iGEM 2020 — Team ZJUT_China_B

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# Team:ZJUT China B/Notebook

Document

[Home](https://2020.igem.org/Team%3AZJUT_China_B)

Team

[Team Members](https://2020.igem.org/Team%3AZJUT_China_B/Team)
[Attributions](https://2020.igem.org/Team%3AZJUT_China_B/Attributions)

Project

[Description](https://2020.igem.org/Team%3AZJUT_China_B/Description)
[Design](https://2020.igem.org/Team%3AZJUT_China_B/Design)
[Implementation](https://2020.igem.org/Team%3AZJUT_China_B/Implementation)
[Contribution](https://2020.igem.org/Team%3AZJUT_China_B/Contribution)
[Engineering](https://2020.igem.org/Team%3AZJUT_China_B/Engineering)
[Proof Of Concept](https://2020.igem.org/Team%3AZJUT_China_B/Proof%20Of%20Concept)
[Results](https://2020.igem.org/Team%3AZJUT_China_B/Results)

Wetlab

[Protocols](https://2020.igem.org/Team%3AZJUT_China_B/Protocol)
[Notebook](https://2020.igem.org/Team%3AZJUT_China_B/Notebook)
[Parts](https://2020.igem.org/Team%3AZJUT_China_B/Parts)
[Wet collaboration](https://2020.igem.org/Team%3AZJUT_China_B/Wet%20collaboration)

HP

[Overview](https://2020.igem.org/Team%3AZJUT_China_B/Human%20Practices)
[Silver](https://2020.igem.org/Team%3AZJUT_China_B/HP-Silver)
[Gold](https://2020.igem.org/Team%3AZJUT_China_B/Integrated%20human%20practice)
[Science communication](https://2020.igem.org/Team%3AZJUT_China_B/Education)
[Collaborations](https://2020.igem.org/Team%3AZJUT_China_B/Collaborations)
[Partnership](https://2020.igem.org/Team%3AZJUT_China_B/Partnership)
[Ethics](https://2020.igem.org/Team%3AZJUT_China_B/Ethics)

Modeling

[Software](https://2020.igem.org/Team%3AZJUT_China_B/Software)
[Modeling](https://2020.igem.org/Team%3AZJUT_China_B/Model)

Hardware

[Overview](https://2020.igem.org/Team%3AZJUT_China_B/Hardware)
[Detecting](https://2020.igem.org/Team%3AZJUT_China_B/Detecting%20)
[Sample processing](https://2020.igem.org/Team%3AZJUT_China_B/Sample%20processing)
[Chip](https://2020.igem.org/Team%3AZJUT_China_B/Chip)
[History](https://2020.igem.org/Team%3AZJUT_China_B/History)

[Safety](https://2020.igem.org/Team%3AZJUT_China_B/Safety)

Judging

[Poster](https://2020.igem.org/Team%3AZJUT_China_B/Poster)
[Judging Form](https://2020.igem.org/Team%3AZJUT_China_B/Judging%20Form)

[![](https://static.igem.org/mediawiki/2020/5/5d/T--ZJUT_China_B--logo-Cas.png)](https://igem2020-zjut.feishu.cn/docs/doccnWz3kWJdTESBOvIunDsW2ac?from=from_copylink)

Team:ZJUT China B/Wetlab/Notebook-2020.igem.org

## **Notebook**

# Jul

| Data | Classification | Experimenter | Experiment | Experimental results |
| --- | --- | --- | --- | --- |
| 2020/7/24 | Cas 13a&b | Qian Rong、Yuanying Wang | Transform | Psm、Lba transformed successfully；Lwa failed |
| 2020/7/25 | Cas 13a&b | Yuzhi Wang、Ziyan Huang | Psm Plasmid extraction | The Psm plasmid concentration is 48.2ng/ul |
| 2020/7/26 | Cas 13a&b | Yuzhi Wang、Ziyan Huang | Prepare *E.coli* BL21 competent cells | The Psm plasmid concentration is 48.2ng/ul |
|  |  | Yuanying Wang、Mengying Wang | Lba and Lwa are transformed into DH5α competence cell | Lba transformed successfully;Lwa only grows a few single colonies |
| 2020/7/27 | Cas 13a&b | Yuanying Wang、Mengying Wang | Vaccinate and transform |  |
|  | Cas 13a&b | Yuzhi Wang | Lba+Lwa Plasmid extraction | Lba：65.2ng/ul；Lwa：49.5ng/ul |
| 2020/7/28 | Cas 13a&b | Ziyan Huang | Prepare *E.coli* BL21 competent cells | 6 tubes |
| 2020/7/29 | Cas 13a&b | Yuanying Wang、Mengying Wang | Lba、Psm and Lwa are transformed into BL21 competence cell | 6 tubes |
| 2020/7/30 | Cas 13a&b | Qian Rong、Mengying Wang | Expression 1.0——Expand cultivation and induction | Shake flask overnight culture OD over 1.0 |

# Aug

| Data | Classification | Experimenter | Experiment | Experimental results | Note |
| --- | --- | --- | --- | --- | --- |
| 2020/8/1 | Cas 13a&b | Yuzhi Wang | Bacterial precipitation collection + fragmentation + Lwa protein purification (1) | No target protein |  |
| 2020/8/3 | Cas 13a&b | Qian Rong、Yuanying Wang | Expression 2.0——Expand cultivation and induction | No target protein |  |
| 2020/8/5 | Cas 13a&b | Qian Rong | Bacterial sediment collection and fragmentation | Bacterial sediment quality——Lba:0.965g;Lwa:0.698g;Psm:0.691g |  |
|  | Cas 13a&b | Yuzhi Wang | Lba, Lwa protein purification (2) +SDS-PAGE | No peak, no target protein |  |
| 2020/8/6 | Cas 13a&b | Qian Rong | Expression 3.0——Expand cultivation and induction |  |  |
| 2020/8/7 | Cas 13a&b | Qian Rong | Bacterial sediment collection | Lwa:0.411g，Lba:0.460g，Psm:0.551g |  |
| 2020/8/8 | Cas 13a&b | Yuzhi Wang | Break bacterias+SDS-PAGE | The target protein band is light |  |
|  | Cas 13a&b | Yuzhi Wang | Lba purification | No peaks |  |
| 2020/8/9 | Cas 13a&b | Yuzhi Wang | Purified green fluorescent protein | Have peaks | Prove that there was no problem with Ni-NTA and buffer |
| 2020/8/12 | Cloning | Ziyan Huang | sumo tag amplification | There is a bright fragment band |  |
| 2020/8/13 | Cloning | Ziyan Huang | SUMO tag purification | Sumo concentrations are 87.6 and 79.6ng/ul |  |
| 2020/8/14 | Cloning | Ziyan Huang | Psm Cas 13b、Cca Cas 13b plasmid linearization and purification | There is a bright fragment band,concentrations are 131.0 and 124.0ng/ul |  |
|  | Cloning | Ziyan Huang | Recombination reaction |  |  |
| 2020/8/15 | Cloning | Ziyan Huang、Yuzhi Wang | Recombinant plasmid transferred into DH5α | failed |  |
| 2020/8/16 | Cloning | Ziyan Huang | Recombinant plasmid transferred into DH5α | failed |  |
| 2020/8/17 | Cas 13a&b | Yuanying Wang | Transform |  |  |
|  | Cloning | Ziyan Huang | Recombination reaction + recombinant plasmid transfer into DH5a | Grow three single colonies |  |
| 2020/8/18 | Cloning | Ziyan Huang | Colony PCR identification 1.0 | Only w3 single colonies have bands |  |
| 2020/8/19 | Cloning | Ziyan Huang | Colony PCR identification 2.0 | Only w3 single colony showed clear bands, w1 bands were mixed. |  |
| 2020/8/20 | Cloning | Ziyan Huang | Recombination reaction | Transform the recombinant plasmid into DH5a |  |
| 2020/8/21 | Cloning | Ziyan Huang | Transform the recombinant plasmid into DH5α |  |  |
|  | Cas 13a&b | Yuzhi Wang | Transform | There are many single colonies | Lba, Lwa transform to Rosetta; Lba, Lwa, Psm transform to DH5alpha |
| 2020/8/23 | Cas 13a&b | Yuzhi Wang | PCR、SDS-PAGE | Most single colonies were successfully transformed |  |
|  | Cloning | Ziyan Huang | Sumo tag amplification and Psm Cas 13b、Cca Cas 13b plasmid linearization + purification + recombination reaction | The Psm Cas 13b、Cca Cas 13b plasmid has obvious bands, the concentration is 10.7ng/ul, and the sumo fragment concentration is 46.4 and 48.7ng/ul. |  |
| 2020/8/24 | Cloning | Ziyan Huang | Transform the recombinant plasmid into DH5α | There are many single colonies |  |
|  | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | 9:30 Lba, Lwa pre-incubation 5ml test tube each | All turbid |  |
|  | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | 18:00 Expansion culture, one bottle each of 100ml shake flask | Turbid before clarification | Stained with phage (maybe shaker, shaker bottle, gauze) |
|  | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | 21:30 Expansion culture, one bottle each of 100ml shake flask | Turbid after 3h | Changed the shaker |
| 2020/8/25 | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | 00:30 Add IPTG to induce expression |  |  |
|  | Cas 13a&b | Xiaojie Zhou | 11:30 Lba and Lwa pre-cultured 5ml test tube each |  |  |
|  | Cas 13a&b | Qian Rong | 16:30 Collect bacterial pellet |  |  |
|  | Cas 13a&b | Yuzhi Wang | 20：00 SDS-PAGE | Lba expressed successful, Lwa failed | The precipitation is more obvious than the supernatant band: it may be related to the loading concentration, or some proteins may form inclusion bodies |
|  | Cloning | Ziyan Huang | Colony PCR identification and recombination | C2, c8, c10 single colonies have obvious bands |  |
|  | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | 23:00 expand culture, 1 bottle of 100ml shake flask each | The bacterial liquid is turbid after 2h |  |
|  | Cas 13a&b |  |  | The bacterial liquid becomes clear after 2.5h |  |
| 2020/8/26 | Cloning | Ziyan Huang | Psm Cas 13b、Cca Cas 13b plasmid extraction | The concentrations of the Psm Cas 13b、Cca Cas 13b plasmids were 46.3, 28.1, 31.9 ng/ul |  |
|  | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | Break bacterias containing Lba protein |  | Since the lysate contains reducing agent, it is not good for Ni-NTA, purification is terminated |
|  | Cas 13a&b |  | 8:00 Lba, Lwa pre-cultured 5ml test tube each 1 tube | After culturing for 10 hours, the bacterial solution is not turbid enough and contains cell debris | The test tube is stained with bacteriophages. Borrow new test tubes and shake flasks to prepare the medium |
| 2020/8/27 | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | 8:30 Lba, Lwa pre-incubation 5ml test tube 2 tubes each | 18:00 Lwa1 tube is turbid, 1 tube is clarified; Lba are all clarified; |  |
|  | Cas 13a&b |  |  | 20:00 Lba has 1 tube also muddy | unusual |
|  | Cas 13a&b |  | 18:30 Lba pre-culture 5ml test tube 5 tubes | After 12h, 1 tube was turbid, 2 tubes were turbid, and 2 tubes were clear |  |
|  | Cas 13a&b |  | 20:30 Lba and Lwa expansion culture 100ml shake flask 2 bottles each |  |  |
|  | Cas 13a&b |  | 21：00 PCR |  | Lba has 13 transformants |
| 2020/8/28 | Cas 13a&b | Xiaojie Zhou | 6：00 SDS-PAGE | Only one has obvious banding |  |
|  | Cas 13a&b | Yuzhi Wang | 6：30 Transfrom |  | Lba has six transformants; Lwa has a transformant |
| 2020/8/29 | Cas 13a&b | Xiaojie Zhou | PCR、SDS-PAGE | No obvious bands |  |
|  | Cas 13a&b | Yuzhi Wang | Lba、Lwa pre-culture |  | one test tube for each of seven transformants |
|  | Cloning | Ziyan Huang | PCR |  |  |
|  | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | SDS-PAGE | Lba has no obvious bands, Lwa has bands |  |
| 2020/8/30 | Cas 13a&b | Yuzhi Wang | Lba，Lwa Plasmid extraction | Concentration is too low |  |
|  | Cas 13a&b | Yuzhi Wang | Lba and Lwa inoculation, two tubes each |  |  |
| 2020/8/31 | Cas 13a&b | Yuzhi Wang | Lba，Lwa Plasmid extraction | Lba1：45.6ng/ml；Lba2：48.9ng/ml；Lwa1：50.9ng/ml；Lwa2：41.4ng/ml |  |
|  | Cas 13a&b | Yuzhi Wang | SDS-PAGE | Lba1, Lba2, Lwa1 have obvious bands, Lwa2 has no obvious bands |  |
|  | Cas 13a&b | Yuzhi Wang | Transfrom | Pam and Lba have many transformants, but Lwa does not | Psm is transformed into DH5α competence cell；Lba、Lwa are transformed into Rosetta |

# Sep

| Data | Classification | Experimenter | Experiment | Experimental results | Note |
| --- | --- | --- | --- | --- | --- |
| 2020/9/1 | Cloning | Ziyan Huang、Yuzhi Wang | PCR |  |  |
|  | Cas 13a&b | Yuzhi Wang | SDS-PAGE | Lba has no band; Lwa has bands; Psm is not ideal |  |
|  | Cas 13a&b | Yuzhi Wang | SDS-PAGE | Lba transformant 8 has a band; Pam transformants 1, 2, 3, 4, 5, 8 have bands; Lwa1 and 4 plasmids have bands |  |
| 2020/9/2 | Cas 13a&b | Yuzhi Wang | 9:00 pre-culture Lba×5 Lwa×2 Psm×2 |  | Lba is used for expression, Lwa and Psm are used for plasmid extraction |
|  | Cas 13a&b |  |  | Lba becamr clarified after 10h | Phage |
|  | Cas 13a&b |  |  | Lba became clarified |  |
|  | Cas 13a&b |  |  | Psm became turbid | normal |
|  | Cas 13a&b | Ziyan Huang、Yuzhi Wang | Lba PCR |  | tdirteen transformants |
|  | Cas 13a&b |  | Psm Plasmid extraction | tde resulting psm plasmid concentration is 39.3 and 52.9ng/ul |  |
|  | Cas 13a&b |  | SDS-PAGE | tdere are obvious bands |  |
| 2020/9/3 | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Lba pre-culture (test tube × 4) | fail | fail |
|  | Cas 13a&b |  | Lwa are transformed into Rosetta |  |  |
| 2020/9/4 | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Lba pre-culture | success |  |
| 2020/9/5 | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Lwa expands training | fail | fail |
|  | Cloning | Xiaojie Zhou | 9：00-12：00 Psm Cas 13b、Cca Cas 13b dHis-1 PCR |  | tde tdree plasmid products of one-step cloning were all subjected to PCR |
|  | Cloning |  | 12:30 PCR products SDS-PAGE | tde band is slightly weaker, but tdree of it are successful |  |
|  | Cloning |  | 13:00-14:00 dHis-1 PCR product digestion |  |  |
|  | Cloning |  | 14：00-16：00 Psm Cas 13b、Cca Cas 13b are transformed into DH5α competence cell |  |  |
| 2020/9/6 | Cloning | Xiaojie Zhou | 13:00 Pick Psm Cas 13b、Cca Cas 13b dHis-1 single colony culture |  | Pick five single colonies |
| 2020/9/7 | Cloning | Xiaojie Zhou | 7：30 Psm Cas 13b、Cca Cas 13b dHis-1 Plasmid extraction | The concentrations are 62.7, 29.8, 33.2, 39.6, 51.8ng/uL |  |
|  | Cloning | Xiaojie Zhou | Streaked live bacteria 106096, 94205, 107838, 330-106 |  | Just for sequencing |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Lwa pre-culture | fail |  |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Psm transformed into Rosetta |  | The original plasmid |
| 2020/9/7-8 | Cloning | Xiaojie Zhou | Psm Cas 13b、Cca Cas 13b dHis-1 delivery test result | 1、2、3、5 has no bands, no sequencing is arranged, 4 is successful and His-1 is successfully removed after comparison | We should do SDS-PAGE after extracting the plasmid to see if the plasmid is actually proposed and the plasmid is the target plasmid |
|  | Cloning | Xiaojie Zhou | Psm Cas 13b、Cca Cas 13b dHis-2 PCR |  | Take the No. 4 template and configure 3 systems (the PCR in the morning was not scheduled and the experiment progress was delayed by another day) |
|  | Cloning | Xiaojie Zhou | TDPs pick single clones into test tubes |  |  |
|  | Cloning | Xiaojie Zhou | Psm Cas 13b、Cca Cas 13b dHis-2 PCR product SDS-PAGE | There are obvious bands |  |
| 2020/9/8 | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Psm pre-culture | Success |  |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | 18:00 Psm expand training | Success |  |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | 20：00 add IPTG | Fail | The next morning, the bacterium liquid became clear, because of the phage |
| 2020/9/9 | Cloning | Xiaojie Zhou | TDPs Plasmid extraction |  |  |
|  | Cloning | Xiaojie Zhou | 13:00 Psm Cas 13b、Cca Cas 13b dHis-2 PCR product digestion |  |  |
|  | Cloning | Xiaojie Zhou | 14：00-16：00 transformed into DH5α competence cell |  |  |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Psm pre-culture | Success | Bacteria is not too thick |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | 18:30 Expanded training×2 |  | In the shaker on the fourth floor, one bottle is successfully stained with phage |
|  | Cas 13a&b | Xinyu Wan、Yuzhi Wang | 21：30 add IPTG |  | The next morning, the bacterial liquid became clear, but slightly turbid; it almost became clear at noon, and the phage infection was severe |
|  | Cas 13a&b | Yuzhi Wang | 21:00 Expanded training × 2 | fail | Shaker on the first floor, both bottles are stained with phage |
| 2020/9/10 | Cloning | Xiaojie Zhou | 12:30 Pick Psm Cas 13b、Cca Cas 13b dHis-2 single colony to test tube |  |  |
|  | Cloning | Xiaojie Zhou | TDPs sequencing results | The result shows that 94205 has a problem |  |
|  | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | gfp、106094、330-106、33020transformed into BL21 competence cell | The transformation was successful, but the coating did not form a single colony | The bacterial solution is not dry, and it does not dry after 1h |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | 13:00 Plasmid PCR |  |  |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | 15:00 cancel template |  |  |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | 18:30 Purification |  |  |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | 20：00 SDS-PAGE | Fail | Suspected of primer |
|  | Cas 13a&b | Yuzhi Wang、Qian Rong、Yuanying Wang | Lwa、Lba transformed into Rosetta |  |  |
| 2020/9/11 | Cas 13a&b | Yuzhi Wang | 9:00 pre-cultivation of Lba and Lwa |  |  |
|  | Cas 13a&b | Yuzhi Wang | 19:00 Expanded culture (Lba×2, Lwa×2) | Lwa1 stained phage |  |
|  | Cas 13a&b | Yuzhi Wang | 21: 00 IPTG induction | 2 bottles of Lba bacteria liquid are more concentrated, Lwa2 becomes slightly clear |  |
| 2020/9/12 | Cloning | Xiaojie Zhou | 7：30 Psm Cas 13b、Cca Cas 13b dHis-2 Plasmid extraction |  |  |
|  | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | 15:00 gfp, 106094, 330-106, 33020 coating | There are satellite colonies, but after consulting the information, it is said that the impact is not significant |  |
|  | Cas 13a&b | Qian Rong | Collect the bacterial pellet Lba, Lwa |  |  |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | 9：00 SARS-CoV-2 PCR and SDS-PAGE | fail |  |
|  |  |  | 14:00 SARS-CoV-2 and H1N1 PCR and SDS-PAGE | SARS-CoV-2 fail, H1N1 success |  |
|  |  |  | 17:00 H1N1 elimination template |  |  |
|  |  |  | 19:00 H1N1 purification and SDS-PAGE | success |  |
|  | Cas 13a&b | Yuzhi Wang | SDS-PAGE | Both Lba and Lwa have target bands |  |
| 2020/9/13 | Cloning | Xiaojie Zhou | Psm Cas 13b、Cca Cas 13b dHis-2 sequencing results | His2 successfully removed |  |
|  |  |  | Psm Cas 13b、Cca Cas 13b transformed into DH5alpha |  |  |
|  |  |  | Lba transformed into Rosetta |  |  |
|  |  |  | Culture Lwa DH5alpha with Streak Plate Method |  | Cooperation with ShanghaiTech University, they need this protein |
|  | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | 1:00 single colony culture | success |  |
|  |  |  | 20:00 Protein induced expression | fail | "It can be cultured for a period of Data before adding new medium before induction (optimization?) Inducer calculation error" |
|  | Cas 13a&b | Yuzhi Wang | Lba purification | fail | May be related to the machine |
| 2020/9/14 | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | 1:00 pick a small single colony shaker culture | fail | Proof that the small colonies are satellite colonies may be correct |
|  |  |  | 6:30 Use 9/13 medium to induce pre-cultivation raise for a period of Data to test | The phenomena are consistent, but GFP has no fluorescence and the induction fails | It’s not clear, even used a microplate reader to test the lure |
|  |  |  | 20:00 Induced expression of two groups of proteins | The phenomena are consistent, but GFP has no fluorescence and the induction fails | The fluorescence of the latter group is lower" |
|  | Sample processing | Yuzhi Wang、Chaonan Fang、Xiaoqi Wang | H1N1 measures DNA concentration |  |  |
|  |  |  | H1N1 transcription and purification |  |  |
|  | Cas 13a&b | Yuzhi Wang | 10:00 Lba pre-culture (×6) |  |  |
| 2020/9/15 | TDPs | Xinzhi Zhou | Save the bacteria after expression at 7:00 am | Successfully saved |  |
|  |  | Jiangxiaojie Zhang、Xinzhi Zhou | 4:00 re-coating | Similar to the previous plate phenomenon, large and small colonies, similar in shape |  |
|  | Sample processing | Yuzhi Wang、Xiaoqi Wang | 8：00 H1N1 PCR |  |  |
|  |  |  | 10:30 H1N1 elimination template |  |  |
|  |  |  | 12:00 H1N1 purification |  |  |
|  | Cas 13a&b | Yuzhi Wang | 9:30 Lba expanded cultivation (×6) |  |  |
|  |  |  | 17:00 IPTG induction |  |  |
| 2020/9/16 | Cas 13a&b | Yuzhi Wang | 7:00 Lba collection |  |  |
|  |  |  | 10：00 SDS-PAGE | No target band |  |
|  |  |  | 9:30 Lba expanded cultivation (×6) |  |  |
|  |  |  | 17：00 SDS-PAGE | The target band of the protein supernatant is not obvious, and the precipitation band is obvious |  |
|  |  |  | 21:30 IPTG induction |  |  |
|  | Sample processing | Yuzhi Wang、Chaonan Fang、Xiaoqi Wang | 11：00 transform |  |  |
|  |  |  | 14:30 RNA purification and concentration measurement |  |  |
|  |  |  | 19:00 RT-RPA, concentration measurement |  |  |
|  | TDPs | Jiangxiaojie Zhang | 7:10 Receive tablet |  |  |
| 2020/9/17 | Cloning | Xiaojie Zhou | 22:30 Pick a single colony of Psm Cas 13b、Cca Cas 13b (BL21) to test tube culture |  |  |
|  |  |  | 22:40 Apply Rosetta tablet |  |  |
|  |  |  | 22:50 Pick a single colony of Rosetta gami into a test tube |  |  |
|  | Cas 13a&b | Yuzhi Wang | 8:00 Broken bacteria |  |  |
|  |  |  | 10:30 Lba purification |  |  |
|  |  |  | 16：00 SDS-PAGE | Only the flow-through liquid has bands, and the rest of the eluates have no bands |  |
|  |  | Qian Rong、Yuanying Wang | 13:30 Collection of bacteria |  |  |
|  |  | Yuzhi Wang | 19：30 SDS-PAGE |  |  |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | 9:00 DNA purification and concentration measurement |  |  |
|  |  |  | 11：00 transform |  |  |
|  |  |  | 14:00 RNA purification and concentration measurement |  |  |
|  | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | 1:00 single colony culture | 106094 bacterial liquid is not very turbid |  |
|  |  |  | 20:00 Protein induced expression | fail | prepare to verify whether the conversion was successful |
| 2020/9/18 | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | Pick four groups of GFP single colonies PCR running gel | Two groups have bands, two groups do not | 9.20 Continue the experiment |
|  | Cas 13a&b | Yuzhi Wang | 1L Lba ultrasonic disruption |  |  |
| 2020/9/19 | Cas 13a&b | Yuzhi Wang、Xiaojie Zhou | Lba first purification |  |  |
|  |  | Xiaojie Zhou | SDS-PAGE |  |  |
|  |  | Ziyan Huang | 3320,cca transformed into BL21 | A large number of single colonies grow |  |
| 2020/9/20 | TDPs | Xinzhi Zhou | Pick 33020, 106094, 330-106 single colony PCR SDS-PAGE |  |  |
|  |  | Jiangxiaojie Zhang、Xinzhi Zhou | Invert the plate, take the transformed colony and streak |  |  |
|  | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | Transform |  |  |
|  |  |  | RNA purification |  |  |
|  |  |  | RT-RPA |  |  |
|  | Cas 13a&b | Yuzhi Wang | 8:00 Cca, 33020 pre-culture (each × 5) |  |  |
|  |  |  | 20:00 Cca, 33020 expanded training (each × 5) |  |  |
|  |  |  | 21:00 Lba pre-culture (×5) |  |  |
|  |  |  | 22:30 Cca, 33020 induction |  |  |
|  |  |  | SDS-PAGE after Lba purification | Lba is eluted in 250mM IM eluate |  |
|  |  |  | 14:30-20:30 MBP digestion |  |  |
|  |  |  | 21: 00-24: 00 Lba secondary purification |  |  |
|  |  |  | 24:00—2:00 of the next day SDS-PAGE after secondary purification | No banding | Too much loss in the first purification, more loss in enzyme digestion, making the gel run after the second purification without bands and no target protein |
| 2020/9/21 | Sample processing | Chaonan Fang、Xiaoqi Wang、Yuzhi Wang | DNA purification and concentration measurement |  |  |
|  |  |  | Transform ×6 |  |  |
|  |  |  | RNA purification and concentration measurement |  |  |
|  | Cas 13a&b | Qian Rong、Yuanying Wang | Cca, 33020 collection |  |  |
|  |  | Yuzhi Wang | 15:00 Lba expansion training (×10) | 4 bottles meet the requirements |  |
|  |  |  | 18:30 Lba induction (×4) |  |  |
| 2020/9/22 | Cas 13a&b | Qian Rong、Yuanying Wang | Lba collection and breakage |  |  |
|  |  | Yuzhi Wang、Qi Rong、Yuanying Wang | SDS-PAGE | There is no target band, and the miscellaneous band is shallow | It may be that the ice was not replaced in Data during the crushing process, and the protein was degraded |
| 2020/9/25 | Cas 13a&b | Xinyu Wan | Culture Cca with Streak Plate Method |  |  |
| 2020/9/26 | Cas 13a&b | Xinyu Wan | Cca pre-culture × 10 |  |  |
| 2020/9/27 | Cas 13a&b | Xinyu Wan | Cca expansion × 20 | fail | Phage |
|  |  |  | Culture Lba with Streak Plate Method |  |  |
|  |  |  | Cca pre-culture × 10 |  |  |
|  | TDPs | Jiangxiaojie Zhang | Select successful single colony to culture | After culturing for 12 hours, there were almost no colonies. After 2 days, GFP formed single colonies, but the PCR results showed that it was not GFP | The tablet is too long to be recoated |
| 2020/9/28 | Cas 13a&b | Xinyu Wan、Yuzhi Wang | Cca expanded culture ×10 |  |  |
|  |  |  | Cca induced |  |  |
|  | TDPs | Jiangxiaojie Zhang | spread plate method | success |  |
| 2020/9/29 | Cas 13a&b | Qian Rong、Yuanying Wang | Cca closed bacteria |  |  |
|  |  |  | SDS-PAGE | Cca does not have any bands; Lba and 33020 bands are small in number and light in color |  |
|  |  | Yuzhi Wang | Lba pre-culture ×15 |  |  |
|  | TDPs | Xinzhi Zhou | PCR vaccination | The PCR results were good and most of the colonies were converted successfully. |  |
|  |  | Jiangxiaojie Zhang | The successful PCR bacteria were preserved |  |  |
| 2020/9/30 | TDPs | Jiangxiaojie Zhang | Adjust the ready protein expression | The arrangement of the experiment was wrong. 3ml should be directly put into the test tube for sterilization, and it should be operated in the super clean table, which is inconvenient and easy to be infected with heterobacteria. Therefore, most protein expression groups were not carried out this Data |  |
|  | Cas 13a&b | Yuzhi Wang | 13:00 Lba expanded culture ×20 |  | One bottles of sterile growth |
|  |  |  | 15:00 Cca ultrasonic sterilization |  |  |
|  |  |  | 16:00 Lba induced ×19 |  |  |
|  |  |  | 19:00 Lba expanded culture ×11 | Two bottles of sterile growth |  |
|  |  |  | 21:00 33020 Pre-culture ×10 |  |  |
|  |  |  | 22:00 Lba induction ×9 |  |  |
|  |  |  | 23:00 Purification of Cca once (1L) |  |  |

# Oct

| Data | Classification | Experimenter | Experiment | Experimental results | Note |
| --- | --- | --- | --- | --- | --- |
| 2020/10/1 | Cas 13a&b | Yuzhi Wang | 2:00 SDS-PAGE after one purification of Cca |  |  |
|  |  |  | 3:30 Cca enzyme digestion and dialysis |  |  |
|  |  | Qian Rong、Yuanying Wang | 9:30 Lba inoculation first batch |  |  |
|  |  |  | 14:00 Lba inoculation of the second batch |  |  |
|  |  | Yuzhi Wang | 21:00 Cca incubated for 1.5h |  |  |
|  |  |  | 23:00 Expand culture ×17 of 33020 |  |  |
|  |  |  | 23:30 Secondary purification of Cca |  |  |
|  | TDPs | Jiangxiaojie Zhang、Yi Fan | Line the bacteria for preservation | 4, 6, 7, 8, 10, 11, 15 growth of preservation bacteria, gun head suspected to be contaminated, only 33020 form a single colony. |  |
|  |  |  | Induced expression after culture on 29 September |  |  |
| 2020/10/2 | Cas 13a&b | Yuzhi Wang | 2:00 Induction 33020 |  |  |
|  |  |  | 3:00 SDS-PAGE after secondary purification of Cca |  |  |
|  |  | Qian Rong | 13:00 Lba ultrasonic crushing |  |  |
|  |  | Qian Rong、Yuanying Wang | 18:30 Collect bacterial precipitation of 33020 |  |  |
|  |  | Yuzhi Wang | 15:00 Lba incubation for 1.5h |  |  |
|  |  |  | 17:00 Lba one purification |  |  |
|  |  |  | 19:00 Cca preculture ×16 |  |  |
|  | TDPs | Jiangxiaojie Zhang | GFP fluorescence detection | There was no green fluorescence, and there was little change in the values measured by the enzyme marker |  |
|  |  |  | Line the bacteria for preservation | GFP, 106094, formed a single colony |  |
| 2020/10/3 | Cas 13a&b | Yuzhi Wang | 2:00 SDS-PAGE after one purification of Lba | Not pure |  |
|  |  |  | 13:00 Lba incubation for 1.5h |  |  |
|  |  |  | 15:00 The purification is repeated Lba |  |  |
|  |  |  | 17:00 Cca expand cultivation ×30 |  |  |
|  |  |  | 21:00 After one purification of Lba SDS-PAGE |  |  |
|  |  |  | 22:00 Cca induction ×30 |  |  |
|  |  |  | 22:30 33020 Incubation for 1.5h |  |  |
|  | TDPs | Jiangxiaojie Zhang、Xiaojie Zhou | Pick the fungus for PCR | GFP1, 2 106094 1 and 2 single colonies may be successfully transformed |  |
|  |  | Jiangxiaojie Zhang | GFP1, 2, 106094 1 and 2 were selected for culture and variable temperature induced expression | All were cultured successfully, and only GFP1 106094 1 induced expression protein was detected visually |  |
|  |  |  | Fluorescence detection (UV lamp, enzyme marker) | GFP was not significantly green, and the enzyme marker increased slightly, but the different concentrations of bacteria could not explain the problem |  |
|  |  | Jiangxiaojie Zhang | 16:00 33020 Ultrasonic sterilization |  |  |
| 2020/10/4 | Cas 13a&b | Yuzhi Wang | 0:00 33020 once purification |  |  |
|  |  |  | 3:00 SDS-PAGE after one purification of 33020 |  |  |
|  |  |  | 4:00 Lba, 33020 dialysis |  |  |
|  |  |  | 22:00 33020 protein concentration |  |  |
|  |  | Qian Rong | 14:00 Collect Cca bacterial liquid |  |  |
|  | Sample processing | Xiaoqi Wang、Yuzhi Wang | 15:00 Culture H1N1 with Streak Plate Method |  |  |
|  | Readout | Xiaojie Zhou | 18:00 Pretreatment of protein virus crRNA and other reagents |  |  |
|  |  |  | 21:00 LwaCas protein activity test | Failure. There was no significant difference in fluorescence between the experimental group and the blank group, but the fluorescence of the negative control group was higher than that of the blank group |  |
| 2020/10/6 | TDPs | Jiangxiaojie Zhang | 3:00 Inoculation 10.2GFP1, culture it in 37℃ |  |  |
|  |  |  | 9 :00 transfer |  |  |
|  |  |  | 11:30 after induction with different IPTG, culture at 30 degrees overnight (12h) | Fluorescence was not visible to the naked eye, and IPTG test showed no obvious induction |  |
|  | Sample processing | Xiaoqi Wang、Yuzhi Wang | 10:00 H1N1 preculture ×2 |  |  |
|  |  |  | 18:00 H1N1 expanded culture ×2 |  |  |
|  |  |  | 21:00 H1N1 induction |  |  |
|  | Cas 13a&b | Yuzhi Wang | 17:00 Cca was incubated for 1.5h |  |  |
|  |  |  | 18:30 One purification of Cca |  |  |
|  |  |  | 22:00 SDS-PAGE |  |  |
|  | Readout | Xiaojie Zhou | Rhodamine, FITC scanning attempt |  |  |
| 2020/10/7 | Cas 13a&b | Yuzhi Wang | 11:00 Cca was incubated for 1.5h |  |  |
|  |  |  | 12:30 Single purification of Cca (2) |  |  |
|  |  |  | 16:00 SDS-PAGE | Only fluid penetration and 10 mM IM have target bands |  |
|  |  |  | 20:00 Cca incubated for 1.5h |  |  |
|  |  |  | 21:30 Single purification of Cca (3) |  |  |
|  |  |  | 24:00 SDS-PAGE | Only fluid penetration and 10 mM IM have target bands |  |
| 2020/10/8 | Cas 13a&b | Yuzhi Wang | 1:00 Condensed 33020 |  |  |
|  |  |  | 2:00 Lba enrichment |  |  |
|  |  |  | 3:00 After enrichment SDS-PAGE | Lba has obvious target bands, but there are miscellaneous bands |  |
|  |  |  | 4:30 Lba digestion | Lba |  |
|  |  |  | 10:00 Cca bactericidal crushing |  |  |
|  |  |  | 11:30 Lba enzyme digestion, 33020 concentration, Cca bacteria - destroying SDS-PAGE |  |  |
|  |  |  | 15:00 Lba standard curve to calculate the concentration |  |  |
| 2020/10/11 | Readout | Xiaojie Zhou | The maximum excitation absorption was determined by rhodamine gradient scan | failure | The gradient is set, but the concentration is still too low |
|  | TDPs | Jiangxiaojie Zhang、Xinzhi Zhou | PCR, 10.3GFP, 10.3/106094, Zxj33020, Zxj106094 | Most transformations are successful |  |
|  |  |  | cultivate GFP, 106094 ,eGFP | Develop success |  |
|  |  |  | Preinduction adaptation | eGFP thin |  |
|  |  |  | Induced expression overnight | eGFP was successfully expressed and GFP had no green fluorescence |  |
| 2020/10/12 | Readout | Xiaojie Zhou | Rhodamine standard curve determination |  | Theoretical maximum excitation launch attempt |
|  | TDPs | Jiangxiaojie Zhang | Culture 330-106 with Streak Plate Method | No single colony was found |  |
|  |  |  | The eGFP and 106094OD values were measured and prepared with 3% glucose solution on the upper layer of the refrigerator |  | OD was measured to prepare 10-4.5 power bacterial solution, which was prepared with glucose solution on the upper layer of refrigerator without bacterial membrane |
| 2020/10/15 | TDPs | Jiangxiaojie Zhang | EGFP and 106094 were cultured for 6h.Treatment before induction for 1h, induction overnight, check for fluorescence | With the fluorescent | After induction, the bacteria solution was put in the refrigerator, packaged and waited for OD freeze-drying test |
|  |  |  | Observe the results of YuZhi Culture 33020 and 33-106 with Streak Plate Method | There are single colonies on the culture medium |  |
|  | Cas 13a&b | Yuzhi Wang | 22:00 Culture 33020 and 33-106 with Streak Plate Method |  |  |
|  |  |  | 24:00 Psm bacteria suspension |  |  |
| 2020/10/16 | Cas 13a&b | Yuzhi Wang | 1:00 Psm ultrasonic demulsification |  |  |
|  |  |  | 3:00 Psm was incubated with Ni column |  |  |
|  |  |  | 3:30 Prepare buffer |  |  |
|  |  |  | 5:00 Psm purification one Data |  |  |
|  |  |  | 22:00 SDS-PAGE |  |  |
|  |  |  | 24:00 Psm bacteria suspension |  |  |
|  | TDPs | Jiangxiaojie Zhang | PCR 33020 and 330-106 had 123 colonies, respectively | fail | High probability operation problem |
|  | Readout | Xiaojie Zhou | Gain value exploration of LWA dynamic curve | About 150 is a good choice |  |
| 2020/10/17 | Cas 13a&b | Yuzhi Wang | 1:30 Psm ultrasonic crushing |  |  |
|  |  |  | 3:30 Psm was incubated with Ni column |  |  |
|  |  |  | 4:00 Prepare buffer solution |  |  |
|  |  |  | 6 :00 Psm purification |  |  |
|  |  |  | 10:30 SDS-PAGE |  |  |
|  |  |  | 14:30 Concentration of Psm protein |  |  |
|  | Readout | Xiaojie Zhou | Lwa probe specificity was determined by X2 |  |  |
|  | TDPs | Xinzhi Zhou |  |  |  |
|  |  | Jiangxiaojie Zhang | IPTG induction group 3 |  |  |
|  |  |  | Inoculation and cultivation, preferential treatment |  |  |
| 2020/10/18 | Cas 13a&b | Yuzhi Wang | 18:00 Psm measurement concentration | 1211.5632ng/ul |  |
|  | Readout | Xiaojie Zhou | Lwa lyophilized protein activity assay | The protein activity decreased obviously |  |
|  |  |  | Lba activity detection and kinetic curve determination | There was a large gap between the parallel groups with little protein activity |  |
|  |  |  | The dynamics curve of lBA experiment was determined after optimization | The data are much more accurate than before, but the protein activity is still very weak |  |
| 2020/10/19 | Readout | Xiaojie Zhou | Lwa virus concentration gradient experiment | Because the group setting is too small, the virus concentration detection limit of LWA is not tested |  |
|  |  |  | PSM dynamics curve |  |  |
|  |  |  | PSM probe specific kinetic curve | The gian value of 150 results in a fluorescence value of extraordinary, and FAM channel has no fluorescence value data, terminating the experiment |  |
|  |  |  | Gain value exploration of PSM probe specificity experiment | The best gain value was 130 when the specificity of PSM was determined |  |
|  |  |  | Lwa, PSM mixed specificity determination 1 | Because the experiment design is wrong, the group is not set clearly, so try again |  |
| 2020/10/20 | Readout | Xiaojie Zhou | Lwa, PSM mixed specificity determination 2 | The grouping of the experiments is still not thoughtful enough |  |

|  |
| --- |
|  |
|  | ---  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | [![](https://static.igem.org/mediawiki/2020/0/0c/T--TPR_China--Acknowledgement2.png)](https://www.mathworks.com/) |  | [![](https://static.igem.org/mediawiki/2020/7/78/T--ZJUT_China_B--sponsors_radobio.png)](http://www.radobio.com/) |  | [![](https://static.igem.org/mediawiki/2020/5/56/T--ZJUT_China_B--sponser-cs.png)](http://www.transcenta.com/ch/) |  | [![](https://static.igem.org/mediawiki/2020/e/ec/T--ZJUT_China_B--sponsors_snapgene.png)](https://www.snapgene.com/) |  | [![](https://static.igem.org/mediawiki/2020/7/78/T--ZJUT_China_B--sponsors_genescript.png)](https://www.genscript.com/) |  | [![](https://static.igem.org/mediawiki/2020/7/78/T--ZJUT_China_B--sponsors_bori.png)](http://www.bioer.com.cn/) |  | [![](https://static.igem.org/mediawiki/2018/e/e8/T--ZJUT-China--%E6%B5%99%E5%B7%A5%E5%A4%A7%E7%94%9F%E5%B7%A5%E5%AD%A6%E9%99%A2.jpg)](http://www.cbb.zjut.edu.cn/) |  | [![](https://static.igem.org/mediawiki/2018/4/4c/T--ZJUT-China--%E6%B5%99%E5%B7%A5%E5%A4%A7.jpg)](http://www.zjut.edu.cn/) |  | ![](https://static.igem.org/mediawiki/2018/b/be/T--ZJUT-China--%E5%85%AC%E4%BC%97%E5%8F%B7%E4%BA%8C%E7%BB%B4%E7%A0%81.jpg) | |  |

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# Team:ZJUT China B/Engineering

Document

[Home](https://2020.igem.org/Team%3AZJUT_China_B)

Team

[Team Members](https://2020.igem.org/Team%3AZJUT_China_B/Team)
[Attributions](https://2020.igem.org/Team%3AZJUT_China_B/Attributions)

Project

[Description](https://2020.igem.org/Team%3AZJUT_China_B/Description)
[Design](https://2020.igem.org/Team%3AZJUT_China_B/Design)
[Implementation](https://2020.igem.org/Team%3AZJUT_China_B/Implementation)
[Contribution](https://2020.igem.org/Team%3AZJUT_China_B/Contribution)
[Engineering](https://2020.igem.org/Team%3AZJUT_China_B/Engineering)
[Proof Of Concept](https://2020.igem.org/Team%3AZJUT_China_B/Proof%20Of%20Concept)
[Results](https://2020.igem.org/Team%3AZJUT_China_B/Results)

Wetlab

[Protocols](https://2020.igem.org/Team%3AZJUT_China_B/Protocol)
[Notebook](https://2020.igem.org/Team%3AZJUT_China_B/Notebook)
[Parts](https://2020.igem.org/Team%3AZJUT_China_B/Parts)
[Wet collaboration](https://2020.igem.org/Team%3AZJUT_China_B/Wet%20collaboration)

HP

[Overview](https://2020.igem.org/Team%3AZJUT_China_B/Human%20Practices)
[Silver](https://2020.igem.org/Team%3AZJUT_China_B/HP-Silver)
[Gold](https://2020.igem.org/Team%3AZJUT_China_B/Integrated%20human%20practice)
[Science communication](https://2020.igem.org/Team%3AZJUT_China_B/Education)
[Collaborations](https://2020.igem.org/Team%3AZJUT_China_B/Collaborations)
[Partnership](https://2020.igem.org/Team%3AZJUT_China_B/Partnership)
[Ethics](https://2020.igem.org/Team%3AZJUT_China_B/Ethics)

Modeling

[Software](https://2020.igem.org/Team%3AZJUT_China_B/Software)
[Modeling](https://2020.igem.org/Team%3AZJUT_China_B/Model)

Hardware

[Overview](https://2020.igem.org/Team%3AZJUT_China_B/Hardware)
[Detecting](https://2020.igem.org/Team%3AZJUT_China_B/Detecting%20)
[Sample processing](https://2020.igem.org/Team%3AZJUT_China_B/Sample%20processing)
[Chip](https://2020.igem.org/Team%3AZJUT_China_B/Chip)
[History](https://2020.igem.org/Team%3AZJUT_China_B/History)

[Safety](https://2020.igem.org/Team%3AZJUT_China_B/Safety)

Judging

[Poster](https://2020.igem.org/Team%3AZJUT_China_B/Poster)
[Judging Form](https://2020.igem.org/Team%3AZJUT_China_B/Judging%20Form)

[![](https://static.igem.org/mediawiki/2020/5/5d/T--ZJUT_China_B--logo-Cas.png)](https://igem2020-zjut.feishu.cn/docs/doccnWz3kWJdTESBOvIunDsW2ac?from=from_copylink)

## **Engineering**

* [A new part](#C1)
* [Characterization](#C2)
* [Success on device](#C3)

# Constructed and characterized a new part： 6xHis/Twin strep -SUMO-PsmCas13b

In order to achieve our multivirus detecting goal, we need to obtain four Cas13 homologues involved in our project. Thus, we constructed biobricks and parts to express the four Cas13 homologues. All the parts we constructed can be viewed in the part registry and are all listed in our >>part page<<. For our favorite part: BBa\_K3406006 , 6xHis/Twin strep -SUMO-PsmCas13b, we successfully demonstrated that it could express PsmCas13b protein as we expected. Besides, we tested the activity and base-cleavage preference of the expressed Cas13 for its characterization.

**## Cloning**

We ordered PsmCas13b gene from a synthesis company, then we cloned the gene into pC0061. Additionally, we attached 6xHis/Twin strep tag and SUMO tag to 5' end of the gene. We transformed the ligation product into *E. coli* Rosetta2(DE3)pLysS and confirmed the cloning by colony-PCR and sequencing.

![](https://static.igem.org/mediawiki/2020/b/b7/T--ZJUT_China_B--Colony_PCR_result_of_6xHis.png)

**# Characterization**

## Collateral cleavage activity of PsmCas13b protein

After we got the expressed and purified PsmCas13 protein, we needed to verify its activity to make sure it folds properly. We tested the kinetic curve of the protein to see the dynamic change of the flourescence. As shown in the figure 3, our protein shows considerable activity in cutting flourescence reporters. We can clearly see that after adding the target, the activity of the protein is greatly improved, which enables us to tell whether the target exists in the detecting system. Interestingly, as same as LwaCas13a protein, the PsmCas13b protein also exhibits "leakage activity(exhibit cleavage activity without target sequence)", which is the main interference to the detecting results.
Furthermore, after 100 mins of detecting, the fluorescence signal gradually became stable and finally decreased. We analysed it on the model page, click >>[Model](https://2020.igem.org/Team%3AZJUT_China_B/Model)<<
to see more details.

![](https://static.igem.org/mediawiki/2020/0/05/T--ZJUT_China_B--The_collateral_cleavage_activity_of_PsmCas13b_protein.png)

Figure 4. The collateral cleavage activity of PsmCas13b protein

Note: "Negative" and "PsmCas13b" are the groups with and without target sequences

**## Cleavage base preference of PsmCas13b protein**

To achieve our multivirus detecting goal, we further investigate the cleavage base preference of PsmCas13b protein. We added 4 kind fluorescence reporters ( The information is shown in Table1 ) into the detecting system and the fluorescence results are shown in Figure 5.

![](https://static.igem.org/mediawiki/2020/3/35/T--ZJUT_China_B--Fluorescence_detecting_results.jpg)
![](https://static.igem.org/mediawiki/2020/c/c5/T--ZJUT_China_B--The_Cleavage_base_preference_of_PsmCas13b_protein_upon_AC%2CAU%2CGA%2CUC.png)

# Success on device

We designed a fluorescence detector for the needs of the project. This device consists of a filter turntable and a controller. It changes the wavelength on the light path through the filter wheel, so as to achieve the purpose of detecting fluorescein of a specific wavelength. Its details will be mentioned on the Detector page, please click >>[Detector](https://2020.igem.org/Team%3AZJUT_China_B/Detecting)<<

We can prove that our device is useful. When there is only one type of fluorescein in the system, only one type of filter can transmit fluorescence. We take off the detector and shoot with the mobile phone facing the light path, as shown in the video below.

>>[click](https://static.igem.org/mediawiki/2020/a/a4/T--ZJUT_China_B--View_through_the_detector.mp4)<< Video 1 View through the detector

We added 10uL fluorescein Cy5 ( 50uM ) to the filter paper and adjusted the exposure of the mobile phone camera to maximum, then we took this real image. In a detection cycle, only a set of filters that are used to detect Cy5 can return visible light signals, which can prove that our device is successful.

We also tested with a lower concentration of fluorescein (Cy5 1uM), and the results are shown in the following table (Table 2):

![](https://static.igem.org/mediawiki/2020/6/6d/T--ZJUT_China_B--detector_return.jpg)

It can be clearly seen that Cy5 is at a positive level, which can prove the success of our device construction. Although we did not test other fluoresceins due to conditions, in principle, our device is totally capable of detecting the existing fluorescein one by one.

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|  | ---  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  |  | | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | --- | | [![](https://static.igem.org/mediawiki/2020/0/0c/T--TPR_China--Acknowledgement2.png)](https://www.mathworks.com/) |  | [![](https://static.igem.org/mediawiki/2020/7/78/T--ZJUT_China_B--sponsors_radobio.png)](http://www.radobio.com/) |  | [![](https://static.igem.org/mediawiki/2020/5/56/T--ZJUT_China_B--sponser-cs.png)](http://www.transcenta.com/ch/) |  | [![](https://static.igem.org/mediawiki/2020/e/ec/T--ZJUT_China_B--sponsors_snapgene.png)](https://www.snapgene.com/) |  | [![](https://static.igem.org/mediawiki/2020/7/78/T--ZJUT_China_B--sponsors_genescript.png)](https://www.genscript.com/) |  | [![](https://static.igem.org/mediawiki/2020/7/78/T--ZJUT_China_B--sponsors_bori.png)](http://www.bioer.com.cn/) |  | [![](https://static.igem.org/mediawiki/2018/e/e8/T--ZJUT-China--%E6%B5%99%E5%B7%A5%E5%A4%A7%E7%94%9F%E5%B7%A5%E5%AD%A6%E9%99%A2.jpg)](http://www.cbb.zjut.edu.cn/) |  | [![](https://static.igem.org/mediawiki/2018/4/4c/T--ZJUT-China--%E6%B5%99%E5%B7%A5%E5%A4%A7.jpg)](http://www.zjut.edu.cn/) |  | ![](https://static.igem.org/mediawiki/2018/b/be/T--ZJUT-China--%E5%85%AC%E4%BC%97%E5%8F%B7%E4%BA%8C%E7%BB%B4%E7%A0%81.jpg) | |  |

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ZJUT China B/Experiments

hello world

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:ZJUT China B/Results

Document

[Home](https://2020.igem.org/Team%3AZJUT_China_B)

Team

[Team Members](https://2020.igem.org/Team%3AZJUT_China_B/Team)
[Attributions](https://2020.igem.org/Team%3AZJUT_China_B/Attributions)

Project

[Description](https://2020.igem.org/Team%3AZJUT_China_B/Description)
[Design](https://2020.igem.org/Team%3AZJUT_China_B/Design)
[Implementation](https://2020.igem.org/Team%3AZJUT_China_B/Implementation)
[Contribution](https://2020.igem.org/Team%3AZJUT_China_B/Contribution)
[Engineering](https://2020.igem.org/Team%3AZJUT_China_B/Engineering)
[Proof Of Concept](https://2020.igem.org/Team%3AZJUT_China_B/Proof%20Of%20Concept)
[Results](https://2020.igem.org/Team%3AZJUT_China_B/Results)

Wetlab

[Protocols](https://2020.igem.org/Team%3AZJUT_China_B/Protocol)
[Notebook](https://2020.igem.org/Team%3AZJUT_China_B/Notebook)
[Parts](https://2020.igem.org/Team%3AZJUT_China_B/Parts)
[Wet collaboration](https://2020.igem.org/Team%3AZJUT_China_B/Wet%20collaboration)

HP

[Overview](https://2020.igem.org/Team%3AZJUT_China_B/Human%20Practices)
[Silver](https://2020.igem.org/Team%3AZJUT_China_B/HP-Silver)
[Gold](https://2020.igem.org/Team%3AZJUT_China_B/Integrated%20human%20practice)
[Science communication](https://2020.igem.org/Team%3AZJUT_China_B/Education)
[Collaborations](https://2020.igem.org/Team%3AZJUT_China_B/Collaborations)
[Partnership](https://2020.igem.org/Team%3AZJUT_China_B/Partnership)
[Ethics](https://2020.igem.org/Team%3AZJUT_China_B/Ethics)

Modeling

[Software](https://2020.igem.org/Team%3AZJUT_China_B/Software)
[Modeling](https://2020.igem.org/Team%3AZJUT_China_B/Model)

Hardware

[Overview](https://2020.igem.org/Team%3AZJUT_China_B/Hardware)
[Detecting](https://2020.igem.org/Team%3AZJUT_China_B/Detecting%20)
[Sample processing](https://2020.igem.org/Team%3AZJUT_China_B/Sample%20processing)
[Chip](https://2020.igem.org/Team%3AZJUT_China_B/Chip)
[History](https://2020.igem.org/Team%3AZJUT_China_B/History)

[Safety](https://2020.igem.org/Team%3AZJUT_China_B/Safety)

Judging

[Poster](https://2020.igem.org/Team%3AZJUT_China_B/Poster)
[Judging Form](https://2020.igem.org/Team%3AZJUT_China_B/Judging%20Form)

[![](https://static.igem.org/mediawiki/2020/5/5d/T--ZJUT_China_B--logo-Cas.png)](https://igem2020-zjut.feishu.cn/docs/doccnWz3kWJdTESBOvIunDsW2ac?from=from_copylink)

## **Results**

* [Sample processing](#C1)
* [Cloning](#C2)
* [Cas13 protein homologues expression and purification](#C3)
* [Fluorescence detecting](#C4)
* [Testing waste recovery](#C5)

# Sample processing

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F5%2F58%2FT--ZJUT_China_B--Table_1_Concentration_of_H1N1_virus_target_sequence.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

**Result:**

To simulate the state in which the viral nucleic acid is wrapped in a protein shell under real conditions, a plasmid with a viral target sequence was constructed and introduced into *E. coli* BL21. Then the recombinant *E. coli* was cultivated and induced to transcribe the target RNA. The resulting cells were used to simulate pathogens for subsequent amplification experiments. It provides an important reference for patient sample detection with the device.

The results were summarized in Table 1. The data was originally intended to be used for modeling, but accurate data could not be obtained due to the instrument and operation reasons. Therefore, the modeling failed, and the experiment can be improved after subsequent improvements.

# Cloning

**PCR amplification and purification of His-SUMO tag**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F6%2F67%2FT--ZJUT_China_B--Result-Cloning-Figure_1.jpg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 1. The agarose gel electrophoresis of His-SUMO tag.

**Note:**

Lane M：Marker DL2000

Lane 1 and Lane 2：His-SUMO tag1. PsmCas13b

**Result:**

As shown in Figure 1，in the range of 250-500 bp, there was a bright band in the product, which demonstrated that the His-SUMO fragment was amplified successfully. The His-SUMO tag concentration was shown in Figure 2.

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Fc%2Fcf%2FT--ZJUT_China_B--Result-Cloning-Figure_2.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 2. His-SUMO tag concentration.

**1. PsmCas13b**

**Linear amplification and purification of Psm plasmid**

![](https://static.igem.org/mediawiki/2020/0/05/T--ZJUT_China_B--Result-Cloning-Figure_3.png)

Figure 3. The agarose gel electrophoresis of Psm plasmid.

**Note:**

Lane M：Marker 1kb plus

Lane 1 and Lane 2：Psm plasmid

**Result:**

As shown in Figure 3，there were bright bands in the range of 8000 to 10000 bp, so the Psm plasmid was linearized and amplified successfully. The Psm plasmid concentration was shown in Figure 4.

![](https://static.igem.org/mediawiki/2020/6/6b/T--ZJUT_China_B--Result-Cloning-Figure_4.jpeg)

Figure 4. Psm plasmid concentration

**The recombinant plasmid His-SUMO-cas13b-Psm was transferred into DH5a**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F9%2F91%2FT--ZJUT_China_B--Result-Cloning-Figure_5.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 5. The transformants of recombinant plasmid His-SUMO-cas13b-Psm

**Result:**

As shown in Figure 5, transformant colonies were found in the Ampicillin resistant medium.

**Identification of recombinant plasmid SUMO-cas13a-Psm by colony PCR**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Fe%2Fe3%2FT--ZJUT_China_B--Result-Cloning-Figure_6.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 6. The agarose gel electrophoresis of His-SUMO-cas13b-Psm transformants.

**Note:**

Lane M：Marker 1kb plus

Lane 1 to Lane 10：His-SUMO-cas13b-Psm transformants

**Result：**

As shown in Figure 6, among the selected single colonies, only the first, second, sixth, eighth and tenth single colonies had bright bands in the range of 8000 to 10000 bp, which demonstrated that the recombinant plasmid was successfully transformed.

**2. CcaCas13b**

**Linear amplification and purification of Cca plasmid**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F2%2F2b%2FT--ZJUT_China_B--Result-Cloning-Figure_7.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 7. The gel electrophoresis of Cca plasmid.

**Note:**

Lane M：Marker 1kb plus

Lane 1 to Lane 4：Cca plasmid

**Result:**

As shown in Figure 7, there were bright bands in Lane 1 and Lane 4 in the range of 8000 to 10000 bp, which demonstrated that the Cca plasmid was linearized and amplified successfully. The Cca plasmid concentration was shown in Figure 8.

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F2%2F2a%2FT--ZJUT_China_B--Result-Cloning-Figure_8.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 8. Cca plasmid concentration

**The recombinant plasmid His-SUMO-CcaCas13b was transferred into *E. coli* DH5a**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Fe%2Fe4%2FT--ZJUT_China_B--Result-Cloning-Figure_9.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 9. The transformants of recombinant plasmid His-SUMO-Cas13b-Cca

**Result：**

As shown in Figure 9, transformant colonies were found in Kanamycin resistant medium.

**Identification of recombinant plasmid SUMO-CcaCas13b by colony PCR**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Ff%2Ffc%2FT--ZJUT_China_B--Result-Cloning-Figure_10.jpeg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 10. The agarose gel electrophoresis of His-SUMO-cas13b-Cca transformants.

**Note:**

Lane M：Marker 1kb plus

Lane 1 to Lane 12：His-SUMO-cas13b-Cca transformants

**Result:**

As shown in Figure 10, among the selected single colonies, only the second and tenth single colonies had bright bands in the range of 250 to 500 bp, which demonstrated that the recombinant plasmid was successfully transformed.

# Cas13 protein homologues expression and purification

**PsmCas13b**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F8%2F89%2FT--ZJUT_China_B--Result-Cloning-Figure_11.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 11. The agarose gel electrophoresis of PsmCas13b purification expression

Lane M：Protein Marker

Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose

Lane 10：10 mM imidazole eluted protein flow through

Lane 50：50 mM imidazole eluted protein flow through

Lane 250-1 to 250-7：250 mM imidazole first to seventh eluted protein flow-through

Lane 500：500 mM imidazole eluted protein flow through

**LbaCas13a**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F3%2F3e%2FT--ZJUT_China_B--Result-Cloning-Figure_12.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 12. The agarose gel electrophoresis of LbaCas13a purification expression

Lane M：Protein Marker

Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose

Lane 10：10 mM imidazole eluted protein flow through

Lane 50：50 mM imidazole eluted protein flow through

Lane 250-1 to 250-5：250 mM imidazole first to fifth eluted protein flow-through

Lane 500：500 mM imidazole eluted protein flow through

**CcaCas13b**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Fb%2Fb7%2FT--ZJUT_China_B--Result-Cloning-Figure_13.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 13. The agarose gel electrophoresis of CcaCas13b purification expression

Lane M：Protein Marker

Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose

Lane 10：10 mM imidazole eluted protein flow through

Lane 50：50 mM imidazole eluted protein flow through

Lane 250-1 to 250-7：250 mM imidazole first to seventh eluted protein flow-through

Lane 500：500 mM imidazole eluted protein flow through

**TDPs-33020**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Fd%2Fd1%2FT--ZJUT_China_B--Result-Cloning-Figure_14.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 14. The agarose gel electrophoresis of TDPs-33020 purification expression

Lane M：Protein Marker

Lane FT：Flow through liquid. After the cell lysate was incubated with Ni-NTA agarose

Lane 10：10 mM imidazole eluted protein flow through

Lane 50：50 mM imidazole eluted protein flow through

Lane 250-1 to 250-7：250 mM imidazole first to seventh eluted protein flow-through

Lane 500：500 mM imidazole eluted protein flow through

**Result：**

Final purification results of four proteins (PsmCas13b, LbaCas13a, CcaCas13b, and TDPs-33020) were shown in Figure 11 to Figure 14, respectively. Among them, PsmCas13b and LbaCas13b are not completely pure proteins, but they are functional and can be used for subsequent fluorescence detection. In addition, CcaCas13b and TDPs-33020 have not been purified to the functional proteins. The proteins were purified with Ni-NTA agarose following the experimental procedures in the literature. Due to the problems of phages and purification equipment, we spent a lot of time o n it in the early stages.

# Fluorescence detecting

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Ff%2Ff3%2FT--ZJUT_China_B--lwa_kinetic_curve.jpg&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 15. Cleavage activity of LwaCas13a protein

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F7%2F76%2FT--ZJUT_China_B--Result-Cloning-Figure_15.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 16. The cleavage activity of LbaCas13a protein

**Result:**

As shown in Figure 16, the activity of LbaCas13a protein is very low. Meanwhile, it seems that the activity of the expressed LbaCas13a protein was independent of the presence or absence of the target sequence. We speculate that the reason is that the soluble tag was not removed. Because the MBP soluble tag is very large, and it may be in the active center of the protein, which may affect the activity of the protein. Moreover, the inaccuracy of protein concentration data may also lead to the low activity of LbaCas13a protein.

We tested the detection limit of LwaCas13a protein (Figure 17). As shown in Figure 17, the LwaCas13a has a sensitivity of 10^- 13, which is quite sensitive.

**Detection limit of LwaCas13a protein**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2F7%2F7b%2FT--ZJUT_China_B--Result-Cloning-Figure_16.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 17. The limitation of LwaCas13a protein

**Lyophilization**

![](https://security.feishu.cn/link/safety?target=https%3A%2F%2F2020.igem.org%2Fwiki%2Fimages%2Ff%2Fff%2FT--ZJUT_China_B--Result-Cloning-Figure_17.png&scene=ccm&logParams=%7B%22location%22%3A%22docs_sdk_comment%22%7D&lang=zh-CN)

Figure 18. The cleavage activity of original and lyophilized LwaCas13a protein

**Result:**

The effect of lyophilization on the LwaCas13a was investigated (Figure 18). As shown in Figure 18, lyophilization can cause a notable loss of LwaCas13a. This will deeply influence the results. Therefore, the protein lyophilization should be optimized. To reduce the influence of lyophilization on protein activity, we plan to add tardigrade intrinsically disordered proteins (TDPs)to lyophilized detecting mix. However, due to the limited time, we did not complete this part before the deadline, so we listed it in our future steps.

# Testing waste recovery

Our project aims to achieve simultaneous detection of multiple viruses. In order to realize our idea, we designed an efficient portable virus detection device based on CRISPR/Cas13 technology that can detect multiple viruses simultaneously.

The device is divided into the reactor part and the detector part. Since the biochemical reagents are sealed in both parts, we plan to recover the used reactor part, disposable gloves and swabs to ensure that the product will not be littered or abused after use. These issues are related to the recycling of medical waste. Since our knowledge in this area is limited, we interviewed the person in charge of Hangzhou Ecology and Environment Bureau to make our project more perfect and safe. Before the investigation and interview, we explained that the project device had not been put into actual production, and the research content was only for communication and learning.

1.Through the interview, we learned about the importance of medical waste recycling. The medical waste can expose medical staff, testers, waste handlers, patients and entire communities to infection and injury, as well as the risk of environmental pollution. Therefore, there are strict qualification requirements for medical waste recycling institutions, which include capital, technology, location, equipments and other requirements.

2.Since domestic medical devices have not been widely used, we learn that there is no domestic medical waste recycling system and relevant policies/regulations in China, which is a blind spot that can be easily ignored. Meanwhile, we also need to arouse people's awareness of the importance of medical waste disposal and follow the correct treatment methods.

3.Respondents advised us to contact a professional medical waste disposal company to solve the recycling and disposal problems. Medical waste recycling begins with the packing, labeling, and storage of the wastes by medical treatment units. Then the wastes were collected from temporary storage centers and transported to the qualified treatment centers. The waste was further treated in the centers.

4.In view of the low toxicity of biochemical reagents in our device to human body and the low content of samples in the reactor part, the interviewees indicated that the waste could be treated as general waste by high-temperature disinfection.

Finally, we learned that the following issues should be paid attention to when recycling waste from devices like ours:

1.Do not take it directly by hand;

2.Strictly mark warning signs and seal them in time;

3.Conduct chemical disinfection treatment or multi-layer package and then collect and treat as infectious waste;

4.The waste recovery and transportation process must be managed by a unified and professional enterprise/government agency.

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