iGEM 2023 — Team shanghaitech-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
{% block page\_content %}

Notebook

Lux Luminescence

BRET Verification

Cyanobacteria

Lux Gene Cluster Luminescence Verif ication

BRET Verif ication

Cyanobacteria Culture and Transformation Verif ication

![](https://static.igem.wiki/teams/4594/wiki/maomao04.png)

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

Overview

Lux Luminescence

BRET Verification

Cyanobacteria

Overview

In order to conduct more experiments to verify the feasibility of the LAMPS project, the students in the wet laboratory group were divided into three groups and carried out three sets of experiments on different themes.

The goals of the three groups of engineering modifications are:

1. To increase the fluorescent substrate of the bacteria through metabolic adjustment;

2. To enhance the luminous intensity and broaden the spectrum through protein engineering;

3. To verify the relevant original components through the recombinant transformation of cyanobacteria.

The entire engineering process is carried out logically under the principles of DBTL (Design, Build, Test, Learn) recommended by iGEM. We continue to carry out transformation and iteration to improve our projects. The following is the experimental content and logic network we have completed.

Lux Gene Cluster Luminescence Verif ication

Research

According to previous studies, Lux CDEABE genes from Photorhabdus luminescens enabled organisms emit luminescence without any additional source of substrates[ Winson, M K et al. “Engineering the luxCDABE genes from Photorhabdus luminescens to provide a bioluminescent reporter for constitutive and promoter probe plasmids and mini-Tn5 constructs.” FEMS microbiology letters vol. 163,2 (1998): 193-202. doi:10.1111/j.1574-6968.1998.tb13045.x], which aligns perfectly with our goal to design a sustainable source of light: LAMPS. So, we utilized this basic lux operon as the base of our design. Additionally, the intensity of light is an essential criterium of LAMPS, leading us to involve two other parts: LuxF(Part:BBa\_K4594002) and LuxG(Part:BBa\_K4594003). Previous study has shown that LuxF would activate luciferases (consists of LuxA and LuxB) by binding to the inhibitor of luciferases[ Brodl, Eveline et al. “The impact of LuxF on light intensity in bacterial bioluminescence.” Journal of photochemistry and photobiology. B, Biology vol. 207 (2020): 111881. doi:10.1016/j.jphotobiol.2020.111881]. And LuxG also contributes to the luminescence with extra substrates FMNH2[ Nijvipakul, Sarayut et al. “LuxG is a functioning flavin reductase for bacterial luminescence.” Journal of bacteriology vol. 190,5 (2008): 1531-8. doi:10.1128/JB.01660-07]. So, both parts are ideal for enhancing LAMPS.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image1.png)

Figure 1 The mechanism of light enhancement by luxF and luxG

Cycle 1: Design pET28a-luxFCDABE and f irst luminescence assay

Design:

In order to enhance the luminescence of LAMPS, we tried to combine basic lux operon, luxG and luxF to form luxCDABEGF(Part:BBa\_K4594004) . And in the first cycle, we took luxF as the first trial. Since we had got pGEN-luxCDABE and pET28a-luxF, we wanted to assemble the luxCDABE fragment into vector pET28a-luxF to create pET28a-luxFCDABE.

Build:

pGEN-luxCDABE was ordered from Addgene and pET28a-luxCDABE was synthesized by GeneScript. PCR and Gibson Assembly were performed to get the final pET28a-luxFCDABE

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image2.png)

Figure 2 Graph of pET28a-luxFCDABE

Test:

We transferred the plasmid into E. coli BL21(DE3) and induced it by IPTG. However, no luminescence was observed, which meant our plasmid didn't work.

Learn:

In pET28a-luxFCDABE, luxF was ahead of the more important part luxCDABE. However, it would be better to put an important gene in a further forward position so that it could be more transcribed and translated. Additionally, luxF had too more tags to function well.

Cycle 2 Remove tags, rearrange genes and assemble luxG

Design:

Based on what we had learnt in the last cycle, we planned to remove the tags after T7 promoter. Also, luxCDABE would be positioned in the first position. Additionally, we assembled luxG into the plasmid to construct pET28a-luxCDABEGF.

Build:

Basic cloning steps were performed to construct pET28a-luxCDABEGF (Figure 3). And we constructed pET28a-luxCDABE as a control.

Test:

Both plasmids were transferred into BL21(DE3). BL21(DE3) were cultured for 16 hours under 23℃ after being induced by 0.2mM IPTG. With excitement, we found the luxF,G did contributed to the enhancement of luminescence by naked eyes and luminescence test.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image3.png)

Figure 3 the final plasmid graph of pET28a-luxCDABEGF

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image4.png)

Figure 4A Comparing luminescence of BL21(DE3) with pET28a-luxCDABE or pET28a-luxCDABEGF. Images were taken by an ordinary phone camera. The left three tubes were BL21(DE3) with pET28a-luxCDABE and the right three tubes were BL21(DE3) with pET28a-luxCDABEGF. It was clear that luminescence can be enhanced by additional luxF and luxG. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image5.png)

Figure 4B The result of luminescence assay for BL21(DE3) with pET28a-luxCDABE and BL21(DE3) with pET28a-luxCDABEGF (n=6). The result showed that the addition of luxF and luxG increased the luminescence by 58%. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG. And the assay was conducted by a plate reader.

Learn:

These results allowed us to conclude that the luxCDABEGF(Part:BBa\_K4594004) we designed was functional to emit a stronger light than basic lux operon, served as an important part of the sustainable source of light LAMPS!

BRET Verif ication

Cycle 1

Design:

Since the bacterial LuxCDABE fluorescence system itself is too low in brightness to meet the requirements of practical applications, we decided to increase the brightness by attaching the yellow fluorescent protein cp157Venus to the C-terminal of LuxB to become the new fusion fluorescent protein LuxB:cp157Venus. [1]Since the emission spectra of the LuxA-LuxB complex overlaps with the excitation spectrum of cp157Venus when the two are close enough (≤10 nm), the LuxA-LuxB complex in the excited state can undergo dipole-dipole resonance with cp157Venus, transferring its energy to the latter in a non-radiative manner, causing the latter to emit light with frequencies and amplitudes that are different from those of LuxA-LuxB complex. Such a principle is called biofluorescence resonance energy transfer (BRET), and its efficiency is related to the sixth power of the distance between donor and receptor. The proper use of BRET can significantly increase the brightness and, incidentally, change the color of the light.

Build:

In order to improve protein expression to allow us to observe the phenomena more easily, we chose the pET-28a plasmid with the T7 promoter. LuxA and LuxB on LuxCDABE from pGEN-luxCDABE source and cp157Venus (synthesis) were cloned by PCR and then recombined onto pET-28a plasmid by Gibson Assembly and transferred into DH5`\alpha` for propagation to get more plasmids. Since the protein is constitutively expressed in the reference, we intentionally deleted the three genes lacIq promotor, lacI, and CAP binding site when constructing the plasmid. The plasmid map is shown below:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili1.png)

Figure 1 : The plasmid of pET-28a(+)\_LuxA, LuxBΔ(lacI)

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili2.png)

Figure 2 The plasmid of pET-28a(+)\_LuxA, LuxB:cp157VenusΔ(lacI)

Test:

The successfully constructed pET-28a(+)\_LuxA, LuxB (BBa\_K4594013) plasmid and pET-28a(+)\_LuxA, LuxB:cp157Venus (BBa\_K4594014) plasmid were transfected into BL21(DE3), cultured at 37°C, and then incubated at 16°C and 24°C, respectively, in an IPTG gradient to Expression was induced by IPTG final concentrations of 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.7 mM, and decanal was added as a substrate for its luminescence. The emission spectra were detected using SpectraMax i3 multifunctional enzyme marker and the protein expression was detected using SDS-page. It was found that there was no luminescence in either, and the bands of the target protein could not be seen in the SDS-page gel, so it seems that there is a problem with the protein expression.

Learn:

We analyzed later that the deletion of the three genes lacIq promotor, lacI, and CAP binding site during the plasmid construction may have led to the impaired expression of the rop genes controlling the number of plasmid replicates, causing the bacteria to use more nutrients for the replication of plasmids and blocking protein expression.

Cycle 2

Design:

We decided to fix the vulnerability described above by adding the three genes lacIq promotor, lacI, and CAP binding site to verify our conjecture.

Build:

We again repaired our plasmid by PCR, Gibson Assembly and other techniques and amplified the plasmid with DH5`\alpha`. The plasmid map is as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili3.png)

Figure 3 The plasmid of pET-28a(+)\_LuxA, LuxB

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili4.png)

Figure 4 The plasmid of pET-28a(+)\_LuxA, LuxB:cp157Venus

Test:

We transferred the modified plasmid into BL21 (DE3), amplified it at 37°C, and then similarly induced the expression using IPTG gradient at 15°C and 24°C, respectively, to make the final concentration as before. But unfortunately, E. coli still did not glow and the target protein was not expressed.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image10.png)

Figure 5 Protein expression after plasmid repair

Learn:

This time we contacted the author of the original article and got a lot of useful information from him(See [Human Practices](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27human-practices%27%29%20%7D%7D) # 11 Academic for details), such as non-inducible constitutive expression leads to better protein expression, adding a linker (Glu-Leu) between LuxB and cp157Venus leads to higher BRET efficiency, etc. In the end, we decided to make the target gene constitutively expressed and add a linker, etc. for better protein expression.

Cycle 3

Design:

Next, we decided to completely delete the lac operator as well as the lacIq promotor, lacI, CAP binding site, rop, and bom genes, and to add a linker between LuxB and cp157Venus in the hope that the protein would be better expressed.

Build:

We reconstructed the plasmid and still amplified the plasmid with DH5`\alpha`, and the plasmid was mapped as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili5.png)

Figure 6 The plasmid of pET-28a(+)\_LuxA, LuxBΔ(AlacO, BlacO)

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili6.png)

Figure 7 The plasmid of pET-28a(+)\_LuxA, LuxB:cp157VenusΔ(AlacO, BlacO)

Test:

We again transferred the modified plasmid into BL21(DE3), cultured under the same conditions for amplification, and still used IPTG gradient to induce expression, and finally performed luminescence assay and protein expression assay. The results were still disappointing, the bacteria still did not luminesce and the corresponding proteins were not expressed.

Learn:

When we analyzed the above results we were able to find that many of the improvements that we made were improving the protein expression, but from the results, our protein was not expressed at all. So in the end, we analyzed that the root of the problem was not in these places, but rather that there was something wrong with our plasmids themselves, or even that the gene was mutated during all of the genetic manipulations, which resulted in the protein not being expressed. It is possible.

Cycle 4

Design:

Next, we are going to abandon the scheme of expressing proteins with dual promoters and change it to express the target gene with a single promoter, delete all the tags that may bring adverse effects to the protein, and express the purest protein. Hopefully, at this time, we can successfully verify the enhancement of the BRET principle for our Lux operon

Build:

This time we constructed single promoter LuxA-LuxB and single promoter LuxA-LuxB:cp157Venus, deleted all tags, and the resulting plasmids were also amplified with DH5`\alpha`. The plasmids were mapped as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili7.png)

Figure 8 The plasmid of pET-28a\_lacO-LuxA-LuxB

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/zhili8.png)

Figure 9 The plasmid of pET-28a\_lacO-LuxA-LuxB:cp157Venus

Test:

After transferring the plasmid pET-28a\_lacO-LuxA-LuxB into BL21(DE3), the expression was induced under the same conditions. The strain of E. coli transfected with the plasmid successfully emitted light, while the protein fused to cp157Venus did not.

Learn:

After several unsuccessful attempts to produce glowing cp157Venus fusion proteins with LuxB, we have come to realize that focusing solely on expressing LuxB:cp157Venus may not be the most effective approach. And relevant literature has proven the role of BRET, so we should focus on more important things. Instead, we need to broaden our search for other fluorescent proteins that can verify the role of BRET and achieve or even surpass the effect of cp157Venus. Additionally, we should explore the possibility of creating or finding brighter fluorescent proteins through BRET.

Cycle 5

Design:

In order to break the limitations of Venus, we expect to find fluorescent proteins with better resonance from the FPbase database on the Internet and try hard to design fluorescent proteins on our own that are not found in nature, in pursuit of better brightness performance and richer colours.

Build:

In order to find a protein with excitation light between 490-520 nm and high fluorescence intensity, we queried the fluorescent protein information in the FPbase database, and screened out the two proteins with the highest fluorescence intensity, named A1 and A2. However, nearly half of the fluorescent proteins in the database only have amino acid sequence information. In order to get the fluorescence data of these information-deficient proteins for screening, we used the fluorescent proteins with complete information to train the Long Short-Term Memory Networks (LSTM) system , established the mapping of amino acid sequence to fluorescence data, and screened the fluorescence of tow proteins named B1, B2. After this, we built a generator model by generative adversarial network to allow the computer to generate sequence information of amino acids as protein candidates that may outperform the function of fluorescent proteins in nature. And then the LSTM model was used as a discriminator to screen the generated protein sequences, and the two proteins named C1, C2 with the best results were obtained.

Test:

We used LuxCDABEGF as a positive control group, and then the four corresponding genes synthesised by genscript A1, A2, B1, B2 were linked to the C-terminal of LuxB to form fusion proteins using the same linker, and then were transferred into BL21(DE3) for expression. Expression was induced by adding a final concentration of 0.2 mM IPTG at 24°C after amplification culture at 37°C. Eventually, E. coli transfected into these plasmids successfully lit up. Here are some photos.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image15.png)

Figure 10 cp157Venus and A1

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image16.png)

Figure 11 A2 , B1 , LuxCDABEGF and Control

Learn:

Unfortunately, E. coli with the fusion protein LuxB:cp157 still did not light up, but E. coli with A1, A2, B1, and B2 all managed to light up with significantly different colours, an experiment that nicely validates the feasibility of our principle and demonstrates the effectiveness of our LSTM system. Additionally, the bacteria expressing the fusion proteins were weaker in brightness than the positive control group as expected, but this is well explained because the efficiency of BRET is correlated with the sixth power of the distance, in other words a small change in the distance can have a big effect on the brightness. So it is most likely that the inappropriate linker led to the weakening of the fusion proteins' brightness. In the future, optimising the linkers between individual fusion proteins is expected to enhance the brightness of the fusion proteins. Last but not least, optimizing proteins is also an important direction.

[1] Kobayashi H, Picard LP, Schönegge AM, Bouvier M. Bioluminescence resonance energy transfer-based imaging of protein-protein interactions in living cells. Nat Protoc. 2019 Apr;14(4):1084-1107. doi: 10.1038/s41596-019-0129-7. Epub 2019 Mar 25. PMID: 30911173.

Cyanobacteria Culture and Transformation Verif ication

Cycle 1

Design:

We designed the sfGFP plasmid because our project from last year also involved genetic modification of cyanobacteria PCC7942. Following last year's protocol and, we designed the transformation experiment.

Build:

We used the transformation plasmid with the imported cscB gene left by last year's team Shanghaitech\_China 2022 as the starting material. We replaced CscB with sfGFP using Gibson Assembly and added the rhythmic output promoter PKaiBC at the front. Then, we mixed the plasmid with the bacterial strain for transformation.

Test:

After transformation, we plated the mixture on agar plates containing kanamycin to check for successful transformation. However, after a week, no cyanobacterial colonies grew. Instead, there were transparent and sparse colonies.

Learn:

There may be contamination in the cyanobacterial culture. We suspect that competition between contaminants and cyanobacteria may have led to a decrease in cyanobacterial activity.

Cycle 2

Design:

We learned that cyanobacteria have some level of ampicillin resistance. We designed a gradient experiment to determine the maximum ampicillin concentration that can kill contaminants while preserving cyanobacterial viability.

Build & Test:

We prepared liquid culture media containing 0, 20, 40, 60, 80, and 100 mg/L of ampicillin and inoculated cyanobacteria. The cultures were continuously grown in a well-lit shaker, and OD750 was measured every 24 hours.

|  | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- | --- |
| 8.3 | 0.10 | 0.105 | 0.11 | 0.13 | 0.08 | 0.08 | 0.11 |
| 8.4 | 0.07 | 0.10 | 0.075 | 0.095 | 0.04 | 0.055 | 0.04 |
| 8.5 | 0.105 | 0.07 | 0.07 | 0.04 | 0.09 | 0.04 | 0.05 |
| 8.6 | 0.155 | 0.015 | 0.07 | 0.065 | 0.05 | 0.05 | 0.015 |
| 8.7 | 0.185 | -0.055 | 0.115 | 0.095 | 0.03 | 0.07 | 0.06 |
| 8.8 | 0.39 | 0.015 | -0.065 | -0.045 | -0.06 | -0.07 | -0.045 |

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-shr-wsb1.png)

Ultimately, we found that, except for the group with no antibiotic concentration, cyanobacteria in all other groups exhibited significant decline, indicating that directly adding antibiotics to the culture medium cannot prevent cyanobacterial contamination.

Learn:

After identification, we found that our cyanobacteria were contaminated by a nitrate-reducing bacterium, Nitratireductor sp., and noted their ampicillin resistance. Furthermore, after consulting Professor Zhao Quanyu from Nan Jing Tech University, he suggested that we could minimize the impact of contaminants by repeatedly centrifuging and resuspending the culture. Additionally, the reason for plate failure may be due to excessive light intensity in the incubator. It was also suggested that we could accelerate colony growth by supplementing the culture medium with NaHCO3 as an inorganic carbon source.

Cycle 3

Design & Build:

Based on previous failures, we redesigned the transformation experiment. We adjusted the light intensity in the incubator as recommended by Professor Zhao. We obtained a pure PCC7942 strain from Professor Luan Guodong from the Institute of Hydrobiology, Chinese Academy of Sciences. We conducted experiments simultaneously with contaminated and pure strains. Additionally, we decided to transform the plasmid containing sfGFP and luxCDABEFG the same time.

Test:

After transformation, colony growth significantly improved, but it was too dense to separate individual colonies.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image18.png)

BG11 plates after transformation and coating

We attempted to pick clustered bacteria for streaking and further isolation. Additionally, due to time constraints, we could not proceed with subsequent rhythm characterization experiments. We aimed to validate successful transformation through colony PCR using the method provided by the Toulose\_INSA 2021 team for Synechococcus colonies.

First, we conducted PCR using primers on both ends of NSIII, which resulted in numerous non-specific bands and blank NSIII bands with lengths similar to the wild-type control group.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image19.png)

blank Neutral site is about 1200bp

Considering that Synechococcus is often polyploid, and there may be untransformed cells, we attempted PCR using exogenous sequences, specifically the upstream and downstream primers for PkaiBC-sfGFP and lux.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/engineering-image20.png)

Almost each sample has a bright target band

Positive bands were also observed.

After some time, we found on another plate culture medium coated at the same time that most of the cyanobacteria originally mixed together had declined, leaving only a small number of single colonies.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-plus01.png)

We believe that this is a single colony that has been successfully transformed. Then select a single colony for PCR and repeat the above operation. The result is as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-plus04.png)

The results are basically consistent with the last time. Considering that Synechococcus PCC7942 is polyploid and the successfully transformed bacteria also contain blank NSIII fragments, we initially believe that we have obtained a single colony with successful transformation.

To further verify that we successfully transferred the gene, we examined the cyanobacteria under a fluorescence microscope.The cyanobacteria transformed into PKaiBC-sfGFP showed obvious green fluorescence under a fluorescence microscope. We used untransformed PCC7942 bacterial fluid as a control group and found that the control group had basically no fluorescence.

![](https://static.igem.wiki/teams/4594/wiki/wsbnum01.png)

Transformed PCC7942

![](https://static.igem.wiki/teams/4594/wiki/wsbnum02.png)

wildtype control

There is a direct significant difference between the two groups. We have sufficient evidence to prove that we successfully transformed the pUC57\_NS3-2-PKaiBC-sfGFP-lacI-KanR-NS3-1 plasmid.

Learn:

Use different primers in PCR experiments. In the first PCR experiment, bands characterizing successful insertion and non-insertion were obtained simultaneously. We hypothesize that wild type and successfully transformed types coexist on agar plates. Moreover, this phenomenon still exists when the PCR experiment is performed again after obtaining a single colony. This may be because cyanobacteria themselves are polyploid, so we were unable to verify the purity of a single colony, but we proved that some site insertions were successful.

The fluorescence photos further proved the successful transformation and also showed that the foreign protein we introduced could be expressed normally in cyanobacteria.

In addition, the time it took for a single colony to grow (about 2 weeks) was much longer than the expected time (5 days), and before the growth of a single colony, patches of cyanobacterial communities with indistinguishable colonies grew, indicating that our agar plates may have The problem of insufficient antibiotic concentration. Compared to standard LB agar, BG11 agar is less fluid and requires the addition of antibiotics at a higher temperature to ensure even mixing and no air bubbles. However, higher temperatures may cause antibiotics to become inactive. We suspect that this may be the reason for the decreased selectivity of our culture medium. Future teams might try preparing BG11 agar plates without antibiotics and, after solidification, lift the gel-like agar and add higher concentrations of antibiotics underneath to obtain plates with greater selectivity.

![](https://static.igem.wiki/teams/4594/wiki/maomao04.png)

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{% endblock %}

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

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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

Results

Lux Luminescence

BRET Verification

Cyanobacteria

Lux Gene Cluster Luminescence Verif ication

Co-expression of luxF and luxG to enhance the brightness of lux operon.

According to previous studies, Lux CDEABE genes from Photorhabdus luminescens enabled organisms emit luminescence without any additional source of substrates, which aligns perfectly with our goal to design a sustainable source of light: LAMPS. So, we utilized this basic lux operon as the base of our design. Additionally, the intensity of light is an essential criterium of LAMPS, leading us to involve two other parts: LuxF(Part:BBa\_K4594002) and LuxG(Part:BBa\_K4594003). Previous study has shown that LuxF would activate luciferases (consists of LuxA and LuxB) by binding to the inhibitor of luciferases. And LuxG also contributes to the luminescence with extra substrates FMNH2. In a conclusion, in this section we combined basic lux operon, luxG and luxF to form luxCDABEGF(Part:BBa\_K4594004) and proved the composite part enabled E.coli to emit stronger luminescence than the ordinary lux operon, which is the basis of our whole design LAMPS.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image1.png)

Figure 1 The mechanism of light enhancement by luxF and luxG

1 The Construction of pET28a\_luxCDABEGF

Initially, the lux operon was available in a pGEN plasmid from ADD-GENE (code: 44918), with an em7 promoter. To increase proteins production for better observation, we selected host cells BL21(DE3), and the vector was changed into pET28a for an efficient T7 promoter. So, we cloned lux operon from the pGEN plasmid by PCR.

And we got the CDS of LuxF(Part:BBa\_K4594002) and LuxG(Part:BBa\_K4594003) in NCBI, which we synthesized in Gene Script within plasmid pET28a.

As we had obtained luxCDABE, luxF, luxG and pET28a vector, we then assembled them together by Gibson Assembly. According to Figure 2A, we obtained two fragments(luxG and luxCDABE) with homology arms added during PCR process, and the linear vector of pET28a-luxF. Then we got pET28a-luxCDABEGF(Part:BBa\_K4594004) as shown in Fig2B.

As the recombinant plasmid had been accessed, we transferred it to E.coli DH5`\alpha` to magnify it. And we extracted the plasmids from it and testified it by Sanger sequencing. The sequencing result reflected that it was correct (Figure 2C).

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image2.png)

Figure 2A Scheme representing the major steps to obtain pET28a-luxCDABEGF. Step1: Perform PCR to linearize the vector, amplify gene frament and add homology arms. Step2: Conduct Gibson Assembly to assemble luxCDABE\_fragment, luxG\_fragment and pET28a-luxF linear vector.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image3.png)

Figure 2B the final plasmid graph of pET28a-luxCDABEGF.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image4.png)

Figure 2C The sequencing result of pET28a-luxCDABEGF

2 Induce Host Cells for Luminescence

Then the plasmid was transferred into BL21(DE3), a kind of competent cells adapted for protein expression. BL21(DE3) with pET28a\_luxCDABEGF was first cultured on LB plates(Kanamycin) for 12h under 37℃. Then three monoclonals were respectively cultured in 5ml LB(Kanamycin) overnight. The overnight cultures were added to new 5ml LB(Kanamycin) with a ratio of 1:100 for about 3 hours under 37℃. And the growing curve was detected before induction.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-table1.png)

When OD600 of cultures reached 0.6-0.8, the induction was initiated by 0.2 mM IPTG, followed by a 16-hour culture under 23℃. After that, we observed luminescence with a 3-second exposure by a camera. Here's the results (Fig 3A). Additionally, we magnified the culture in a 200ml LB for a better phenomenon. Both culture and detection manners were the same as above (Fig 2B).

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image10000.png)

Figure 3  The luminous E.coli BL21(DE3) with pET28a-luxCDABEGF, which was cultured for 16 hours under 23℃ after being induced by 0.2mM IPTG. The photo was captured by an ordinary phone camera with an exposure time for 3 seconds.

3 The Luminescence test

We detected the luminescence not only by naked eyes, but also by a luminous spectrum assay. The result was shown in Fig 4. The first column was luminous BL21(DE3) with an OD600 adjusted to 2.2 by additional LB; the second column was the negative control (BL21(DE3) transformed with original pET-28a with an OD600 of 2.2); and the third column was luminous BL21(DE3) with an unchanged OD600 of 4.2. It was obvious that the luminescence was successfully detected.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image6.png)

Figure 4 Luminescent test by a multimode plate reader. The first column was luminous BL21 with an OD600 adjusted to 2.2 by additional LB; the second column was the negative control (BL21 transformed with original pET-28a with an OD600 of 2.2); and the third column was luminous BL21 with an unchanged OD600 of 4.2. The test clearly showed that BL21(DE3) with pET28a-luxCDABEGF emitted strong luminescence.

4 The Determination of Best IPTG Concentration

Also, we conducted an experiment to determine the best IPTG concentration for luminescence. So, we set a gradient of 0, 0.05, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2(mM). Both culture and detection manners were the same as above. Here comes the result (Figure 5). Results showed that the best IPTG concentration for induction was between 0.05mM and 0.2mM.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image7.png)

Figure 5 Effect of the inducer concentration on light emission. 3 replications were set for each IPTG concentration, and the main value was used. OD600 was adjusted to 2 for each sample. The first 8 columns were luminous BL21(DE3) with different IPTG concentration and the negative control was BL21(DE3) transformed with an original pET28a plasmid. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG.

5 The Proof of Light Enhancement by luxG and luxF

In order to confirm whether luxG and luxF can enhance the brightness, we constructed plasmids with and without luxF,G and compared the luminescence of them. Here are the plasmid graphs of them. And after culturing and induction, manners of which were the same as those above, we got photos and luminescence data for comparison (Fig5A, 5B) Results clearly showed luxF and luxG strongly enhanced the brightness of designed BL21(DE3) (Figure 6A). And luminescence assay excitingly showed the addition of luxF and luxG increased the luminescence by 58% (Figure 6B). So, our design is definitely feasible.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image8.png)

Figure 6A The result of luminescence assay for BL21(DE3) with pET28a-luxCDABE and BL21(DE3) with pET28a-luxCDABEGF (n=6). The result showed that the addition of luxF and luxG increased the luminescence by 58%. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG. And the assay was conducted by a plate reader.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image9.png)

Figure 6B Comparing luminescence of BL21(DE3) with pET28a-luxCDABE or pET28a-luxCDABEGF. Images were taken by an ordinary phone camera. The left three tubes were BL21(DE3) with pET28a-luxCDABE and the right three tubes were BL21(DE3) with pET28a-luxCDABEGF. It was clear that luminescence can be enhanced by additional luxF and luxG. All BL21(DE3) underwent a 16-hour culture under 23℃ after being induced by 0.2 mM IPTG.

Conclusion

In this part, we successfully constructed pET28a-luxCDABEGF, and the host cell was enlightened as we have designed. Also, we determined the best concentration of IPTG to induce expression. And excitingly, we testified the capacity of luxF and luxG to enhance brightness, with a convincing data of 58%! It's undeniable that to enhance the brightness of LAMPS by luxF and luxG is feasible.

BRET Verif ication

In order to increase the brightness and change the colour of the Lux operon system using the principle of biofluorescence resonance energy transfer (BRET). We ligated the yellow fluorescent protein cp157Venus [(Part: BBa\_K4594005)](http://parts.igem.org/Part%3ABBa_K4594005) at the C-terminal of LuxB with a linker (Glu-Leu) to become a new fusion fluorescent protein LuxB:cp157Venus [(Part: BBa\_K4594006)](http://parts.igem.org/Part%3ABBa_K4594006), and in this way constructed pET-28a\_lacO-LuxA-LuxB [(Part: BBa\_K4594013)](http://parts.igem.org/Part%3ABBa_K4594013) and pET-28a\_lacO-LuxA-LuxB:cp157Venus [(Part: BBa\_K4594014)](http://parts.igem.org/Part%3ABBa_K4594014) two plasmids, they were plasmids amplified by DH5`\alpha` strain and transfected into BL21 (DE3) strain, cultured at 37°C and induced to express at 24°C. E. coli transfected with pET-28a\_lacO-LuxA-LuxB successfully luminesced, but E. coli with the fusion protein LuxB:cp157Venus did not luminesce. We speculate that the target protein expression was unsuccessful due to the occurrence of a gene mutation.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results100.jpg)

pET-28a\_lacO-LuxA-LuxB

Since the BRET principle has been proven in various literature, we firmly believe that our experimental principle is correct. Therefore, we will now turn our attention to our ultimate goal. Immediately after that, in order to find brighter fluorescent proteins after passing BRET, we first queried and screened the FPbase database for the two brightest proteins with fluorescence data and with excitation light between 490-520 nm and named them as A1 [(Part: BBa\_K4594007)](http://parts.igem.org/Part%3ABBa_K4594007) and A2 [(Part: BBa\_K4594008)](http://parts.igem.org/Part%3ABBa_K4594008). We also mapped the fluorescence data of the fluorescent proteins with incomplete information from amino acid sequences to fluorescence data by using the trained Long Short-Term Memory (LSTM) system and then filtered out the brightest two proteins named B1 [(Part: BBa\_K4594026)](http://parts.igem.org/Part%3ABBa_K4594026) and B2 [(Part: BBa\_K4594027)](http://parts.igem.org/Part%3ABBa_K4594027) from the mapped fluorescent proteins.

We synthesised the four genes A1, A2, B1, and B2 by GenScript and fused them at the C-terminal of LuxB in LuxCDABEGF using the same linker (Glu-Leu) to form four different fusion fluorescent proteins, and then the constructed plasmids were transferred into BL21(DE3) for induced expression.

After expression, it was found that the four LuxCDABEGFs fused with the new proteins A1, A2, B1, and B2 emitted light of significantly different colours, thus proving the feasibility of the BRET system. However, since the BRET efficiency is related to the sixth power of the distance, the brightness of the fusion proteins is weaker compared to the original LuxCDABEGF at non-optimal distances (all four proteins use the same linker as before).

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image11.png)

Afterwards, we conducted emission spectroscopy. The spectral data revealed that all four fused fluorescent proteins exhibited either a bimodal trend or a flat-head peak trend. The latter was due to the excitation light and emission light being close in frequency, and the brightness levels were fairly consistent across the board. The results of the spectral data proved the effectiveness of the LSTM system.

A1:

LuxB:A1 fluorescent protein after BRET the highest peak of emission spectrum should be around 525nm. The experimental results show a double peak. The peak at 490nm is the highest peak of emission light of the first protein. The experimental results are in accordance with the theoretical values.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results101.png)

The spectrum of LuxCDABEGF fused with A1

A2:

LuxB:A2 fluorescent protein should have the highest peak of the emission spectrum after BRET around 512nm. The experimental results show a double peak. The peak at 490nm is the highest peak of the emission light of the first protein. The experimental results are in accordance with the theoretical values.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results102.png)

The spectrum of LuxCDABEGF fused with A2

B1:

LuxB:B1 fluorescent protein after fluorescence resonance energy transfer the highest peak of the emission spectrum should be around 507nm, the peak at 490nm is the highest peak of the emission light of the first protein, but due to the first highest peak of the emission light is close to the second one, so the experimental double peaks are not obvious, and it becomes a flat head peak at 490-510nm. The experimental results are in line with the theoretical values. As far as the vertical axis RLU value is concerned, it is not much different from the A series fluorescent proteins and is in the same order of magnitude, which is in line with the prediction of the LSTM system and proves that the model prediction is successful.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results103.png)

The spectrum of LuxCDABEGF fused with B1

B2:

LuxB:B2 fluorescent protein after fluorescence resonance energy transfer the highest peak of the emission spectrum should be around 506nm, the experimental results show a double peak, the peak at 490nm is the highest peak of the emission light of the first protein. The experimental results are in line with the theoretical values. As for the RLU value of the vertical axis, it is not much different from the A series fluorescent proteins and is in the same order of magnitude, which is consistent with the prediction of the LSTM system and proves that the model prediction is successful.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results104.png)

The spectrum of LuxCDABEGF fused with B2

Finally, in order to obtain fluorescent proteins with brighter excitation light between 490-520 nm, we decided to break away from nature and create fluorescent proteins by ourselves using computer technology. We used Generative Adversarial Network to generate the amino acid sequences of the candidate fluorescent proteins, and then screened the generated sequences by LSTM system, and finally got the two brightest fluorescent proteins and named them as C1 [(Part: BBa\_K4594028)](http://parts.igem.org/Part%3ABBa_K4594028) and C2 [(Part: BBa\_K4594029)](http://parts.igem.org/Part%3ABBa_K4594029) . Since the effectiveness of the LSTM system has been proved in the previous experiments, the fluorescent proteins created as C1 and C2 have a certain degree of credibility.

Cyanobacteria Culture and Transformation Verif ication

Culture

Exploration of Optimal Antibiotic Concentration

After consulting the literature, we found that PCC7942 has some degree of ampicillin resistance. We inoculated algal culture in 7 groups of 5 ml liquid media with ampicillin concentrations ranging from 0-120 μg/ml, monitoring the growth. The goal was to identify a concentration of the antibiotic that prevents contaminants and allows good cyanobacterial growth. The specific data is as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-table2.png)

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-table3.png)

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-shr-wsb1.png)

We found that, except for the group with a concentration of zero antibiotics, all other groups of cyanobacteria exhibited significant decline in growth. This indicates that directly adding antibiotics to the culture medium cannot prevent cyanobacterial contamination.

Plate Purif ication

We inoculated algal culture in 8 groups of 1 ml liquid media with ampicillin concentrations ranging from 0-100 μg/ml and monitored their growth. We then streaked the culture from groups 6 and 7 onto antibiotic-free BG-11 agar plates. After 6-7 days, green colonies were observed, and single colonies were picked and cultured in 5 ml of antibiotic-free BG-11 liquid media while monitoring their growth. We observed poor growth, with OD750 reaching its highest value of 0.26 after 16 days. We also validated this by streaking on antibiotic-free LB agar and found that contaminants were still present.

After identification, we found that our cyanobacteria were contaminated by a nitrate-reducing bacterium, Nitratireductor sp.

Optimization Conditions

Light Intensity

The original light intensity used was 10,000 lux. After consulting with Professor Zhao Quanyu (see Human Practice for details), we learned that excessive light intensity, combined with antibiotic-containing culture medium, could induce stress in cyanobacteria. This may have been one of the reasons for the long-term transformation failures. Ultimately, we adopted a light intensity of 4,000-6,000 lux, and we were able to achieve normal growth of cyanobacteria on solid culture media.

Inoculation Density

Based on previous experiments and discussions with Professor Zhao Quanyu from Nan Jing Tech University, we speculated that the initial cell density of inoculation could affect the growth rate of cyanobacteria. This may have been a reason why cyanobacterial growth was poor when isolating colonies in liquid culture media. Therefore, we inoculated HL7942 cyanobacterial strains from solid culture media into liquid media with volumes of 0.5, 1, 2, and 5, and measured growth curves.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image13.png)

We assumed that OD750 was linearly related to cell density and multiplied OD750 by the volume to obtain the relative quantity of cells. The graph is as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image14.png)

It seems that inoculated in 1mL or 2mL is optimal.

This experiment provided preliminary validation, but it was not repeated, and the grouping was coarse. Further investigation is needed.

Recombination plasmid construction

We started with the plasmid pUC57-NS3-2-lacUV-cscB-lacI-KanR-NS3-1[BBa\_K4115045] left by the shanghaitech\_China 2022 team as the basis and used Gibson Assembly to construct our transformation plasmid, as shown in the diagram.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image15.png)

original plasmid pUC57-NS3-2-lacUV-cscB-lacI-KanR-NS3-1

Our plasmid construction underwent two rounds of Gibson Assembly. We replaced CscB with sfGFP(with LVA tag)[BBa\_K4115001] containing a degradation tag, and at the same time, we replaced the lacUV promoter with the final output promoter PKaiBC[BBa\_K4594011], which is an intrinsic rhythm promoter in cyanobacteria. Finally, we constructed the plasmid pUC57\_NS3-2-PKaiBC-sfGFP-lacI-KanR-NS3-1.[BBa\_K4594019]

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image16.png)

constructed plasmid pUC57\_NS3-2-PKaiBC-sfGFP-lacI-KanR-NS3-1.

After constructing the plasmid, we attempted to insert them into the cyanobacterial genome. Although the filamentous cyanobacteria can naturally take up foreign DNA, they cannot stably maintain plasmids.

After mixing the culture with the plasmid and overnight incubation, we plated the mixture on solid agar medium containing kanamycin to select for recombinant cyanobacterial colonies. After multiple experiments, we finally obtained cyanobacterial colonies on kanamycin-containing solid agar medium. Unfortunately, these cyanobacteria grew too densely, making it impossible to pick single clones.

First, we designed primers at the NS3 ends and performed PCR experiments, revealing that the transformed HL7942 did not have the desired fragment inserted, but PCC7942 showed a different pattern from the negative control group. Therefore, we scraped a bit of cyanobacteria transformed with the pUC57\_NS3-2-PKaiBC-sfGFP-lacI-KanR-NS3-1[BBa\_K4594019] plasmid as a template and designed primers on the left side of PkaiBC and the right side of sfGFP for PCR. After electrophoresis, we observed a clear and single band of the correct length (approximately 1236 bp).

Similarly, using cyanobacteria transformed with the pUC57\_NS3-2-PKaiBC-luxCDABEFG-KanR-NS3-1[BBa\_K4594020]. plasmid as a template, we designed primers at both ends of the lux gene cluster, and we also obtained the correct band (approximately 7212 bp). This indicates that there were successfully transformed PCC7942 colonies on several plates, but possibly due to antibiotic inactivation or insufficient concentration, these solid agar plates did not provide adequate selection, leading to the co-growth of wild-type cyanobacteria with the successfully transformed ones.

Obtaining Genetic Elements

Since neutral integration sites, PKaiBC, and other elements are present in the cyanobacterial genome, we aimed to obtain these gene fragments from cyanobacteria using PCR technology. We attempted various methods, including repeated freeze-thawing, sonication, and genome extraction, to prepare PCR templates. Although the sonication method achieved some success in obtaining the NS2 element (as seen in the figure below), it may be because sonication randomly disrupts the cyanobacterial genome, and this method of preparing templates was not successfully reproduced.

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-image17.png)

The first lane to the eighth lane represents ultrasonication for 0s, 20s, 30s, 60s, 90s, 120s, 180s, and 240s, respectively. Only the result of ultrasonication for 30s was positive.

Ultimately, we referenced the PCR method from Toulouse\_INSA-UPS in 2021 and attempted to prepare PCR templates by repeated freeze-thawing at -80°C/60°C. We successfully obtained the NS3 fragment from HL7942, and this method produced clear and bright bands in PCR for three different samples.

Transform Verif ication

We obtained a pure and improved PCC7942 strain from Luan Guodong of the Institute of Hydrobiology for use in transformation experiments. Use the natural transformation method (see protocol for details) to perform the transformation operation, and spread the bacterial solution on a BG11 solid plate containing 10 μg/mL kanamycin. Colonies grew after about 2 weeks. As shown below:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-plus01.png)

Due to lack of time, we were unable to perform characterization experiments on PKaiBC, so colony PCR was performed to verify whether the target gene was transferred.

We first used primers at both ends of NSIII and primers at both ends of the PkaiBC-sfGFP fragment to perform colony PCR, which were set as group 1 and group 2 respectively. Group 1 is expected to obtain a fragment band inserted into NSIII by homologous recombination, with a length of approximately 7000 bp, while Group 2 is expected to obtain a PKaiBC-sfGFP fragment band with a length of approximately 1200 bp.

The results of gel electrophoresis after PCR are as follows:

![](https://static.igem.wiki/teams/4594/wiki/wetlab-static/results-plus04.png)

Lanes 1, 2, and 3 from left to right are the selected successfully transformed colonies, lane 4 is the blank control, and the far right is the DNA Marker.

It can be seen that compared with the control group, the two groups have obvious bands of about 1200 bp, which proves that the transformation plasmid we constructed has been successfully transferred.

However, almost no correct bands were obtained in group 1. Article evidence shows that Synechococcus PCC7942 is polyploid [1]. We hypothesized that there may still be a blank NSIII fragment (approximately 1800 bp) in the successfully transformed strain, and the blank The fragment binds to the primer more readily than the inserted foreign fragment and undergoes chain growth catalyzed by the polymerase.

However, we obtained single-colony transformants and demonstrated the success of the transformation.

Reference

[1] Watanabe S. Cyanobacterial multi-copy chromosomes and their replication. Biosci Biotechnol Biochem. 2020 Jul;84(7):1309-1321. doi: 10.1080/09168451.2020.1736983. Epub 2020 Mar 11. PMID: 32157949.

![](https://static.igem.wiki/teams/4594/wiki/maomao04.png)

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