect the amount of relE protein synthesis. Competent host cell: DH10B. Fluorescence spectrum: 579/616nm. 0-8h: data collection time after UVC induction.

    #### Plan B: T4 lysis Device and beta-galactosidase synthesis

    We designed another suicide pathway based on T4 lysis Device, a system derived from bacteriophage T4, which has two main components: T4 holin (BBa\_K112805) and T4 endolysin (BBa\_K112806), that cause the bacteria to rupture and die [3,4].

    ![](https://static.igem.wiki/teams/4226/wiki/wiki-de/dna1.png)

    ![](https://static.igem.wiki/teams/4226/wiki/g12.jpg)

    According to the records of the team SZ-SHD (2020), the competent cells could died rapidly after the introduction of T4 Holins and lysozyme genes. Therefore, in our project, we first used the inducible pBad/araC promoter (BBa\_I0500) to suppress expression, preventing the recombinant cells from dying immediately.
    At the same time, we inserted the lacZ gene (BBa\_I732019) in the T4 lysis Device (Gene circuit 6). The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color [5]. This design provides great inspiration for the artistic design of this project.

    ![](https://static.igem.wiki/teams/4226/wiki/g13.jpg)

    - Gene circuit 6: pSB1C3-pBAD-lacZ-T4 lysis

    As shown in figure 7, the OD600 of recombinant cells with pBAD-lacZ-T4 lysis gene circuit reduced significantly by 2-3 times than non-recombinant cells after induced by different concentrations of arabinose.

    ![](https://static.igem.wiki/teams/4226/wiki/g14.jpg)

    Figure 7: The result of T4 lysis Device after induced by different concentrations of arabinose. (pSB1C3: non-recombinant DH10B without arabinose induction; pSB1C3-pBAD-lacZ-T4 lysis: recombinant DH10B without arabinose induction and with different concentrations of arabinose).

    In order to test the effect of lacZ gene and Beta-galactosidase synthesis, the recombinant cells (pSB1C3-pBAD-lacZ-T4 lysis) were placed in M9 medium and cultured for 12 hours. Subsequently, x-gal was added to the M9 medium, and the recombinant cells were induced by arabinose. The experimental phenomena are as follows:

    ![](https://static.igem.wiki/teams/4226/wiki/g15.jpg)

    Figure 8: The lacZ gene controls the synthesis of β-galactosidase, which can react with X-Gal in blue color.

    We used this experimental phenomenon to create a piece of artwork. [Please click to see more details.](https://2022.igem.wiki/cafa-china/implementation)

    #### To sum up

    These engineering cycles (design -> build -> test -> learn -> design...) greatly promoted the development of our project.
    Both designs achieved great engineering success in synthetic biology!

    For more details, please see the [Parts page](https://2022.igem.wiki/cafa-china/parts)

    #### References:

    [1] Christensen S K, Mikkelsen M, Pedersen K, et al. RelE, a global inhibitor of translation, is activated during nutritional stress[J]. Proceedings of the National Academy of Sciences, 2001, 98(25): 14328-14333.
    [2] Wan, X., Volpetti, F., Petrova, E. et al. Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals. Nat Chem Biol,2019, 15, 540-548.
    [3] Ramanculov E, Young R. Functional analysis of the phage T4 holin in a λ context[J]. Molecular Genetics and Genomics, 2001, 265(2): 345-353.
    [4] Young R, Bläsi U. Holins: form and function in bacteriophage lysis[J]. FEMS Microbiology Reviews, 1995, 17(1-2): 191-205.
    [5] Diao W, Guo L, Ding Q, et al. Reprogramming microbial populations using a programmed lysis system to improve chemical production[J]. Nature Communications, 2021, 12(1):1-14.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

# E X P E R I M E N T S

## Experiments

## CAFA: experiment protocols

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### Culture medium:

#### 1. LB medium:

Peptone 10g/L, yeast extract 5g/L and sodium chloride 10g/L . After dissolving in water, autoclaved at 121C for 15min.

(If antibiotics are needed: add 1/1000 of the volume of antibiotic solution when the temperature is below 60℃)

#### 2. LB medium (solid):

Peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L and agar 15g/L. After dissolving in
water, autoclaved at 121℃ for 15min.

(If antibiotics are needed: add 1/1000 of the volume of antibiotic solution when the temperature is below 60℃)

#### 3. M9 medium

![](https://static.igem.wiki/teams/4226/wiki/ex1.png)

### Antibiotics

#### 1. Chloramphenicol

Weight 510 mg anhydrous chloramphenicol and dissolve it in 15mL pure ethanol, filter and sterilize it to obtain 1000X solution, which should be stored at -20℃ after separation.

#### 2. Ampicilin

Weight 500 mg ampicilin in 5ml pure water, filter and sterilize it to obtain 1000X solution, which should be stored away from light at -20℃.

### Basic operation

#### 1. Plasmid extraction

Note: The following information is based on the instructions of TIANGENR Rapid Plasmid Extraction Kit.
1. Collection of bacteria: Take 1-4 mL of bacterial solution into 2 mL EP tube, centrifuge at 12000 RPM for 1 min, and discard the supernatant.
2. Resuspended thallus: Add 150 uL of P1 solution to the thallus precipitation
(with RNaseA, purple), blown with pipette or resuspended with vortex oscillator.
3. Lyse thalli: Add 150 uL of P2 solution and mix gently upside down 6-8 times until the solution turns a slightly transparent rose red.
4. Neutralizing and alkaline: add 350 uL P5 solution, immediately and gently invert the mixture 6-8 times, until the light yellow flocculent is completely formed.
5. Separation of cell debris and macromolecular impurities: centrifuge at 12000 RPM for 2min to fully settle flocculent.
6. Assemble the adsorption column: Install the adsorption column CP3 on the collecting tube and mark the number on the cover of the adsorption column tube.
7. Plasmid DNA collection by adsorption column: Transfer the supernatant obtained in Step 5 to the assembled adsorption column, centrifuge at 12000 RPM for 30-60 s, and discard the filtrate.
8. Rinse: Add 300 uL of PWT(ethanol must be added before use) to the adsorption column, centrifuge at 12000 RPM for 30-60s, and discard the filtrate.
9. Removal of ethanol ①: Put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2 min, and try to remove the bleaching liquid.
10. Remove ethanol ②: Put the adsorption column into a clean 1.5mLEP tube (mark on the tube cover), place at room temperature (or metal bath 50C) for 5-10 min, make ethanol volatilization thoroughly, to prevent residual ethanol from affecting the next experiment.
11. Solubilizing plasmid DNA: Add 30-50 uL ddH2O to the silica gel membrane in the middle of the adsorption column (ddHO at 50° C has a higher recovery rate), and let stand at room temperature for 1 min.
12. Plasmid DNA collection: centrifuge at 12000 RPM for 1 min. The filtrate in 1.5mL EP tube is the obtained plasmid DNA solution, and the adsorption column can be discarded.

#### 2. Agarose gel electrophoresis

1. Put the rubber mold on a horizontal workbench surface, and put the rubber plate (divided into 25mL/ 50ml / 100ml) into the corresponding rubber mold. Note that there should be no air and debris between the bottom of the rubber mold and the bottom of the rubber plate, and then insert the sample comb to ensure that the bottom edge of the comb teeth will not contact with the rubber mold, otherwise it will lead to leakage of liquid in the sample holes.
2. Weigh 0.25g /0.5g/1.0g agarose into a triangular bottle, add 1×TAE buffer (25ml / 50ml / 100ml) to make 1% gel, heat in microwave oven on high heat for 0.5-1min, during which you can observe and shake well (boiling several times during the process) until the liquid in the bottle is clear and homogeneous.
3. Add the dye SYBR Safe DNA Gel Stain (10000×) 2.5ul / 5UL / 10ul, shake and mix well, carefully pour into the glue making plate (to avoid bubbles), and let stand at room temperature for 20-60 min. 4. After the solidification is complete, gently pull out the sample comb with both hands, and then form the spot sample holes separated from each other.
5. Put the gel together with the glue making plate into the electrophoresis tank and pour
1×TAE buffer until the gel surface is immersed 2-3 mm, and gently move the glue making plate and gel to eliminate bubbles in the spot sample Wells.
6. Slowly add the sample into the spot sample hole with the pipette, paying attention to avoid overflow and contamination of adjacent samples.

#### 3. DNA recovery from agarose gel

The following information is based on the TIANGEN RUniversal DNA Purification and Recovery Kit instructions.
1. Cutting glue: Take a clean 1.5ml EP tube, mark the number on the tube cover, and add the strip containing glue block cut with a rust-free blade.
2 sol: to glue block to add 1~3 times the volume of solution PC(such as gel weight 0.1g, the volume can be regarded as 100 uL, then add 100 uL solution PC), 50 ℃ metal bath placed about 10 min, from time to time turn EP tube, to ensure that the glue block fully dissolved.
3. Assemble the adsorption column and column balance: put the adsorption column CB2 into the collection tube, add 500 uL of equilibrated BL to the adsorption column, centrifuge at 12000rpm for 1 min, and discard the filtrate. A balanced adsorption column can only be used on the same day.
4. DNA collection by adsorption column: Add the solution obtained in Step 3 to the adsorption column, place it at room temperature for 2min, centrifuge it at 12000 RPM for 30-60 s, and discard the filtrate. If the sample is larger than the adsorption column volume (800 uL), batch addition can be performed.
5. Rinse: Add 600 uL of PW to the adsorption column (ethanol should be confirmed before use), centrifuge at 12000 RPM for 30-60s, and discard the waste liquid.
6. Repeat Step 6.
7. Removal of ethanol ①: Put the adsorption column back into the collection tube, centrifuge at 12000rpm for 2 min, and try to remove the bleaching liquid.
8. Remove ethanol ②: Put the adsorption column into a clean 1.5ml EP tube, mark it on the EP tube, room temperature (or metal bath)
50℃) for 1-3 min to make ethanol volatilize.
9. Dissolve DNA: Add 30-50 uL ddHzO to the silica gel membrane in the middle of the adsorption column (ddHzO at 50° C has a higher recovery rate), and let stand at room temperature for 1 min. 1200 RPM centrifugal
After 1min, the DNA solution was collected.

#### 4. Chemical transformation

1. Remove 50 uL of competent E. coli cells and thaw them slowly on ice.
2. Add the DNA to be transformed (plasmid or ligation/assembly product), gently mix, and place in the ice bath for 20 min.
3.After 60 seconds of heat shock at 42° C, quickly ice bath for 2 min. Do not shake EP tube during this process.
4. 300 uL of antibiotic free LB liquid medium was added and resuscitated in a shaker at 37℃ for 60 min.
5. For plasmid transformation (also known as "positive transformation", the amount of plasmid DNA added in the transformation can be >5ng), 100 uL of resuscitate bacterial solution was taken, coated on solid plates containing antibiotics, and incubated upside down at 37℃ for about 16 h.
6. For the transformation of the connection/assembly products, the resuspended bacterial solution should be centrifuged at 4000 RPM for 1 min, and 250 uL of the supernatant should be aspirated, and the remaining supernatant medium should be resuspended with the bacteria precipitation, and then all of them should be coated on the solid plate containing antibiotics, and the culture should be inverted at 37° C for about 16 h.

#### 5. Colony identification by PCR

1. Prepare a PCR reaction system according to the table on the following page, and blow with a pipette
Suction or briefly oscillate with a vortex oscillator to mix the system. When multiple colonies need to be identified at the same time, they can be combined and prepared and then separated into PCR tubes:

![](https://static.igem.wiki/teams/4226/wiki/ex2.png)

2. Prepare fresh LB solid medium plates containing antibiotics, and mark and number the plates and PCR tubes.
 3. In the ultra-clean working table, single colonies on the transformation plate were selected with sterilized toothpicks, a line was drawn on the LB plate as a copy of the original colonies, and then immersed into the corresponding PCR tube containing PCR system. After several rotations of the toothpicks, the toothpicks were removed and thrown into the waste liquid tank.
4. Repeat the above operations to select multiple candidate single colonies.
5. Run the PCR program according to the table below:

![](https://static.igem.wiki/teams/4226/wiki/ex3.png)

### Construct the recombined plasmid

#### 1. T4 lysis Device and beta-galactosidase synthesis

![](https://static.igem.wiki/teams/4226/wiki/ex4.png)

#### 2. Amp30E Amplification Device

![](https://static.igem.wiki/teams/4226/wiki/ex5.png)

#### 3. relE characterization and reporting systems

![](https://static.igem.wiki/teams/4226/wiki/ex6.png)

{% endblock %}

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

# R E S U L T S

## Results

## Summary, difficulties and prospects:

---

difficulties：

- Our work was doubly restricted by the epidemic and the team could not meet, so we had to work online. It wasn't until October that all the team members were finally able to meet. Because of the epidemic, our hp program was very limited, and our results were affected in some ways: our offline social research was put on hold, the exhibition needed to be strictly limited to not cause crowds to gather, and education and communication were mostly conducted online.

- The shortcoming of our experimental visualization is that it is too figurative and the subject matter is too specific and culturally contextualized, which can be improved in terms of artistic presentation.

In terms of visual effect, our work attempts to cause a shock with a large volume to achieve the effect of a spectacle. However, the actual landing does not achieve the desired effect due to various practical considerations.

Outlook and conclusion：

Our project is believed to be only an initial exploration of the combination of art and technology, but at the same time it will always exist as a platform, a channel for art and technology collaboration.
We were greatly affected by the epidemic in the process of exploring and realizing hp, and we encountered a series of difficulties as a result. Our project was not perfect, but we ultimately persevered and believe that our work is just the beginning. The epidemic will not stop the combination of technology and humanities, and it will not stop the light of beauty from spreading on the rational earth, and technology and art will eventually work together to produce the flowers of human wisdom and offer them to the future world.

{% endblock %}