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.

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.

Figure 1 The mechanism of light enhancement by luxF and luxG

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.

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

Figure 2 Graph of pET28a-luxFCDABE

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.

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.

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.

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

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.

Figure 3 the final plasmid graph of pET28a-luxCDABEGF 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. 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.

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!

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.

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:

Figure 1 : The plasmid of pET-28a(+)_LuxA, LuxBΔ(lacI) Figure 2 The plasmid of pET-28a(+)_LuxA, LuxB:cp157VenusΔ(lacI)

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.

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.

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

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:

Figure 3 The plasmid of pET-28a(+)_LuxA, LuxB Figure 4 The plasmid of pET-28a(+)_LuxA, LuxB:cp157Venus

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.

Figure 5 Protein expression after plasmid repair

This time we contacted the author of the original article and got a lot of useful information from him(See Human Practices # 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.

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.

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

Figure 6 The plasmid of pET-28a(+)_LuxA, LuxBΔ(AlacO, BlacO) Figure 7 The plasmid of pET-28a(+)_LuxA, LuxB:cp157VenusΔ(AlacO, BlacO)

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.

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.

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

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:

Figure 8 The plasmid of pET-28a_lacO-LuxA-LuxB Figure 9 The plasmid of pET-28a_lacO-LuxA-LuxB:cp157Venus

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.

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.

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.

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.

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.

Figure 10 cp157Venus and A1 Figure 11 A2 , B1 , LuxCDABEGF and Control

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.

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.

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.

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.

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.

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.

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

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.

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.

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.

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

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.

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.

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.

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:

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.

Transformed PCC7942

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.

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.