iGEM 2018 — Team Tsinghua

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# Team:Tsinghua/Notebook

Neon Coli-Necessary Expression Only

[![](https://static.igem.org/mediawiki/2018/b/be/T--Tsinghua--logo.png)](#page-top "Neon Coli")

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  + [Description](https://2018.igem.org/Team%3ATsinghua/Description)
  + [Design](https://2018.igem.org/Team%3ATsinghua/Design)
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  + [Results](https://2018.igem.org/Team%3ATsinghua/Results)
  + [Model](https://2018.igem.org/Team%3ATsinghua/Model)
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  + [Attributions](https://2018.igem.org/Team%3ATsinghua/Attributions)
* Parts
  + [Basic Parts](https://2018.igem.org/Team%3ATsinghua/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ATsinghua/Composite_Part)
  + [Improve](https://2018.igem.org/Team%3ATsinghua/Improve)
* HP
  + [Overview](https://2018.igem.org/Team%3ATsinghua/Human_Practices)
  + [General HP](https://2018.igem.org/Team%3ATsinghua/GHP)
  + [Intergrated HP](https://2018.igem.org/Team%3ATsinghua/IHP)
  + [Safety](https://2018.igem.org/Team%3ATsinghua/Safety)
* [Collaboration](https://2018.igem.org/Team%3ATsinghua/Collaborations)

## Lab Notebook

###

#### Week1 20180325-20180331

Moving in our new lab! The first week Huang Tianze, Shen Yunhao, Wang Tingxuan, Zhou Manxuan, Wu Yan, Ge Xiaofei and Zhou Liqun worked on the biobrick part transformations and sequencing. Also we received some help from our PI， Professor Chen’s lab. They provided us with sfGFP and RFP reporters. We prepared the necessary equipment and materials, like LB medium and plates. For the first batch of plates we used agarose instead of agar. The plates turned to stone afterwards hahaha.

#### Week2 20180401-20180407

The parts we intended to use were amplified and sent for sequencing by Huang Tianze, Shen Yunhao, Li Yibo and Wang Tingxuan. We started the pilot studies for Neon Coli and divided the work. Three groups of people started working on characterization of the quorum sensing system Lux and Las, the red light sensing system and the CRISPRi system.

#### Week3 20180408-20180414

Still doing transformation…Some parts are just unbelievable. Some sequencing results were inconsistent so we had to find another copy. Huang Tianze, Shen Yunhao, Li Yibo and Wang Tingxuan, Zhou Manxuan, Wu Yan and Zhou Liqun started designing the pilot test plans. We decided to use InFusion ligation technique because it sounds much faster than restriction cloning. (We were so wrong…)

#### Week4 20180415-20180421

Primers were flying in! Tsinghua iGEM lab is on full capacity. Huang Tianze, Wang Tingxuan, Zhou Manxuan, Wu Yan and Shen Yunhao were doing PCRs and gel recovery, building the pilot plasmids. The Phusion polymerase we were using was quite fickle. We spent half of the time debugging the PCRs.

#### Week5 20180422-20180428

First batch of InFusion! Huang Tianze and Wang Tingxuan tried to ligate the CRISPRi test plasmids. Zhou Manxuan, Wu Yan and Shen Yunhao repeated the PCRs for light sensing and quorum sensing plasmids

#### Week6 20180429-20180505

We tested the CRISPRi plasmids but they had no effect on sfGFP expression. They were definitely not right because enzyme cutting revealed inconsistent bands. Retested the recovered PCR products. Shen Yunhao built the first lux QS plasmids. Huang Tianze and Wang Tingxuan decided to order new primers for CRISPRi construction.

#### Week7 20180506-20180512

Retried CRISPRi InFusion. Huang Tianze and Wang Tingxuan spent loads of time on PCR. Some primers were just too long. Zhou Manxuan and Zhou Liqun finished the light sensing reporter plasmid.

#### Week8 20180513-20180519

Retried InFusion, still no CRISPRi plasmids…Zhou Manxuan, Zhou Liqun and Wu Yan used restriction enzymes to test the light sensing reporter plasmid OG, the results were inconsistent. Redid PCR and InFusion for OG and the sensor plasmid HPTC. Shen Yunhao transformed the lux reporter plasmid, on LB plates the colonies seemed quite green. Lux pR reporter must be leaky.

#### Week9 20180520-20180526

Still trying… CRISPRi and light sensing system colony PCRs generated no proper ligated plasmid. Shen Yunhao started building the positive feedback circuit. Dr. Li Peng suggested we use overlapping extension PCR to ligate smaller parts together before InFusion.

#### Week10 20180527-20180602

Overlapping PCR worked! Huang Tianze and Wang Tingxuan built the strong expressed CRISPRi plasmids that successfully repressed the production of sfGFP. Even though some colonies still leaked. Started to build medium and weak dCas9 generators. Zhou Manxuan and Zhou Liqun also began to use overlapping extension to build HPTC plasmid.

#### Week11 20180603-20180609

Weaker dCas9 generator showed better repression of sfGFP. M0.3, W1.0, W0.6 and W0.3 CRISPRi plasmids eliminated co-transformed sfGFP generator, while S1.0, S0.6 and S0.3 still showed some leakage. We were very puzzled. Shen Yunhao finished the lux positive feedback plasmid. Even before extraction the broth shone very brightly. We decided to build an IPTG induction device above the quorum sensing system.

#### Week12 20180610-20180616

Light sensing still had no results. We decided to divide and conquer, separating the sensor HPTC to several parts and then ligate. However sequencing and colony PCRs indicated that the construction of smaller parts were no lighter work than the entire plasmid. We decided to change the construction plan.

#### Week13 20180617-20180623

Sequencing of the S1.0 dCas9 plasmid revealed multiple mutations. Strong expression of dCas9 may be too toxic to the bacteria that it has to be mutated so that the bacteria can survive. Therefor we decided to use W0.6 dCas9 generator in the following experiments.

#### Week14 20180624-20180630

Summer holiday (also finals) were creeping in! Nothing really changed. Phusion was still fickle, gel recovery was still lacky and InFusion was still very difficult. We started the PCRs for IPTG inducible quorum sensing devices.

#### Week15 20180701-20180707

Summer holiday officially starts! We asked around other iGEM teams and found out that nearly everyone had encountered troublesome promotors, leaky, unreliable or otherwise had low intensity. We finally finished light sensing reporter LSP. But even without its sensors the reporter sfGFP still expressed. We reconsidered our position and decided to focus more on expression control instead of artistic presentation. Also we welcomed the high school teams SHSBNU\_China and RDFZ\_China. They settled into the lab next door.

#### Week16 20180708-20180714

Wang Tingxuan and Huang Tianze stayed behind… Life was all about PCRs, overlapping PCRs and gel recovery. In order to improve our PCR success rate we ordered Q5 premix. IPTG induced las QS plasmid was constructed. We sprayed some IPTG on the plate but there was no effect. We finished the design of the NEON system.

#### Week17 20180715-20180721

IPTG induction still had no results. Our advisor Wang Xuan suggested that maybe lacI expression was too strong. Preliminary model simulation indicated that lux pR was a knot where loads of biochemical reactions intersects. We decided to build a less leaky but more sensitive lux pR promotor and started the design of lux pR-HS.

#### Week18 20180722-20180728

We realized that previous plasmid construction was not efficient, so Huang Tianze designed a new set of plasmid construction plans. The parts are InFusioned into the smallest transcription units and individually amplified, then use 3A assembly to assemble the units together to form the test plasmids. So…even more PCRs and gel recoveries.

#### Week19 20180729-20180804

The new plans worked out quite well. This week we sequenced the unit plasmids and confirmed most of them. Wang Tingxuan and Chen Xinyi ligated the lux pR test plasmid. Yao Liang designed the lux pR mutants. Started to build Neon and Safety Catch.

#### Week20 20180805-20180811

Wang Tingxuan went to the regional meet up hosted in Peking University. We sent out the first batch of our HP surveys. Pilot lux pR testing showed that AHL induction intensity was quite high. But IPTG induction of lacI+pI promotor still showed no results. RDFZ\_China suggested that we use Tac promotor.

#### Week21 20180812-20180818

Wang Tingxuan designed new quorum sensing test systems of Rhl and Cin. IPTG induction still generated no results. Started Ptac test plsmid construction. We considered the effect of lacI dosage and decided to use three different levels of induction.

#### Week22 20180819-20180825

We tested lux pR and mutants AHL induction with Thermo Varioskan Flash. The preliminary results lead us to design the sequence of lux pR-HS. Designing plasmids for IPTG induction of CRISPRi. Ordering more primers hahaha.

#### Week23 20180826-20180901

Finished construction of Neon plasmids. Positive feedback was indeed very strong, the fluorescent intensity was way stronger than lux pR AHL induction. CRISPRi plasmids were difficult to assemble because dCas9 was too long. We aborted the original plan and decided to build dCas9 and gRNAs into separate transcription units and ligate them later. Also Ge Xiaofei and Chen Xinyi attended CCiC in Shanghai and sent out our questionnaires.

#### Week24 20180902-20180908

Uploaded our safety form! Finished Neon-HS construction and all the lux pR mutants. Discovered that dCas9 contained an illegal EcoRI site and gRNA contained illegal SpeI sites. Ordered primers for point mutation.

#### Week25 20180909-20180915

Uploaded the final version of our abstract. Finished construction of dCas9 generators. IPTG induction of Ptac promotors were still unsuccessful. Finished the construction of different levels of lacI generators.

#### Week26 20180916-20180922

Began building the plasmids for part registry. IPTG induction finally had results. Indeed too much lacI protein made Ptac not respond to IPTG. Lower lacI concentration would suffice. Repeated lux pR mutation analysis.

#### Week27 20180923-20180929

Finished construction of plasmids for IPTG induced CRISPRi, Safety Catch and Safety Catch-HS. Started lacI dosage analysis. Nearly finished the plasmids for part registry.

#### Week28 20180930-20181006

Prepared for the exhibition at China Science and Technology Museum. Tested the IPTG induced CRISPRi system. Prepared the test of NEON system.

#### Week29 20181007-20181013

Sent out the plasmids just in time. While transforming the NEON plaids we discovered that unbalanced copy number can cause variation in the outcome of the circuits. We decided to try to ligate them together.

#### Week30 20181014-20181017

Working hard on Wiki! We repeated other experiments for more data and continued the NEON characterization.

Copyright © Tsinghua iGEM 2018


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# Team:Tsinghua/Experiments

Neon Coli-Necessary Expression Only

[![](https://static.igem.org/mediawiki/2018/b/be/T--Tsinghua--logo.png)](#page-top "Neon Coli")

* [Home](https://2018.igem.org/Team%3ATsinghua)
* [Team](#Team)
* Project
  + [Description](https://2018.igem.org/Team%3ATsinghua/Description)
  + [Design](https://2018.igem.org/Team%3ATsinghua/Design)
  + [Experiments](https://2018.igem.org/Team%3ATsinghua/Experiments)
  + [Results](https://2018.igem.org/Team%3ATsinghua/Results)
  + [Model](https://2018.igem.org/Team%3ATsinghua/Model)
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  + [Attributions](https://2018.igem.org/Team%3ATsinghua/Attributions)
* Parts
  + [Basic Parts](https://2018.igem.org/Team%3ATsinghua/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ATsinghua/Composite_Part)
  + [Improve](https://2018.igem.org/Team%3ATsinghua/Improve)
* HP
  + [Overview](https://2018.igem.org/Team%3ATsinghua/Human_Practices)
  + [General HP](https://2018.igem.org/Team%3ATsinghua/GHP)
  + [Intergrated HP](https://2018.igem.org/Team%3ATsinghua/IHP)
  + [Safety](https://2018.igem.org/Team%3ATsinghua/Safety)
* [Collaboration](https://2018.igem.org/Team%3ATsinghua/Collaborations)

## EXPERIMENTS

###

#### lux pR Mutation Analysis

 With the hope to find an optimal promotor for our NEON system, we designed 9 mutations on sites -35, -36, -37 near the luxR binding site and tested another on -10 site that TUST 2017 reported to show decreased leakage. We conducted experiments to evaluate these lux pR mutants' reaction to AHL stimulation and their leakage level. The test devices we designed include a constantly expressed luxR and a lux pR (or mutant) driven sfGFP (like [BBa\_K2558211](http://parts.igem.org/Part%3ABBa_K2558211) with original lux pR promotor , and [BBa\_K2558212](http://parts.igem.org/Part%3ABBa_K2558212) with lux pR-HS promotor).
The protocol is as follows:
1. Transform the plasmids into *E. coli* DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add AHL to final concentrations of 0, 10-10,10-9,10-8 M. Fresh LB medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and measure the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. Each group should be repeated for at least 3 times..

#### lacI Dosage & IPTG Induction

During pilot studies we found that high level of lacI expression severely decrease IPTG induction efficiency. In order to investigate how lacI dosage affects IPTG induction, we used Anderson promotor J23100, J23110 and J23114 to design three constitutive lacI generators of different intensities. The three lacI generators were then ligated with Ptac driven reporter sfGFP to make three IPTG induction devices ([BBa\_K2558203](http://parts.igem.org/Part%3ABBa_K2558203), [BBa\_K2558204](http://parts.igem.org/Part%3ABBa_K2558204),[BBa\_K2558205](http://parts.igem.org/Part%3ABBa_K2558205)). By measuring sfGFP fluorescence we tested how these devices react to IPTG.
The protocol is as follows:
1. Transform the plasmids into *E. coli* DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 5, 10, 20 mM. Fresh M9 medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 for each well every 30 minutes.
5. Each group should be repeated for at least 3 times.

#### IPTG Induced CRISPRi

 After we clarified the correlation between lacI dosage and its induction efficiency, we furthered the experiments by adding CRISPRi to the system. Instead of sfGFP, the IPTG induction devices with different levels of lacI generators would produce a gRNA that targets the promotor of a constitutive sfGFP generator ([BBa\_K2558206](http://parts.igem.org/Part%3ABBa_K2558206), [BBa\_K2558207](http://parts.igem.org/Part%3ABBa_K2558207), [BBa\_K2558208](http://parts.igem.org/Part%3ABBa_K2558208)). By adding IPTG, we induced the transcription of gRNA. Binding with constantly expressed dCas9, gRNA could inhibit the expression of sfGFP. We were able to observe how this process proceeded under different levels of lacI expression. This is the experiment design: (Table.1)

**Table.1. IPTG induced CRISPRi experiment**

|  | Plasmid 1 (Amp+) | Plasmid 2 (Chl+) | Group |
| --- | --- | --- | --- |
| Bacteria 1 | sfGFP generator | [dCas9 generator](http://parts.igem.org/Part%3ABBa_K2558201) | Positive control |
| Bacteria 2 | sfGFP generator | [CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI](http://parts.igem.org/Part%3ABBa_K2558206) | Test 1 |
| Bacteria 3 | sfGFP generator | [CRISPRi device with Ptac expressed gRNA and Anderson medium (J23110)- expressed lacI](http://parts.igem.org/Part%3ABBa_K2558207) | Test 2 |
| Bacteria 4 | sfGFP generator | [CRISPRi device with Ptac expressed gRNA and Anderson weak (J23114)- expressed lacI](http://parts.igem.org/Part%3ABBa_K2558208) | Test 3 |
| Bacteria 5 | Empty backbone | [CRISPRi device with Ptac expressed gRNA and Anderson strong (J23100)- expressed lacI](http://parts.igem.org/Part%3ABBa_K2558206) | Negative control 1 |
| Bacteria 6 | Empty backbone | [dCas9 generator](http://parts.igem.org/Part%3ABBa_K2558201) | Negative control 2 |

The protocol is as follows:
1. Transform the plasmids into E. coli DH5α according to the design in Table 1.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 3, 5, 10 mM. Fresh M9 medium serves as blank control. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. Each group should be repeated for at least 3 times.

#### NEON System Characterization

Assisted with the experience we gained form the experiments above, we built and tuned the NEON system. We designed this experiment to characterize how Neon the positive feedback plasmid ([BBa\_K2558214](http://parts.igem.org/Part%3ABBa_K2558214)), and Safety Catch the CRISPRi plasmid ([BBa\_K2558215](http://parts.igem.org/Part%3ABBa_K2558206),[BBa\_K2558216](http://parts.igem.org/Part%3ABBa_K2558206)) work together.
This is the experiment design: (Table.2)

**Table.2. NEON system characterization experiment design.**

|  | Plasmid 1 (Amp+) | Plasmid 2 (Chl+) | Group |
| --- | --- | --- | --- |
| Bacteria 1 | [Neon](http://parts.igem.org/Part%3ABBa_K2558214) | [dCas9 generator](http://parts.igem.org/Part%3ABBa_K2558201) | Positive control |
| Bacteria 2 | Empty backbone | [dCas9 generator](http://parts.igem.org/Part%3ABBa_K2558201) | Negative control (to see how dCas9 interfere with bacteria growth) |
| Bacteria 3 | [lux pR test device](http://parts.igem.org/Part%3ABBa_K2558211) | [dCas9 generator](http://parts.igem.org/Part%3ABBa_K2558201) | Non-positive feedback control 1 |
| Bacteria 4 | [lux pR-HS test device](http://parts.igem.org/Part%3ABBa_K2558212) | [dCas9 generator](http://parts.igem.org/Part%3ABBa_K2558201) | Non-positive feedback control 2 |
| Bacteria 5 | [Neon](http://parts.igem.org/Part%3ABBa_K2558214) | [CRISPRi Safety Catch device with lux pR promotor](http://parts.igem.org/Part%3ABBa_K2558215) | Test 1 |
| Bacteria 6 | [Neon](http://parts.igem.org/Part%3ABBa_K2558214) | [CRISPRi Safety Catch device with lux pR-HS promotor](http://parts.igem.org/Part%3ABBa_K2558216) | Test 2 |
| Bacteria 7 | [lux pR test device](http://parts.igem.org/Part%3ABBa_K2558211) | [CRISPRi Safety Catch device with lux pR promotor](http://parts.igem.org/Part%3ABBa_K2558215) | Non-positive feedback test 1 |
| Bacteria 8 | [lux pR-HS test device](http://parts.igem.org/Part%3ABBa_K2558212) | [CRISPRi Safety Catch device with lux pR-HS promotor](http://parts.igem.org/Part%3ABBa_K2558216) | Non-positive feedback test 2 |

The protocol is as follows:
1. Transform the two plasmids into *E. coli* DH5α according to the design in Table 2.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Place the 96-well plate into an automatic microplate reader. Incubate at 16 ℃ overnight and record the fluorometric value at 510 nm and OD600 of each well every 30 minutes. Or Fix sample with 1.5 mg/ml kanamycin at one hour intervals. Then use flow cytometry to measure the fluorescent intensity at 488 nm of each sample.
5. Each group should be repeated for at least 3 times.

Copyright © Tsinghua iGEM 2018

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Tsinghua/Results

Neon Coli-Necessary Expression Only

[![](https://static.igem.org/mediawiki/2018/b/be/T--Tsinghua--logo.png)](#page-top "Neon Coli")

* [Home](https://2018.igem.org/Team%3ATsinghua)
* [Team](#Team)
* Project
  + [Description](https://2018.igem.org/Team%3ATsinghua/Description)
  + [Design](https://2018.igem.org/Team%3ATsinghua/Design)
  + [Experiments](https://2018.igem.org/Team%3ATsinghua/Experiments)
  + [Results](https://2018.igem.org/Team%3ATsinghua/Results)
  + [Model](https://2018.igem.org/Team%3ATsinghua/Model)
  + [Notebook](https://2018.igem.org/Team%3ATsinghua/Notebook)
  + [Attributions](https://2018.igem.org/Team%3ATsinghua/Attributions)
* Parts
  + [Basic Parts](https://2018.igem.org/Team%3ATsinghua/Basic_Part)
  + [Composite Parts](https://2018.igem.org/Team%3ATsinghua/Composite_Part)
  + [Improve](https://2018.igem.org/Team%3ATsinghua/Improve)
* HP
  + [Overview](https://2018.igem.org/Team%3ATsinghua/Human_Practices)
  + [General HP](https://2018.igem.org/Team%3ATsinghua/GHP)
  + [Intergrated HP](https://2018.igem.org/Team%3ATsinghua/IHP)
  + [Safety](https://2018.igem.org/Team%3ATsinghua/Safety)
* [Collaboration](https://2018.igem.org/Team%3ATsinghua/Collaborations)

## Results

###

#### Lux pR Mutation Analysis

##### >>>>Design

With the hope to find an optimal promotor for our NEON system, we designed 9 mutations on sites -35, -36, -37 near the luxR binding site and tested another on -10 site that TUST 2017 reported to show decreased leakage. We conducted experiments to evaluate these lux pR mutants’ reaction to AHL stimulation and their leakage level. The test devices we designed include a constantly expressed luxR and a lux pR (or mutant) driven sfGFP (like [BBa\_K2558211](http://parts.igem.org/part%3ABBa_K2558211) with original lux pR promotor, and [BBa\_K2558212](http://parts.igem.org/part%3ABBa_K2558212) with lux pR-HS promotor).

##### >>>>Results

We conducted the experiment first with 9 mutations on sites -35, -36, -37 near the luxR binding and another on -10 site that TUST 2017 reported to show decreased leakage. We transferred the plasmids with lux pR driven sfGFP into *E. coli* DH5α. In theory, AHL can induce gene expression by activating lux pR promotor. We measured the value of Fluorescence/OD without AHL stimulation by microplate reader to show the leakages of different lux pR mutants, as lux pR promotor is not supposed to be activated without AHL. Meanwhile, we also regard the value of Fluorescence/OD with 10-10 M AHL stimulation to observe the sensitivity and expression intensity of different lux pR mutants. Experiment data is shown in the figures below. (Figure.1)

![](https://static.igem.org/mediawiki/2018/9/98/T--Tsinghua--AHL1.png)

**Figure.1. Leakage and 10-10 M AHL stimulation of lux pR promotor and its mutations.**

The fluorescence strengths and OD values are measured by microplate reader respectively at 510 nm and 600 nm wavelength. Plasmids with lux pR promotor and its mutants are transferred into *E.coli* DH5α. **A** The bacteria were treated without AHL inducing. High (Fluorescence/OD) ratio indicates strong leakage of promotor. Among all the strains, lux pR promotor has the strongest leakage and the largest error bar as well. **B** The bacteria were treated with 10-10 M AHL induction. High (Fluorescence/OD) ratio indicates high expression intensity. Mutation 5 has almost the same expression intensity as the wild type lux pR promotor.

It is observed that high expression intensity is accompanied by high level of leakage in lux pR promotors. Wild type lux pR represented with black strand in the figures has the highest level of both leakage and intensity. The next three are Mutant 5 (G-36T), Mutant 6 (G-36C), Mutant 9 (T-35G). Mutant 5 has almost the same intensity as wild type, but the leakage is significantly smaller. Mutant 6 and Mutant 9 have a little bit less leakage and medium intensity. We also did the experiment with 10-9 M and 10-8 M AHL, all of the promotors show high expression levels and therefore unable to compare. (Data not shown)

![](https://static.igem.org/mediawiki/2018/f/f2/T--Tsinghua--AHL2.png)

**Figure.2. Leakage and AHL stimulation of lux pR Mutant 5 and Mutant 10.**

fluorescence strengths and OD values are measured by microplate reader respectively at 510 nm and 600 nm wavelength. **A** Plasmid with lux pR Mutant 5 is transferred into *E.coli* DH5α. The bacteria were treated without or with 10-10, 10-9, 10-8 M AHL inducing. **B** Plasmid with lux pR Mutant 10 is transferred into E.coli DH5α. The bacteria were treated without or with 10-10, 10-9, 10-8 M AHL inducing.

We repeated AHL stimulation test on Mutant 5 and Mutant 10 (Figure.2) and decided to combine the two mutations together to form a new promotor with high intensity and small leakage. We named it lux pR-HS. We tested lux pR-HS under the same conditions. We can see that the lux pR-HS has similar leakage to Mutant 10, while it has much higher expression intensity. (Figure.3) The statistics of relative induction and leakage intensity of the 10 Mutants and lux pR-HS are shown in Table.1

![](https://static.igem.org/mediawiki/2018/6/66/T--Tsinghua--AHL3.png)

**Figure.3. Leakage and 10-10 M AHL stimulation of lux pR promotor WT, Mutant 5, Mutant 10 and HS.**

The fluorescence strengths and OD values are measured by microplate reader respectively at 510 nm and 600 nm wavelength. Plasmids with lux pR promotor WT, Mutant 5 and Mutant 10 are transferred into *E.coli* DH5α. A The bacteria were treated without AHL inducing. High (Fluorescence/OD) ratio indicates strong leakage of promotor. B The bacteria were treated with 10-10 M AHL induction. High (Fluorescence/OD) ratio indicates high expression intensity.

**Table.1. Results of lux pR mutation analysis.**

![](https://static.igem.org/mediawiki/2018/7/7c/T--Tsinghua--AHLtable.png)

As for why some mutations on -35 to -37 sites would decrease the leakage of lux pR without severely influencing the expression level, we have some hypotheses and explanations. It is reported that -35 to -37 sites are last three bases of lux box which is upstream of the lux promotor and bound specifically by luxR, one of regulatory protein involved in lux expression system.[1] G-36T mutation has less influence than other mutation on sites -35 to -37, which is same to our result. Besides, G-36T is not involved in the two regions of nucleotides -52 to -50 and -39 to -37 which directly contacted with luxR protein. Therefore, this single substitution of nucleotide would decrease the leakage of lux promotor without influencing the expression level too much.[2]

##### >>>>Protocol

1. Transform the plasmids into *E. coli* DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add AHL to final concentrations of 0, 10-10, 10-9, 10-8 M. Fresh LB medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and measure the fluorometric value at 510 nm and OD600 of each well every 30 minutes.
5. Each group should be repeated for at least 3 times.

[1] Antunes, L. C., et al. "A mutational analysis defines Vibrio fischeri LuxR binding sites." Journal of Bacteriology 190.13(2008):4392-4397.
[2] Zeng, Weiqian, et al. "Rational design of an ultrasensitive quorum-sensing switch." Acs Synthetic Biology 6.8(2017).

#### IPTG Induction and lacI Dosage

##### >>>>Design

In order to investigate how lacI dosage affects IPTG induction, we used Anderson promotor J23100, J23110 and J23114 to design three constitutive lacI generators of different intensities. The three lacI generators were then ligated with Ptac driven reporter sfGFP to make three IPTG induction devices ([BBa\_K2558203](http://parts.igem.org/part%3ABBa_K2558203),[BBa\_K2558204](http://parts.igem.org/part%3ABBa_K2558204),[BBa\_K2558205](http://parts.igem.org/part%3ABBa_K2558205)). By measuring sfGFP fluorescence we tested how these devices react to IPTG.

##### >>>>Results

![](https://static.igem.org/mediawiki/2018/3/31/T--Tsinghua--IPTG1.png)

**Figure.4. The effect of varied lacI-LVA ([BBa\_C0011](http://parts.igem.org/part%3ABBa_C0011)) promotor strength on IPTG induction of Ptac ([BBa\_K2558004](http://parts.igem.org/part%3ABBa_K2558004)).**

Relative fluorescent intensity is fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at Time=0, IPTG=0. The promotors we used are from the Anderson collection: BBa\_J23100 for strong lacI-LVA expression (pink), BBa\_J23110 for medium expression (green), BBa\_J23114 for weak expression (orange).

With high level of lacI expression ([BBa\_K2558203](http://parts.igem.org/part%3ABBa_K2558203)), sfGFP fluorescence had almost no response to IPTG induction. Weak lacI expression ([BBa\_K2558205](http://parts.igem.org/part%3ABBa_K2558205)) had the most significant IPTG induced sfGFP expression. With medium lacI expression level ([BBa\_K2558204](http://parts.igem.org/part%3ABBa_K2558204)), the induction efficiency lay in between. Therefore, the result proves that high level of lacI expression severely decreases IPTG induction efficiency [1]. Furthermore, IPTG concentration can affect the regulation part performance. The figure shows that without IPTG the sfGFP florescence intensity remained low. After IPTG addition, fluorescence signal immediately began to climb, forming a peak at five hours after induction, then sfGFP florescence intensity decreased and maintained at a lower level afterwards. IPTG concentration did not significantly affect the height of the peak or the expression level after the peak, but rather the peak width and expression stability of the system. Figures indicate that 5-10 mM IPTG had the most stable induction results.

##### >>>>Protocol

1. Transform the plasmids into *E. coli* DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 5, 10, 20 mM. Fresh M9 medium serves as blank control. Positive control is colony constantly expressing sfGFP and negative control is colony without sfGFP expression. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 for each well every 30 minutes.
5. Each group should be repeated for at least 3 times.

[1] Szabolcs Semsey, Sandeep Krishna. "The effect of LacI autoregulation on the performance of the lactose utilization system in Escherichia coli" Nucleic Acids Res 2013 Jul; 41(13): 6381–6390

#### IPTG Induced CRIPSRi

##### >>>>Design

After we clarified the correlation between lacI dosage and its induction efficiency, we furthered the experiments by adding CRISPRi to the system. Instead of sfGFP, the IPTG induction devices with different levels of lacI generator is now going to produce a gRNA that targets the promotor of a constitutive sfGFP generator ([BBa\_K2558206](http://parts.igem.org/part%3ABBa_K2558206), [BBa\_K2558207](http://parts.igem.org/part%3ABBa_K2558207), [BBa\_K2558208](http://parts.igem.org/part%3ABBa_K2558208)). By adding IPTG, we can induce the transcription of gRNA. Binding with constantly expressed dCas9, gRNA is going to inhibit the expression of sfGFP. We will be able to observe how this process can operate under different levels of lacI expression.

##### >>>>Results

![](https://static.igem.org/mediawiki/2018/4/47/T--Tsinghua--IPTG2.png)

**Figure.5. The effect of varied lacI-LVA ([BBa\_C0011](http://parts.igem.org/part%3ABBa_C0011)) promotor strength on IPTG induction of CRISPRi.**

 The Y axis values are fluorescence per OD600 standardized with fluorescence per OD600 value of each test group at Time=0, IPTG=0 M. The promotors we used are from the Anderson collection: BBa\_J23110 for medium expression (orange), [BBa\_J23114](http://parts.igem.org/part%3ABBa_J23114) for weak expression (blue).

In this experiment, the expression of sfGFP was inhibited by IPTG induced CRISPRi system. (Figure.5) Without CRISPRi the Fluorescence/OD600 increased steadily. With medium lacI expression, the addition of IPTG caused the fluorescence intensity to decrease at three hours after addition. With different IPTG concentration the final sfGFP fluorescence intensity remained approximately the same, which is consistent with previous IPTG induction results. However, with weak lacI expression, sfGFP expression was inhibited from the beginning. With the direct IPTG induction experiment we performed before, we hypothesize that with the weak Anderson promotor, basal gRNA expression will be above the threshold that is required for suppressing the sfGFP expression.[1]

##### >>>>Protocol

1. Transform the plasmids into *E. coli* DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml M9 medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0, 1, 3, 5, 10 and 20 mM. Fresh M9 medium serves as blank control. Place the 96-well plate into an automatic microplate reader. Incubate at 16℃ overnight and record the fluorometric value at 510 nm and OD600 of each well every 30 minut
es.
5. Each group should be repeated for at least 3 times.

[1]Mückl A, Schwarz-Schilling M, Fischer K, Simmel FC. “Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach.” PLoS One. 2018 Sep 11;13(9):e0198058. doi: 10.1371/journal.pone.0198058. eCollection 2018.

#### Neon System Characterization

##### >>>>Design

Assisted with the experiences we gained form the experiments above, we built and tuned the NEON system. We designed this experiment to characterize how Neon the positive feedback plasmid ([BBa\_K2558214](http://parts.igem.org/part%3ABBa_K2558214)), and Safety Catch the CRISPRi plasmid ([BBa\_K2558215](http://parts.igem.org/part%3ABBa_K2558215), [BBa\_K2558216](http://parts.igem.org/part%3ABBa_K2558216)) work together.

##### >>>>Results

We are still calibrating the NEON system. The results are preliminary, however from Figure.6 we can conclude that the system works to some extent. The positive feedback plasmid Neon ([BBa\_K2558214](http://parts.igem.org/part%3ABBa_K2558214)) had the highest expression due to uncontrollable leakage. Original lux pR ([BBa\_R0062](http://parts.igem.org/part%3ABBa_R0062)) and the new lux pR-HS ([BBa\_K2558001](http://parts.igem.org/part%3ABBa_K2558001)) we designed had lower basal expression. The addition of Safety Catch ([BBa\_K2558215](http://parts.igem.org/part%3ABBa_K2558215)) and Safety Catch-HS ([BBa\_K2558216](http://parts.igem.org/part%3ABBa_K2558216)) almost eliminated the leakage of both positive feedback and the non-positive feedback systems. It is foreseeable that with appropriate parameters NEON system can be activated to almost 104 fold.

![](https://static.igem.org/mediawiki/2018/6/6b/T--Tsinghua--Neon1.png)

**Figure.6. Basal expression of lux quorum sensing systems.**

 The fluorescence intensity was measured by flow cytometer at 488 nm. Neon, Safety Catch, Safety Catch-HS and lux pR, lux pR-HS test plasmids were transferred into *E.coli* DH5α. High fluorescence intensity suggests high level of basal expression in the system.

##### >>>>Protocol

1. Transform the plasmids into *E. coli* DH5α.
2. Pick a single colony by a sterile tip from each of the LB plates for all the experimental and control groups. Add the colony into 5ml LB medium with ampicillin at 100 ng/µl and chloramphenicol at 34 ng/µl. Incubate for 6-8 h at 37℃ in a shaker.
3. Measure OD600 of the culture medium with photometer. Dilute the culture medium until OD600 reaches 0.6.
4. Add 100 µl bacteria culture medium into a sterile 96-well plate. Add IPTG to final concentrations of 0 or 10 mM and AHL to final concentrations of 0, 10-9, 10-8 M. Fresh LB medium serves as blank control. Fix sample with 1.5 mg/ml kanamycin at one hour intervals. Then use flow cytometry to measure the fluorescent intensity at 488 nm of each sample.
5. Each group should be repeated for at least 3 times.

[1]Afroz, T., & Beisel, C. L. (2013). Understanding and exploiting feedback in synthetic biology. Chemical Engineering Science, 103(11), 79-90.
[2]Qi, L., Larson, M., Gilbert, L., Doudna, J., Weissman, J., & Arkin, A., et al. (2013). Repurposing crispr as an rna-guided platform for sequence-specific control of gene expression. Cell, 152(5), 1173.

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