iGEM 2019 — Team Tsinghua

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# Team:Tsinghua/Notebook

iGEM Tsinghua

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[iGEM
Tsinghua](https://2019.igem.org/Team%3ATsinghua)

* [Home](https://2019.igem.org/Team%3ATsinghua)
* Team
  + [Members](https://2019.igem.org/Team%3ATsinghua/Team)
  + [Collaborations](https://2019.igem.org/Team%3ATsinghua/Collaborations)
  + [Notebook](https://2019.igem.org/Team%3ATsinghua/Notebook)
  + [Attributions](https://2019.igem.org/Team%3ATsinghua/Attributions)
  + [Design](https://2019.igem.org/Team%3ATsinghua/Design)
* Project
  + [Description](https://2019.igem.org/Team%3ATsinghua/Description)
  + [Experiments](https://2019.igem.org/Team%3ATsinghua/Experiments)
  + [Results](https://2019.igem.org/Team%3ATsinghua/Demonstrate)
  + [Measurement](https://2019.igem.org/Team%3ATsinghua/Measurement)
* Model
  + [Part I](https://2019.igem.org/Team%3ATsinghua/Model)
  + [Part II](https://2019.igem.org/Team%3ATsinghua/Model2)
  + [Part III](https://2019.igem.org/Team%3ATsinghua/Model3)
* Parts
  + [Improvement](https://2019.igem.org/Team%3ATsinghua/Improvement)
  + [Characterization](https://2019.igem.org/Team%3ATsinghua/Characterization)
  + [Chart Form](https://2019.igem.org/Team%3ATsinghua/Chart)
* Human Practices
  + [General](https://2019.igem.org/Team%3ATsinghua/Human_Practices)
  + [Integrated](https://2019.igem.org/Team%3ATsinghua/Integrated)
  + [Safety](https://2019.igem.org/Team%3ATsinghua/Safety)

# Notebook

### June 28th

![](https://static.igem.org/mediawiki/2019/2/2c/T--Tsinghua--nb10.jpg)

A little struggle during these days. We had very fancy ideas and constructed a great system, but there was almost no existing biochemical reactions that could be test in LLPS. In order to find more information, we carried out a lot of talks with LLPS experts since then.
We went to Pilong Li's Lab and met Liang Wang, who was an expert in LLPS. He was very interested in the LLPS phenomenon happed in bacteria and wondered whether the phenomenon of forming phase at both end of a bacteria could mean something. He also suggested us to utilize some critical biochemical reactions that naturally happen in E. coli, like DNA replication and double strand isomerization.

### June 29th

Zikang Huang, Chunyu Chen, meng Fang, Ji Gao went to Peking University to meet iGEMer from Peking 2018. He told us about the three classical experiments to verify liquid liquid phase separation, as well as the criteria of choosing a biochemical reaction whose efficient could be modified by LLPS.

Zikang Huang talked with Pilong Li online, using wechat. Dr. Li also showed great interest in our program and promise to offer more help in the next few weeks. He mentioned a powerful phase separation system composed of PixE and PixD, which would sense light by themselves and dissociate when being stimulated. Furthermore, a two-step luciferase reaction was proposed by him.

### June 30th

![](https://static.igem.org/mediawiki/2019/0/0c/T--Tsinghua--nb15.jpg)

We have the last lab meeting today evening. We confirm the last version of working time in summer vacation. What's more, the rough schedule of wet experiment, modelling, wiki design and hardware design in summer are outlined. We still do not have much idea of HP.

### July 1st

Summer begins!
Zikang Huang designed subcloning process, constructing vectors using FUS as phase separation element induced by light. Everyone in the group started to search for papers utilizing luciferase assay to prepare for our enzymatic reaction.

### July 2nd

![](https://static.igem.org/mediawiki/2019/0/0b/T--Tsinghua--nb11.jpg)

Zikang Huang designed the primer for PCR of 3HSB, FUS, Cry-2.
talk with Min Zhou in Pilong Li's lab. Firstly try one-step luciferase.

### July 3rd

Zikang Huang did PCR for 3HSB, FUS, Cry-2, as well as retried the ligation of 3HSB and (SIM-SUMO)3.
The result of electrophoresis seemed normal, and we expected the success of this cloning!
Also, Zikang Huang talked to Ru Li in Pilong Li's lab, who shared her result of light induced phase separation system working in mamalian cells. Zikang Huang got the information of PixLL system from her, which was a fancy system sensitive to light without coupled to light element.
A lot of luciferase paper had been collected by the great team members, the luciferase assay seemed more realistic to us than before!
Modeling by Meng FANG
• A model based on three-dimensional grid has been initially established.
• Different types of protein particle movement has been modeled.

### July 4th

![](https://static.igem.org/mediawiki/2019/d/da/T--Tsinghua--nb14.jpg)

Zikang Huang did gibson assembly of both FUS, Cry2 and CIB1 it self, using HiFi from NEB. Then the products are transformed to DH5alpha. The latter one managed to form colonies. However, the first one failed. 106-(s-s)3-2 has been amplified, which was the only vector contained (s-s)3 and correct promoter.
Zikang Huang went to Pilong Li's lab and get PixE and PixD back, which were special light element coupled to FUS, dissociated when stimulated with light and aggerated in dark.
All team members at school met with two of our advisors Dr. Chen and Dr. Sun. Zikang Huang showed the results and plan for wet lab experiments, as well as the brainstorming for possible hardware detecting light intensity. Meng, Fang showed the outline of modelling. Jiale, Fu presented the blueprint of wiki design. Chunyu, Chen exhibited HP plan. Zihan, Zhai integrated all contents into a powerpoint.

### July 5th

![](https://static.igem.org/mediawiki/2019/8/82/T--Tsinghua--nb7.jpg)

Modeling by Meng FANG
• The structure of a protein particle has been well-defined.
• Parameters to be determined are clarified.
• The brief idea of simulation algorithm is formed.
To-do list
• Draw figures to illustrate the idea more clearly. (already in the PPT, but some adjustment remains to be made)
• Write down the complete simulation algorithm and arbitrarily choose parameters to run it.
• Clarify the methods to determine the parameters from experimental data.
• To be continued...
PDF has been updated. Part 1.3, 2.1 are yet to be finished.

### July 6th

Zikang Huang did plasmid extraction of CIB1-3HSB, 106 (SIM-SUMO)3 expression 2, 106 (SIM-SUMO)3 expression 1-3HSB, which are verified by bacteria PCR. PixE, PixD are also extracted, but the PCR primers would arrive tomorrow. Though the positive bacteria colonies have been seen on all four plates of cry2-FUS ligation using two ligation strategy, ligation free kit and HiFi Assembly kit, and the bacteria were amplified in LB with ampicilin, bacteria PCR did not work for two times. Therefore, he designed new probes for this PCR and waited to do it again tomorrow.
Also, the ligation of 3HSB AscI PacI and 106 (SIM-SUMO)3 expression AscI PacI had been carried out again, since Chunyu Chen's ligation did not work yesterday.

### July 7th

![](https://static.igem.org/mediawiki/2019/e/eb/T--Tsinghua--nb2.jpg)

We had our first group meeting during summer vacation.
Huang summarized the progress of the experiment this week.
Chunyu Chen taught us how to use PowerPoint to record presetation. Everyone in the meeting presented papers they read about luciferase during this week.
Meng Fang talked about our basic ideas of modelling on phase separation. Sounds cool.
Chen ordered a huge pizza too, but it lost most of its yummy taste when it got cold. What a pity!
We had our first summer group meeting today, and most of team members were present. Except for the unqualified HiFi assembly result, the wet lab experiment went very well the first week. Meng Fang gave a brief overview of the model we were going to use. The model covered most process of phase separation. We were looking forward to the simulation of phase separation by this model. Also, we were eager to check out the simulation coupling phase separation and light induction as well as enzymatic reactions. All team members present gave a brief talk of their investigation of luciferase and phase separation element paper, which were very informative. We believed that through more discussion of these paper, we would found out more information about how we could involve luciferase assay and light induction in our phase separation system.
Zikang Huang verified the result of amplification of PixE and PixD, which were both positive. However the bacteria PCR did not work for verification of cry2-FUS ligated by HiFi assembly kit. This result was a little weird because there were a lot of positive bacteria colonies on the plate, which indicated that the ligation had a large possibility to succeed. So more colonies were amplified, and more verification would be done tommorow.

### July 8th

Zikang Huang PCRed HiFi assembly products and verified the success of this experiment. Well done! Up to the moment, FUS-cry2-CIB1 light induced phase separation system had been constructed. We could not wait to test its ability to react towards light.

### July 10th

Zikang Huang did PCR of PixD and PixE and digested them to prepare products for ligation.

### July 11st

The first microscopic observation in summer! Though the result might be a little disappointed, we still harvested a lot. The light induction did not work at all in this system. Trying to find out what happenned to our system, we noticed that the phase in today's result did not look as usual. They did not form spheres at the end of bacteria, instead of that, they were observed in different patterns, moon-like, ring-like, and so on. This may indicated the solid-like behaviour of them, which might explain for the phenomenon happened: without light induction, 3HSB-CIB1 already formed a green phase (coupled with GFP), and in most case, FUS (mcherry) could not be included in this phase. We did FRAP assay for the two phases, the green one did not recover at all but the red one, in a few phase-existing cases, show swift recover after bleaching.
We also got familiar with the manipulation of confocol system, with data analysis and so on.

### July 12nd

![](https://static.igem.org/mediawiki/2019/a/a4/T--Tsinghua--nb6.jpg)

We communicated our ideas and results with Pilong Li in the evenning, who was an expert in phase separation. I must say, he was so helpful. He gave comments about our first microscopic result in summer, and told us about the state of 3HSB-CIB1 in mamalian cells, which also had relatively strong background aggeragates. Therefore, he suggested other self-assembly proteins to establish a high local concentration of phase separation elements. Also, he proposed the possibility of using (SIM)3 and (SUMO)3 to couple with light induced elements. He said he would like to help us out with our dilemma of lacking elements but would leave the space of application to ourselves. What a respectful and helpful professor!
Zikang Huang rePCRed PixE and PixD and transferred them into competent cells.
Ji Gao tried add a MCS to 106-(SS)3-3HSB-lacO vector. Repeat sequence brought many unexpected trouble to PCR.
Transform new SS3 plasmid with MCS to DH5a anyway.

### July 13rd

No colonies on the plate of yesterday's transformed DH5a. Sad.
Modeling by Meng FANG:
• Finished the coding of the models of protein particles, grids and protein movement in Python.
• Plan to give the code for simulation tomorrow.
Modeling by Bowen CHEN:
Start to seek for methods of visualization.

### July 14th

Zikang Huang and Bowen Chen went to Peking University to meet the team members of Peking iGEM 2018, who were also interested in using phase to control reactions. They talked about their modelling process to us, which was helpful. Since not much information about parameters can be acquired from experiments, to provide insight for experiment was the main goal of our modelling work. Although we could not determine the detailed state of in vivo reactions, we could still verify the higher efficiency of controlling two-step reactions than the one-step reaction, and the distribution of substrate matters a lot in this process. However, there were certainly a lot of puzzles remained there to be solved, like how to make hypothesis about quick response and rather slow response, and whether it was a must to consider the diameter about our proteins toward substrate. Also, some issue about wet experiments were discussed. A very important thing to know was that our work should mainly focus on the proposed function of this system, rather than clarify all characteristics about this system.
I must say, they were very helpful! They told us what they know and gave us some useful suggestions. I am looking forward to talk with them again.
The ligation of PixD and PixE finally worked today, but still need to be proved.
According to the sequencing results, only 100 (SIM-SUMO)3 plasmid contain correct sequence. So Ji did PCR of plasmid 100. Also increased template concentration in PCR since Huang suggested that more DNA is need in transformation.
Modeling by Meng FANG:
• Simulation code done, but it runs a little bit slow. I'll try to look for ways to accelerate it, but it won't be the main job in the following days.
• Oh, I just forgot the velocity decay and random increment! I'll add them to my code tomorrow.
• Parameter adjustment and taking the enzyme into consideration will be put on the agenda.

### July 15th

Modeling by Meng FANG:
• The velocity decay and random increment have been added to the code.
Ji and Zikang used confocal to examine the inducing condition of Cry2-FUS/CIB1-3HSB and Cry2/FUS-CIB1-3HSB. No good results but we learned a lot about how to operate the heavenly expensive microscope.

### July 16th

\* PCR of FusePixD, FusePixE and LacO-Amp
\* Design Hifi assmembly primers

### July 17th

PCR of 3HSB-GFP. We want to see if solely 3HSB can induce some kind of phase separation and confirm our previous imaging results.
PCR of Hifi assembly fragments of FUS-FusionRed-PixD and FUS-Citrion-PixE.
Hifi assembly above and transformation into DH5a.
Bowen who did the modeling work before come to help wet experiment today!
Our 2018-member and 2019-advisor Bobo, who is Bowen's roommate too, got sick last night. We hope everyone could just take care of themselves and stay healthy in such a lovely vacation…

### July 18th

Extract FUS-FusionRed-PixD. We hope we can perform microscopy examination of this system in vivo on Firday!
Plate of FUS-Citrion-PixE showed no colonies. Maybe it is the template. PCR again and tried again today.
PCR of FUS-FusionRed-PixD colonies proved its intergrity.
PCR of pmGFP to test all oligomerizing tags we have, like 3HSB.
PCR of new oligomerizing tags we got from Prof. Li's lab. Problem in primer design… Try again tomorrow.
Can we get a red light filter or something so that we will not have to prepare our samples for microscopy in the dark…? Mr. Peng Li said we might get a red lamp.
Get rid of contaminated garbages in the lab. These things need to be done in a proper way. Thank Chunyu!

### July 19th

![](https://static.igem.org/mediawiki/2019/0/04/T--Tsinghua--nb8.jpg)

Extract FUS-FusionRed-PixD and FUS-Citrion-PixE plasmids from DH5a and transform them into BL21 which can be induced by IPTG. We hope we can perform microscopy examination of this system in vivo on Firday!
Plate of FUS-Citrion-PixE showed no colonies. Maybe it is the template. PCR again and tried again today.
PCR of FUS-FusionRed-PixD colonies proved its intergrity.
PCR of pmGFP to test all oligomerizing tags we have, like 3HSB.
PCR of new oligomerizing tags we got from Prof. Li's lab. Problem in primer design… Try again tomorrow.
Can we get a red light filter or something so that we will not have to prepare our samples for microscopy in the dark…? Mr. Peng Li said we might get a red lamp.
Get rid of contaminated garbages in the lab. These things need to be done in a proper way. Thank Chunyu!

### July 20th

GCN4-4, GCN4-3 and li4k show colonies on the plates, which we pick and culture in liquid LB with kanamycin, but there is still no colonies on the plate of FUS-PixE-Citrion.

### July 21st

Ji designed a new hifi assembly approach for FUS-PixE-Citrion with backbone pRSF.
We had our 3rd group meeting in summer and discussed current problems and future plans. Frankly, we are now short of hands in wet experiment… Gladly, next week, Gengzhe, Zikang and Site will all come back to our lab, supposedly.

### July 22nd

We went to perform microscopy of Cry2-FUS/CIB1-3HSB and Cry2/FUS-CIB1-3HSB, trying to reproduce the good inducible result done by Hongrui in May. Sadly, it proved difficult.
Ji did redesigned hifi assembly of PixELL system. We could see it under microscope in Thursday if we are lucky.
We measured the growth curve of BL21 under 37 celsius, 220 rpm, which will help us determine the best time to add IPTG for inducing.

### July 23rd

![](
https://static.igem.org/mediawiki/2019/3/31/T--Tsinghua--nb9.jpg)

Modeling by Meng FANG:
• Light-induced conformational changes, simulated FRAP experiments, and visualizations are being added to the code. The idea is simple, but it's horrible to change the code. It takes time to find out how to use various Python packages. In short, I have to take it slowly.
• Some parameters may be handed over to the neural network for training and learning. This is a crazy idea that only stays in my mind. If I work fast enough these days, I might try it. Maybe it is quite cool.
Gladly, the PixE plate showed many colonies. Ji picked one of them and cultured in rotater, and PCR to identifiy its fuse protein sequence. It seems right and the plasmid was extracted.
However, when Ji tried cotransforming two plasmids of PixD and PixE, he chose the wrong antibiotic plate, and an evening came to waste…
The first day for Feng Site to work in the lab during the vacation! To begin with, the previously failed transformation of pRSF-li4k-GFP was repeated, as well as the transformed GCN4-3 and GCN4-4 was conformed by PCR

### July 24th

Modeling by Meng FANG:
I made up some code today and made the visualization. Although since there are still some important parts that have not been written, the results obtained may not be correct, the results are still somewhat confusing. Over time, proteins seem to like moving toward a wall of the cube, and I don't know why. I should be able to complete the key code tomorrow, and see if this will go on happening. If so, I have to do some investigation.
In short, the results are horrible, and I will continue debugging tomorrow.
Ji finished cotransforming PixELL plasmids to BL21 strain in the morning. And our ordered red light finally came. Let's prepare for the next day's microscopy.
Site begin to construct vectors containing GCN4-3, GCN4-4 or li4k. First, Vectors and inserts for Hifi assembly are amplified. However, unexpected electrophoretic band appeared when creating hifi parts.

### July 25th

Chunyu, Site and Ji went to microscopy examination of the newly designed PixELL system, unfortunately, the microscope we used before did not support the 450nm light we need for PixELL stimulation… We may find some microscope else that meets our need for this system.
Modeling by Meng FANG:
Modules simulating intramolecular interactions and FRAP experiments have been built. But the bug in the boundary condition setting drives me crazy. Still have to continue debugging.
Above is the scene after 1000 iterations of 1000 homologous protein particles. They still like to gather toward the boundary instead of show phase separation inside. It has been basically confirmed that this is because that there is a bug in the setting of the boundary conditions, but it is really a headache to find out what kind of bug it is.
Above is the scene after 1000 iterations of 500 yellow particles (representing the PixELL parts in light) and 500 green particles (representing the PixELL parts in dark). The green ones do not show phase separation as expected.
Another strange thing is that after adding the module for protein interaction, the program runs extremely slow. I will see if any constant optimization can be done.
Site tried to hifi assemble. As the fragments purified by gel extraction was low, she worried if the assembly can work.

### July 26th

odeling by Meng FANG:
I made a video to show the process of protein particle movement. It now seems that the problem is the parameter setting is improper.
The hifi assembly was not successful. Only GCN4-4 received a positive result. Site prepared hifi assembly fragments for pRSF-li4k/GCN4-4/GCN4-3-GFP-CIB1 for the second time.

### July 27th

![](https://static.igem.org/mediawiki/2019/6/68/T--Tsinghua--nb12.jpg
)

Site, Zikang and Ji did hifi assembly of the new oligomerizing tags to pRSF vector again.
Chunyu got the firefly luciferase DNA from iGEM parts and sent it to sequencing.
Zikang got the Renilla luciferase gene from teacher in our school.
Hifi assembly for the second time did not succeed either, Sad! Zikang pointed out that the ratio of fragments utilized in the previous assembly was unresonable. Thus, the third hifi assembly was implemented using recalculated parameters.

### July 28th

We had our 4th group meeting in summer, in which we summarized the week's work and plan for the coming weeks. Meng introduced his tryouts in modeling these days. And we plan to examine our PixELL system and oligomerizing tags under microscopy next day, with otpimized condition.
The third assembly failed again, so we had to repeat the experiment one more time. Fortunately, the fourth assembly seemed to be succeed, for the E.coli appears on the plate in 12h
After the 4th group meeting, we started to prepare for the next microscopy experiment.
We should have prepared for it earlier! cry2-fus system, cry2 system and li4k system had not been transformed into fresh BL21 yet, which would impede our doing microscopy. We should start to prepare those material 2 days before next microscopy experiment.
In this group meeting, though we still do not have exciting wet lab results, we started to try multiple strategies. We have two kinds of phase separating elements, five kinds of self aggaregating elements, three kinds of luciferase systems. Hope one or more of them would work some day!
Although there are still some bugs in modelling process, we can see the improvement in every modification. Hope wet lab experiments would catch up with the stimulation of phase separation process and then start to check out enzymatic reactions together.

### July 29th

Genzhe Lu:
1. Circular PCR for MCS insertion in 1Xsim-sumo plasmid. Failed. I'll design new primers for this work.
2. Restriction enzyme cleavage: mutant-(ss)3 plasmid with NcoI and AvrII, synthetic gene plasmid with the same enzymes. And did ligation of synthetic gene 3Xsumo and plasmid in order to generate redundant sumo sites. The gel pattern is strange. Refer to the data.
Zikang, Site, Chunyu and Ji went to perform microscopy. We found it still hard to find the correct focus plane and it took us a lot of time. Generally, we got some data, but it was below our expectation. What's worse, it was raining cats and dogs when the experiment ended and we did not bring any umbrellas. It is very kind of Chunyu for running in the rain and fetching umbrellas for us.

### July 30th

Genzhe: Circular PCR for MCS insertion in 1Xsim-sumo plasmid. Failed. I'll design new primers for this work.
Restriction enzyme cleavage: mutant-(ss)3 plasmid with NcoI and AvrII, synthetic gene plasmid with the same enzymes. And did ligation of synthetic gene 3Xsumo and plasmid in order to generate redundant sumo sites. The gel pattern is strange. Refer to the data.
Zikang Huang found lux part in 2019 distribution and transformed it into DH5alpha.
He also found renilla luciferase gene from inter-lab communication and designed HiFi strategy to do subcloning to make its expression compatible in E.coli.

### July 31st

Genzhe extracted plasmid: Mut-(SS)3-3Xsumo.

### August 1st

Zikang Huang did HiFi assembly to get renilla luciferase on the right vector.
Also, he amplified CIB1-3HSB-GFP pRSF.
Though iGEM provided the part sequence of lux operon and the backbone sequence of pSB1C3, the exact linker between them was unknown. Therefore, after plasmid extraction, Zikang Huang send the original plasmid to do sequencing.

### August 6th

Zikang Huang Jiale Fu and Lize Sun went to BIT to attend a meeting
Tend to use traditional method to clone CIB1-li4k/GCN4-3/GCN4-4
Induce expression of CIB1-li4k-GFP/CIB1-li4k-GFP&mcherry-cry2-FUS/CIB1-GCN4-3-FUS-GFP&mcherry-cry2/CIB1-GCN4-4-FUS-GFP&mcherry-cry2/Pixell/CIB1-li4k-FUS-GFP&mcherry-cry2/CIB1-3HSB-FUS-GFP&mcherry-cry2,with 0.5 mM 10h

### August 7th

Modeling by Meng FANG:
Good news from modeling part 1: phase separation image effects have been made!
The mathematical outline of part 2 have been written. Still working on the details.
This time on confocal get some results, which will be reconfirmed on Saturday
Doing bacteria PCR to find CIB1-GCN4-3-GFP and CIB1-GCN4-4-GFP is constructed successfully

### August 8th

![](https://static.igem.org/mediawiki/2019/9/91/T--Tsinghua--nb5.jpg)

CIB1-li4k/GCN4-3/GCN4-4-GFP are extracted and transformed into BL21. Also bacteria contains CIB1-li4k-GFP/CIB1-li4k-GFP& mcherry-cry2-FUS/CIB1-GCN4-3-FUS-GFP & mcherry-cry2/CIB1-GCN4-4-FUS-GFP& mcherry-cry2/Pixell/CIB1-li4k-FUS-GFP & mcherry-cry2 are cultured.

### August 9th

Induce the expression of bacteria cultured in 8.08 and find bacteria contains CIB1-li4k/GCN4-3/GCN4-4-GFP& mcherry-cry2-FUS didn't grow.

### August 16th

Zikang, Lize, Zihan further confirmed the light stimulation of GCN4-4 and this is the third replicate. Cry-2 mcherry can be incorporated into the FUS-CIB1 phase quickly, not longer than 8 seconds, at which we captured the first picture after light stimulation. Also, we completed a few control groups to make our experiments more rigorous.

### August 17th

Our poster for ccic came out today, which surprised most of us. We must appreciate our designer of this poster, Zihan. It was amazing!

### August 18th

We had our 7th group meeting in summer vacation. Zikang summerized the finished work and pointed out the main tasks of this stage is to find the reactions that can elucidate our principle.

### August 19th

Ji and Genzhe arrived in Shenzhen for 6th Conference of China iGEMer Community (CCiC). We printed our poster and prepared our powerpoint for presentation. We visited Bluepha, which is a synthetic biotech company founded by former iGEMers. Its CEO, Dr Zhang shared his iGEM experience, entrepreneurship in biotech field and devotion in biology education.

### August 20th

CCiC formally opened today. We found it interesting to learn other Chinese iGEMers' project and listen to their presetation and debates with judges. Explaining our own project in front of our poster to a lot of other iGEMers proved to a tough work since everyone kept asking questions. Maybe there are too many words on our poster so that few people are willing to read them…

### August 21st

Zikang, Lize, Zihan saw the luminescence of lux and renilla luciferase today. The blue light emit from lux solution is really beautiful though rather weak. We are so happy that our first downstream reaction is going to be tested in the next few weeks. In the afternoon, advisor Xuan Wang taught us about how to use microplate reader in Guoqiang Chen's lab. Though with some fluctuation in results, we still verified that our parts can be used in E.coli, especially Rluc and lux.
The desserts they provided in Tea Rest of CCiC were very testy! Genzhe found project of NTHU iGEMers in Taiwan very appealing and hoped to collabrate with them. Sadly, these girls left by the sunset and Genzhe lost the chance to deeply communicate with them. One of the judges suggested that the title of our project 'Light controled phase separation' is somehow too abstract and said it would be better if we found a good downstream reaction and rename according to it.

### August 22nd

We gave our presetation on CCiC today. Generally it went well, and the judges gave many practical advices to our project. Basically, our story is very complete but more verification in experiment and modeling need to be done. And a good downstream reaction is necessary, which will be best if we find another group to colabrate in this part.

### August 23rd

Nothing happened today. No primers designed. No gel run. No codes debugged. No papers summarized. No microscope used. No photograph taken. No powerpoint made. No poster drawn. No group meeting holden. What a peaceful Friday.

### August 24th

![](https://static.igem.org/mediawiki/2019/9/95/T--Tsinghua--nb13.jpg)

Zikang designed hifi assembly primers for luxA and luxB system today. It seemed that he was in a haste, because the order form he sent to the synthesis company showed a date of 8.20, which is a cute little mistake that no one would notice unless he is desperately bored.

### August 25th

We had our 8th group meeting in summer. Work data of the past week was displayed. Genzhe and Ji reported feedback about our project from CCiC.

### August 26th

![](https://static.igem.org/mediawiki/2019/e/ea/T--Tsinghua--nb4.jpg)

Ji designed primers for hifi assembly of xylE gene (catechol dioxygenase) into cry2-mcherry element. Ji also transformed original xylE plasmid into TOP10 competent bacteria, in order to test its enzyme activity.

### August 27th

Zikang designed primers for hifi assembly of Rluc into cry2-mcherry element.
Ji tested transformed original xylE gene in TOP10 competent bacteria. The activity of enzyme proved to be very effective.

### August 29th

Zihan got into trouble about hifi assembly. At last, she found it was the primers designed by Lize that may cause all these ado. Now Zihan had to redesign the primers.
Cheng Li from Peking University has provided us with more insight about the application of phase separation. Since the distribution of phase in a cell is not uniform, while division, the filial cells may have different component in their cytoplasma. Furthermore, we found some phase separating protein can form aggregate only on one side of the cell which could fulfill the possibility of differentiation of E.coli.

### August 30th

Genzhe, Zikang and Ji went to the group meeting of Prof. Chen's lab and talked with PhD students there about our project. Xuan Wang and Jianwen Ye suggested us to use E. coli with polymorphism to look into the locating of phase in bacteria cell.
We got a new phase-separation element, Tudor(SMN) from Hongrui's lab, which has less tendency to form a new phase than FUS-LCD. We hope this element can help us to do the reversible-phase-separation thing, since the phase formed by FUS-LCD proved to be too stable to dissolve.

### September 1st

aft of our website, which was so cool that we can not wait to put something hardcore in it:)
Jiayi introduced her plan for human practice, and some of the ideas seems really interesting and meaningful.
Ji co-transformed a pack of plasmids, mainly Rluc, xylE and Tudor, which were supposed to be checked under microscope in Wednesday.
Our brand-new autoclave beeped error twice today and we could not find out the reason. Wonder why the administrator made the good old one retired.

### September 3rd

Zikang and Ji measured enzyme activity of Renilla luciferase fusion protein by microplate reader in Prof. Chen's lab.
Zikang came into bug with PCR again, and he had his python project work to hand in the next day… God bless him.
Renilla luciferase does not work well if fused with cry2-mcherry. How to improve the performance of this fused protein is still a question.
Genzhe, Ji and Hongrui went microscopy today.(Zikang had to fight with his python homework) We checked out a lot of new things, like FUS phase in vitro (bacteria cell lysis), phase-element Tudor and Renilla luciferase fused phase element.
Generally, we had not observed a single successful FRAP experiment today… And the light induction showed weird results, too.
Maybe we should take Lize with us to microscopy next time, since it was she who first got the reproducible light induction result after so many failures of others…?

### September 8th

Zikang tried to do PCR of dcas9 plasmid from the iGEM group last year, but failed, presumbly because repeated sequence.
In fact, they had used restrictive enzyme a lot last year, but we mainly used hifi assembly this year…
Hongrui, Zikang, Genzhe and Ji went microscopy today. The software had been updated and some problem occurred so considerable time was wasted.
Generally, we got some vague positive results of Tudor and fused xylE and a clear negative result of fused Rluc. Not bad, huh.
Ji also measured enzyme activity of fused xylE and fused Rluc by microplate reader, and it seemed that fused Rluc needed to be reconstructed.
Site tried to continue Zikang's work on dcas9 plasmid, but failed in PCR again.
Ji co-transformed cry2-mcherry-xylE and CIB1-GCN4-4-GFP, as a control group that does not form phase separation.
Zihan did PCR for SIM-SUMO oligomer construction.
Chuheng did PCR.
Ji extracted plasmid of cry2-mcherry

### September 10th

We had our first group meeting in autumn today.
Hongrui summarized results of microscopy this week.
Meng introduced his new progress in modeling recently.
Ji displayed data of fusion enzyme activity measurement.
And the new title, PhASE, has came out.
There is little time left until the deadline, so we'd better hurry up.
Site and Lize were assigned to work on improvement part. We targeted to Cry2, our most commomly used light controled part, aiming to decrease its ability to oligomerize
Site designed the primers of Cry2 improvement. There 2 sites to mutate, each sites has several ways to improve, so there are totally 5 pairs of primer. The plasmid she used was Cry2-mcherry-Fus
Another microscopy was performed today, by Genzhe, Ji, Zikang and Hongrui. Gladly, we found good results in light induction of fusion XylE and lux system.
Ji measured enzyme activity of fusion XylE with different IPTG induction concentration.
Site began to PCR. In the attempt, there are 2 kinds of mutations on the one mutation site, as well as 4 kinds on the other, thus there are 2\*4=8 patterns in total; Each experimental group need 2 framents, so there are totally 16 fragments to PCR. About half of the PCRs failed. So sad!

### September 12nd

![](https://static.igem.org/mediawiki/2019/a/ab/T--Tsinghua--nb3.jpg)

Ji prepared to repeat the measurement of XylE's enzyme activity tomorrow.
Zikang went to Peking University to discuss projects of both teams. Friends from Peking would help us do the micro-flow experiment to observe the asymmetric division of E.coli. Also, they discussed the influence of dcas9, binding on the target plasmid. Whether it would attenuate the transcription and replication of this plasmid is unknown. In addition, they found our phase separation system may be a counter of the number of genome in their engineered E.coli. This was the charm of collaboration and communication!
Site did Hifi experiments with the succeed fragments, and transformed it into DH5alpha.
For the failed fragment, Site tried for the second time, and a few more parts came out. However, there are still some fragments which did not successfully amplified.

### September 15th

Ji repeated measuring enzyme activity of fusion XylE with different IPTG induction concentration.
Site did Hifi experiments with the succeed fragments, and transformed it into DH5alpha.
For the failed fragment, Site tried for the third time, and a few more parts came out. However, there are still some fragments which did not successfully amplified. Through the communication with Zikang and Ji, Site discovered that the plasmid sequence map file is not correct, effecting her judgement in setting PCR perameters. Site was glad that she finally found out the problem.
The molecular cloning of dcas9 and sgDNA onto FUS and cry2 has been completed by Zikang. Also, the targeting plasmid containing CFP as a reporter gene and targeting sequence of sgRNA was finished. We are waiting for the results from Peking University and then we would observe this system under confocal.
Today was a big day for iGEMers, since a lot of things were frozen. Finally, our team members, safety form, track, title, abstract were all submitted. We are now on a team with a clear goal and would work even hard to prove our system in the next month!
Genzhe went microscopy tonight. In spite of a festival night when people are supposed to go moon-seeing, it was cloudy in Beijing tonight.
We had our 2nd group meeting in autumn.
New data of the week were presented, but little progress had been made.
We examined the medal critiria and allocated missions for everyone to work towards it.
Jiale made a little game for the Mid-Autumn Day (yesterday, an important Chinese traditional festival).
Chunyu helped us in a number of financial chores.
Site did Hifi experiments with the succeed fragments, and transformed it into DH5alpha.
For the failed fragment, Site tried for the fourth time. This time, she set longer elongtion time, and a few more parts came out. However, there are still some fragments which did not successfully amplified.

### September 21st

Site did Hifi experiments with the succeed fragments, and transformed it into DH5alpha.
Site finally worked out all the PCR fragments, and did Hifi experiment.
At the same time, the previous mutated groups are able to implement plasmid extraction, which serves for the transformation of BL21.
Site further dealed with the mutated bacteria, amplifying them.
Ji measured enzyme activity of fusion xylE under 20C.
An abundant bottles of bacteria were handled to be speculate under confocal. The improved-plasmids-transformed-bacteria did not present differences between wild type and mutated type.
Ji did PCR to mutate and weaken T7 promoter on our petL8 vector.
Lize handled IPTG induction of many many bottles of bacteria, which was truly a lot of work!
Measuring their OD, addition of IPTG, cover all bottles with silver paper, etc.
New plasmids without FUS are used to mutated in the same ways, in order to elimunate the effect of Fus in the aggregation. This time, PCR was successful!
Zikang, Lize, Genzhe, Ji went confocal microscopy.
Ji verified weaken T7 promoter by colony PCR and sequencing, and construct mcherry-xylE plasmid by Hifi assembly.
We had our 3rd group meeting in autumn.
Although not as expected, enzyme activity measurement data and microscopy result of xylE system could be explained with former assumption and modeling insight, in which improvement is still need.
Meng presented his modified plan for modeling.
Stymied by failure some time ago, Rluc system is ready to restart on Zikang's hand.
Jiale took photo for a lot of team members.

### September 26th

Ji co-transformed cry2-mcherry-xylE with weaken T7 promoter with CIB1-GCN4-4-FUS-GFP, and mcherry-xylE as control.
Site, Lize and Genzhe went to microscopy of improved cry2 element today, but there seemed no difference between aggregating condition of original and improved protein.
The sequencing results were sent to us. Surprisingly, only a few mutations appears to presented correctly. As we were running out of time, we decided to measure the bacteria
We run out of agarose…
Our team logo was finally finished designing by Zihan, which was so pretty. We cannot wait trying our team shirts on!
Previously, Rluc does not work if linked to cry2-mcherry. We decided to change the linker between Rluc and mcherry. We prolonged the soft linker and designed rigid linker. The two new fused protein came out today and hope they will work under microplate reader.
Both Rluc with soft and rigid linker got into phase in our system with 1mM IPTG induction. We would try lower concentration and shorter time course later.
We will observe the improved cry2, which would be more smear in dark in our expectation, under confocal microscope tomorrow. We hope the result would be better than that of Monday.

### September 30th

Zihan turned in our banner design, that was wonderful!
Ji measured enzyme activities of all constructed xylE types by microplate reader.
Zikang measured re-constructed enzyme activity of Rluc fusion protein, which proved to work, though worked with less activity compared to wild type Rluc. However, the results of experimental groups with different IPTG induction seemed a little weird.
We had our 4th group meeting in autumn.
Jiayi discussed human practice plan with us in detail.
Zikang, Lize, Hongrui and Genzhe went microscopy before and after the meeting.
Lize measured enzyme activity of firefly luciferase fusion protein by microplate, and found no activity.
Zikang, Lize, Site worked till mid night to complete today's moleculer cloning and induction of E.coli by IPTG. 25 groups of sample was really a lot of work! And more work would be done tomorrow under microscope. God knows how long it would take us! Anyway, wish a wonderful result!

### October 1st

It is National Day of China today. Genzhe and Hongrui watched Celebration Gala at Tian'anmen Square via smartphone while doing microscopy experiment. How touching!
Lize, Zikang and Ji found a new microplate reader since the former cannot be used during the golden week, but the new one was somehow difficult to operate. And we still cannot get a satisfying result of mcherry luminescence intensity measurement.
Site began to construct mCherry.
Still mystifying results from microplate reader. We suspect that our bacteria cells may lose too many plasmids.
We finally found why co-transformed bacteria keeping losing plasmids.
Genzhe messed up with the proper concentration of stock solution of antibiotics and Lize did, too, so these data concerned with the plate and culture were all strange…
To make up his fault, Genzhe made 100 new culture plates today.

### October 7th

![](https://static.igem.org/mediawiki/2019/5/50/T--Tsinghua--nb1.jpg)

Zihan shoot a fantastic video with candy and card paper to elucidate the idea of our project.
Ji tried measuring enzyme activity of all 4 types of xylE system from former liquid culture tubes but there was no reaction observed. Strange.
Zikang and Ji tried changing T7 promoter on pETL8 plasmid to Anderson promoter but failed in PCR.
Ji deleted cry2 domain from original cry2-mcherry-xylE vector, and tried deleting MBP element from fusion protein, hoping these could solve our current problem.
It is the end of National Day Golden Week today.
We had our 5th group meeting in autumn.

### October 11st

Bad news! We found that we run out of clz, substrate of Rluc. What was more, there would be three or more weeks for Invitrogen to pass a new tube of clz. We might have to draw conclusions based on the insufficient data obtained up to now.
Ji tried to delete MBP domain from xylE fusion protein and succeed in experimental group plasmid.
Zikang and Lize found there is still a tube of clz in the refrigerater…
Ji found it hard to get a good result from PCR aiming to replace T4 promoter for Anderson promoter.
That tube of clz had been used up loog ago…
Fortunately, a parcel of new clz has arrived, though it is not the same brand as before.
Zikang and Lize found it producing far less luminescence than the former one.
Lize helped Ji to PCR the hifi fragment for Anderson promoter replacement of xylE plasmids.
Ji co-transformed xylE experiment and control group with Anderson promoter.
Anderson promoter on xylE plasmid proved it can express the gene when induced with IPTG, and can almost repress the expression with lacO without IPTG. Thank Prof Anderson!

### October 16th

![](https://static.igem.org/mediawiki/2019/3/3e/T--Tsinghua--nb0.jpg
)

We had our third last group meeting today.
We generally focused on the website and the final judging form.
However, hopefully some more good data can be squeezed out of our downstream reactions.
Zikang finished his summary of results in Rluc downstream reaction.
Site, Genzhe and Hongrui went for microscopy to find a disappointment, where a lot of samples showed few bacteria cells.
Lize and Zikang tested Rluc with new Anderson promoter, and found it work well.
It is Final Judging Form deadline today.
Bowen contacted last year Tsinghua Team's leader Tianze for advices, and started uploading website to Wiki.

### October 20th

Site came to summarize her part of experiment.
Genzhe, Chuheng and Ji went microscopy, mainly with E.coli with fusion biliverdin reductase, but got few good images.
Zikang, Lize, Chuheng and Ji prepared the last set of experiment before the Wiki freeze. Behold! Here come our final data!!
We had our last group meeting before Wiki freeze.
Medal criteria are examined again, and everyone present their work in their final draft.
Bowen, Zihan and Jiale stayed up late for the website uploading. Let's hit the wire!!

#### Thanks for your support !

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# Team:Tsinghua/Experiments

iGEM Tsinghua

![](https://static.igem.org/mediawiki/2019/1/15/T--Tsinghua--loading.gif)

![](https://static.igem.org/mediawiki/2019/b/be/T--Tsinghua--logo.png)

[iGEM
Tsinghua](https://2019.igem.org/Team%3ATsinghua)

* [Home](https://2019.igem.org/Team%3ATsinghua)
* Team
  + [Members](https://2019.igem.org/Team%3ATsinghua/Team)
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* Project
  + [Description](https://2019.igem.org/Team%3ATsinghua/Description)
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* Model
  + [Part I](https://2019.igem.org/Team%3ATsinghua/Model)
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* Parts
  + [Improvement](https://2019.igem.org/Team%3ATsinghua/Improvement)
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* Human Practices
  + [General](https://2019.igem.org/Team%3ATsinghua/Human_Practices)
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  + [Safety](https://2019.igem.org/Team%3ATsinghua/Safety)

# Experiments / Protocols

### Protein expression in E. coli

We exploited E. coli BL21(DE3) cells to transiently express protein elements. The proteins of interest are cloned into IPTG-inducible systems under distinct promoters.

1. Inoculate a single colony in 3 mL LB medium, incubate at 37℃, 220 rpm overnight.

2. Inoculate 1 mL bacteria into 50 mL LB medium in bottle, incubate at 37 ℃, 220 rpm for about 2~5 h until O.D.600 reaches 0.6~1.0.

3. Add IPTG of final concentrations 10-3 to 2 mM according to different assays.

4. Express protein at 37℃, 220rpm for 2h or at 16℃, 220 rpm for 10h.

### Microscopy

We used NIKON A1 microscope with 100 × oil immersion lens for imaging. And the microscopic slides of bacteria samples were made for confocal imaging.

##### **Fluorescence recovery after photobleaching (FRAP).**

We photobleached green fluorescence puncta within E. coli with a 488 nm laser pulse (10%-20% intensity; dwell time 0.125-0.5 s). And we record the intensity change with time.

##### **Light induction.**

For light induction, we used a 488 nm laser pulse (2%-3% intensity; dwell time 8 s) to stimulate a specific region. We then recorded its change for 10-15 min after stimulation.

##### **3D imaging.**

For 3D imaging, the sample was scanned at every 10 μm focal plane within a range of 100 μm. Then the images of different focal planes were integrated by the software and a 3D image of the sample was generated.

### Rluc

Materials

##### › LB culture

##### › Coelenterazine (clz)

##### › dissolved in ethanol (10mM) or stay freeze-dried and store in -80C, heat before using if there is precipitate.

##### › Spontanously oxidize anyway.

##### › PBS

### Rluc

Procedure

1. Coelenterazine (clz, from Invitrogen) dissolved in ethanol (10mM) or stay freeze-dried and store in -80C, heat before using if there is precipitate.

2. Bacteria grown to an OD 0.6-0.8, and add IPTG. Shake for 4 h at 22℃ on a rotary shaker to an OD~1. The culture was washed twice with PBS (pH 6, slightly acidic to avoid oxidization of clz), adjusted to an OD of 0.4–0.5, and aliquoted 100ul/well into a black 96-well plate in triplicate (or 2ml in a cuvette).

3. Prior to each assay, a single aliquot of clz was thawed, diluted in PBS to a final concentration of 1 mM, and incubated in the dark at room temperature for a few minutes.

4. Clz was added to a final concentration of 30 μM to the PBS-washed cells, which were subsequently incubated in the dark for 30 min at room temperature to allow the luciferase-generated signal to stabilize (stir to make bubbles if in cuvette).

5. The total luminescence of each well was then measured every minute for a total of 5 min with an integration time of 1 sec using a Microplate Reader.

### Rluc

Microplate Reader measurement

1. Incubate samples to reach 37C.

2. First OD600 of the bacteria is measured to determine the amount.

3. Intensity of florescence is measured to see if Rluc is expressed.

4. Luminance of sample is measured 3 times to determine the background values of the samples.

5. Add substrates according to protocol.

6. Luminance of sample is measured 10 times to determine velocity of the reaction.

### Rluc

Data analysis

Average all luminance value(after addition of clz) and normalize the average to OD600 of the sample.

### HiFi DNA Assembly

[https://international.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix#](https://international.neb.com/products/e2621-nebuilder-hifi-dna-assembly-master-mix)

1.Set up the following reaction on ice:

|  | Recommended Amount of Fragments Used for Assembly | | |
| 2–3 Fragment Assembly\* | 4–6 Fragment Assembly\*\* | Positive Control✝ |
| Recommended DNA Molar Ratio | vector:insert = 1:2 | vector:insert = 1:1 |  |
| Total Amount of Fragments | 0.03–0.2 pmols\* | 0.2–0.5 pmols\*\* | 10 μl |
| X μl | X μl |
| NEBuilder  HiFi DNA Assembly Master Mix | 10 μl | 10 μl | 10 μl |
|
| Deionized H2O | 10-X μl | 10-X μl | 0 |
| Total Volume | 20 μl✝✝ | 20 μl✝✝ | 20 μl |
| \* | Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of inserts.Use 5 times more insert if size is less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. | | |
| \*\* | To achieve optimal assembly efficiency, design ≥ 20 bp overlap regions between each fragment with equimolarity (suggested: 0.05 pmol each). | | |
| † | Control reagents are provided for 5 experiments. | | |
| †† | If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix. |
|  |  |  |  |

2.Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.

Note: Extended incubation up to 60 minutes may help to improve assembly efficiency in some cases.

### Q5-PCR

[https://international.neb.com/products/m0492-q5-high-fidelity-2x-master-mix#](https://international.neb.com/products/m0492-q5-high-fidelity-2x-master-mix)

Reaction setup:We recommend assembling all reaction components on ice and quickly transferring the reactions to a thermocycler preheated to the denaturation temperature (98°C). All components should be mixed prior to use.

| Component | 25 µl Reaction | 50 µl Reaction | Final Concentration |
| Q5 High-Fidelity 2X Master Mix | 12.5 µl | 25 µl | 1X |
| 10 µM Forward Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| 10 µM Reverse Primer | 1.25 µl | 2.5 µl | 0.5 µM |
| Template DNA | variable | variable | < 1,000 ng |
| Nuclease-Free Water | to 25 µl | to 50 µl |  |
|  |  |  |  |

Notes: Gently mix the reaction. Collect all liquid to the bottom of the tube by a quick spin if necessary. Overlay the sample with mineral oil if using a PCR machine without a heated lid.

Transfer PCR tubes to a PCR machine and begin thermocycling.

Thermocycling Conditions for a Routine PCR:

| STEP | TEMP | TIME |
| Initial Denaturation | 98°C | 30 seconds |
| 25–35 Cycles | 98°C | 5–10 seconds |
| \*50–72°C | 10–30 seconds |
| 72°C | 20–30 seconds/kb |
| Final Extension | 72°C | 2 minutes |
| Hold | 4–10°C |  |
|  |  |  |

\*It depends on the sequence of primers.

General Guidelines for templates:

Use of high quality, purified DNA templates greatly enhances the success of PCR. Recommended amounts of DNA template for a 50 µl reaction are as follows:

| DNA | AMOUNT |
| DNA Genomic | 1 ng–1 µg |
| Plasmid or Viral | 1 pg–10 ng |
|  |  |

General Guidelines for primers:

Oligonucleotide primers are generally 20–40 nucleotides in length and ideally have a GC content of 40–60%. The best results are typically seen when using each primer at a final concentration of 0.5 µM in the reaction.

### Taq-PCR

<https://www.takarabio.com/products/pcr/pcr-master-mixes/routine/takara-taq-hs>

##### General reaction mixture for PCR (total 50 μl)

Premix Taq Hot Start Version＊ 25 μl

Template< 500ng

Primer 1 0.2 - 1.0 μM (final conc.)

Primer 2 0.2 - 1.0 μM (final conc.)

Sterile purified water up to 50 μl

＊ Please mix gently to be uniform and then use.

##### PCR conditions

This enzyme can be used in general PCR conditions, since the monoclonal antibody is denatured in the initial DNA-denaturation step. No need for a special step to denature the antibody to Taq DNA polymerase.

Note :
Denaturation condition varies depending on the thermal cycler and tubes used for PCR. The recommendtion is for 5 - 10 sec at 98℃ or 20 - 30 sec at 94℃.

### Competent Cell Information

[DH5-alpha](http://www.tiangen.com/?productShow/t1/6/id/160.html)

##### Features:

A strain commonly used for plasmid cloning. The product of the φ80lacZ△M15 gene is alpha complementary with the amino terminal of the beta-galactosylase encoded by the pUC vector, which can be used for blue-white spot screening. The mutation of recA1 and endA1 was beneficial to the stability of clone DNA and the extraction of high purity plasmid DNA.

##### Genotype:

F- φ80lacZ△M15△ (lacZYA-argF) U169

endA1 recA1hsdR17(rk-,mk+)

supE44λ- thi-1 gyrA96 relA1 phoA

### Competent Cell Information

[BL21](http://www.tiangen.com/?productShow/t1/6/id/162.html)

##### Features:

The strain used T7 RNA polymerase as the protein expression host of efficient exogenous gene expression system. Expression of the T7 phage RNA polymerase gene is controlled by the lambda phage DE3 region of the lacUV5 promoter, which is incorporated into the BL21 chromosome. The strain is suitable for the expression of non-toxic proteins.

##### Genotype:

F- ompT hsdSB(rB- mB-) gal dcm（DE3）

### Activity of biliverdin reductase (in vivo)

Materials

1)LB liquid medium

2)1 mM Biliverdin solution (in 100% methanol)

3)100 mM IPTG

![](https://static.igem.org/mediawiki/2019/3/3d/T--Tsinghua--protocol_qch.png)

### Activity of biliverdin reductase (in vivo)

Procedures

1)Culture the E. coli cells in 5ml LB liquid medium (37℃, 220 rpm) until OD600 of the culture reaches 0.6 ~ 0.8.

2)Add 25 μl IPTG (100 mM) to the cell culture.

3)Add 5 μl biliverdin (1 mM) to the cell culture. For control group, add biliverdin after step (5).

4)Culture at 22℃, 220 rpm for 3 h.

5)Centrifuge at 10,000 rpm for 1 min.

6)Turn on the microplate reader and computer. Run the software to set the protocol and plate layout.

7)Add 100 μl of supernatant to each well on the microplate.

8)Measure the absorbance of cell culture at 600 nm as the estimation of cell amounts.

9)Measure the absorbance of cell culture at 450 nm as the amount of the product, bilirubin.

### Measurement-of-xyle-catechol-2-3-dioxygenase

#### Thanks for your support !

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# Team:Tsinghua/Results

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)