Part:BBa_K4782006

Profile

Name: ALPaGA (Codon-optimised LldRPD promoter) + RBS+ Colicin E1 from E.coli
Origin: Escherichia coli, synthetic
Properties: Colicin E1 expression induced by sodium lactate in anoxia and glucose rich environment
Design: codon optimized LldRPD promoter for glucose-rich and anoxic environment
Subparts: BBa_K4244000, BBa_K2922024

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 1535
  Illegal AgeI site found at 1592
  Illegal AgeI site found at 1724
  
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Biology

It has been a hot topic on how to make chemotherapy less invasive. Synthetic biology proposes a solution by synthesizing anti-tumor drugs only in response to the tumor microenvironment. In this system, the widely used chemotherapeutic agent Colicin E1 is expressed in response to the presence of L-lactate (sodium lactate). Tumor microenvironment (TME) is widely associated with high lactate concentrations. It is related to the increased glucose uptake and lactate production as a result of hypoxia and glycolysis as a metabolic pathway recruited by cancer cells to obtain energy, known "Warburg effect". This leads to the acidification of TME to pH 6.3-6.9, which favors tumor promotion, angiogenesis, and immunosuppression and leaves lactate as the metabolic end product. Along with the Warburg effect, the production of lactate and protons primarily stems from glutaminolysis. ^[1] It is reported that while in normal physiological cells have a lactate concentration of 1.5-3 mM, in TME, it is 15-30 mM and can reach up to 40 mM.^[2] Hence, lactate is a reliable reporter for the TME and could be used as a condition for the synthesis of anti-tumor drugs.

lldPRD is an operon used by E.coli cells to detect, grab, and metabolize L-lactate as a secondary carbon source when the cell is scarce on its primary carbon source, glucose. Therefore, the wild-type E.coli shows that the lactate utilization operon has lower induction by 70% in the presence of glucose. Moreover, wild-type promoter is expressed in high copies and oxic conditions (oxygen presence).^[3]

Difference between PLldPRD and ALPaGA biosensors:

Experimental data indicates that wild-type PLldPRD has a significantly lower performance compared to ALPaGA in both glucose-containing anoxic ^[4,5]
and glucose-containing aerobic experimental settings ^[6]. As for the succinate-containing aerobic environments, both systems perform quite similarly.Functional analysis of both l-Lactate biosensors in E. coli cells indicates that leakage RPU (reference promoter units) of PLldPRD equals 0.8 ± 0.0, while for ALPaGA, leakape RPU = 1.0 ± 0.1. As for the glucose-rich aerobic settings, leakage RPU for PLldPRD = 0.2 ± 0.0 and for ALPaGA it is equal to 0.9 ± 0.0. Similar trend was observed in glucose-rich anoxic conditions: Leakage RPU is equal to 0.2 ± 0.0 for PLldPRD and 0.9 ± 0.0 for ALPaGA. Therefore, protein of interest production by PLldPRD-based systems is low in the cancer microenvironment due to anoxic and glucose catabolite-rich settings.

While posing metabolic stress on a host cell, wild type poses many limitations for its applications for tumor microenvironment, which is both hypoxic and glucose-rich. Moreover, the llDR is genetically unstable и could be aborted from the host cell due to metabolic burden. ALPaGA is expressed in pET9a, a low-copy plasmid that could be used in hypoxic, anoxic (absence of oxygen), and aerobic conditions.^[6]

Molecular Mechanism

ALPaGA Promoter

For lactate sensing, the two main components are lldP and lldR. lldP is a transmembrane protein that imports L-lactate from the environment into the cell, while LldR is an intracellular protein that binds to two DNA sites, O1 and O2. The binding of lldR to these sites leads to the formation of a DNA loop that “hides” the region between O1 and O2 from the transcription activation complex. On the other hand, once L-lactate is present, lldR binds it and undergoes a conformational change as a consequence. This results in the loop opening whereby intervening regions are exposed again.

^[8]

Colicin E1

Colicins are a group of highly specific bactericidal toxins which are produced by different strains of Enterobacteriaceae; Colicins reduce the viability of the same species strains or the strains of the same genus ^[6]. Colicin synthesis is based on the presence of Col-like plasmids located in the cytoplasm of the bacteria. One of the examples of the bacteria that can produce Colicins is E.coli ^[9]. Overall, there are two types of Colicins: Colicins A and B. Colicins from the A groups exhibit pore-forming activity, disturbing the bacteria's cell wall and cell membrane structure, thus causing an efflux of vital intracellular components and nutrients. Examples of Colicins of the A group are Colicin E1, Colicin A, and Colicin B. On the other hand, Colicins from the B group have a nuclease activity and allow them to destroy nucleic acids after the translocation into the nucleus. The examples of Colicin from the B group are: Colicin E2, E3, E4 ^[10].

The cytotoxic activity of ColE1 in tumor cells is explained due to the fact that tumor cells exhibit higher production of the vitamin B12 receptors, BtuB, which is a receptor for Colicin E1 binding to the plasma membrane ^[11]. When ColE1 is diffused from the producing bacterial cell, it dissociates from its Immunity protein and becomes active. When in its active form, ColE1 possesses a highly cationic character, thus, it is easily attracted to cancer cells, the cell membrane of which is negatively charged due to the Warburg effect ^[12]. The structure of Colicin is composed of three domains: T-domain (translocation domain), R-domain(receptor-binding domain) and C-domain (catalytic domain)^[13]. Initially, when ColicinE1 molecules approach the cell membrane of a cancer cell in close proximity, they bind to the BtuB receptor via the R-domain, which have extremely high affinity to each other. When bound to the BtuB receptor, the T- and C-domains become available for transfer across the membrane. After being translocated into the cytoplasmic environment, the T- and C-domains dissociate, and the C-domain exhibits its cytotoxic activity. It has been experimentally proven that Colicin E1 exhibits cytotoxic activity in low concentration on all cancerous cell lines. For example, fibrosarcoma HS913T growth was inhibited by 50% upon treatment with Colicin E1 ^[14].

Experience: Experiment Lists and Applications

Transformation and Cultivation with Kanamycin

The batch of competent cells DH5a with optical density 20 and plasmid were unfrozen in ice for 5-10 minutes. The concentration of plasmid of interest was measured in NanoDrop (ng/μL). Under the microbiological hood, 200 ng of plasmid was added to competent cells. They were incubated on ice for 30 min, and heat shock in 42°C for 1 min. They were then transferred to ice and incubated for 1 min. Using aseptic techniques, 900 uL sterile LBbroth media was added. The cells were incubated in a 37°C shaker incubator at 180-200 rpm for 40 min. After incubation, 1/5 of the batch with transformed cells was seeded on a 10 cm agar plate with Kanamycin 50 µg/ml and 4/5 in another dish. The cells were spread with an L-shaped spreader until the surface was dry. They were left to incubate overnight (12-18 hours) at 37°C.

Purification of plasmid and gel electrophoresis

Heat the solution of 500 mg agarose to 50 mL Tris Acetate EDTA (1%), after it is cool enough add EtBr (working concentration of 0.2-0.5 µg/mL), pour the solution in the electrophoresis tray and put respecetive sized comb. After it has solidified, add 300 ng of plasmid and LoadingDye 5X and top up with DI water. Remove the comb, fill the tray with TAE so it covers gel, and load 6ul of sample to each well and DNA ladder. The gel is run on 120V for 30 min.

Expression of ColE1 by sodium lactate

The expression is conducted by mentioned protocol of transfromation in E.coli BL21 cells. The ColE1 is expressed in response to sodium lactate solution (1:1 D- and L- lactate). DIfferent overnight cultures are induced via 0, 10, 15, 20, 30, 40 mM of L-lactate (twice as much is needed if sodium lactate is used). This will report Colicin E1 expression in lactate concentrations in physiological conditions as well as in the TME.

Cultivation with L-lactate and SDS-PAGE

Transformed E.coli BL21 were incubated overnight at a temperature of 37C on a shaking incubator. The cultures were then diluted to OD600 = 0.1-0.2, and cultivated at 37C until OD600 = 0.5-0.6. Following that, bacteria were induced with 20 mM lactate and further incubated at 37 C for 8 hours. The expression cultures were then collected using a centrifuge at 5700 rpm for 30 minutes at 4C. As bacteria pellet was obtained, it was resuspended with a lysis buffer in an ice bath, in a ratio of 3 ml of buffer per 1 gram of bacteria. Lysates were collected using a centrifuge at 5700 rpm for 10 minutes. The obtained supernatant was further centrifuged at 20200 rpm for 1 hour at 4C. As samples were centrifuged, they were passed through a 0.22 micron filter. For SDS-PAGE gel electrophoresis the gels were prepared first. After that, 25 uL of protein supernatant were taken, and 5 uL of 6x Loading buffer was added to the protein. The samples were then bathed at 95C for 10 minutes. As the SDS-PAGE gel electrophoresis chamber was assembled, 5 uL of protein ladder were transferred to the first well of the gel, and 30 uL of protein were transferred to the second well. The samples were run at 100 V for 15 minutes, and then voltage increased to 220 V for 30 minutes. The samples then were carefully extracted and washed using a destain solution for 10 minutes on a shaker. Coomassie Blue was then applied to the gel for 20 minutes on a shaker. As samples were stained, Coomassie Blue stain was discarded, and samples were washed with dH2O, and washed with destain solution several times until gel got transparent. The proteins were visualized using ChemiDoc Imaging System.

The concentration of Colicin E1 synthesized by E.coli BL21 (DE3) transformed with this our plasmid. The model simulated on MATLAB Simbiology software proposes the synthesis of Colicin E1 in efficient concentrations to kill cancer cells.

Ion-Exchange Chromatograpy

A syringe is filled with a start buffer and is connected to a column. The cap should be removed and the column should be washed with 5ml of Start buffer at a flow rate of 1ml per minute. Then, the column should be washed with 5ml of Elution buffer at the same flow rate. The column should be brought to equilibrium with 5-10ml of Start Buffer. The prepared bacterial lysate sample is applied to the column and the eluted liquid is collected. The Start buffer should be applied until no eluent is seen, using 5ml. The eluted liquid is to be collected and the eluent is to be saved. After the elution, the column should be regenerated with 5ml of Regeneration buffer and the eluent should be saved. The column should be washed with 5-10ml of Start buffer and the eluent should be saved. The protein concentration of each collected liquid should be checked on nanodrop, and the presence of the protein of interest should be determined on SDS-PAGE. A start buffer-filled syringe is linked to a column. Wash the column with 5ml of Start buffer at 1ml per minute after removing the lid. Wash the column with 5ml of Elution buffer at the same flow rate. The column should be balanced with 5-10ml Start Buffer. Apply bacterial lysate to the column and collect the eluted liquid. 5ml of Start buffer should be added until no eluent is observed. Collect and preserve the eluted liquid. After elution, renew the column with 5ml of Regeneration buffer and preserve the eluent. Wash the column with 5-10ml of Start buffer and preserve the eluent. Nanodrop should measure the protein content of each liquid, and SDS-PAGE should detect the protein of interest.

Applications of ALPaGA biosensors

ALPaGA has a wide range of possible applications in research, medicine, and industry. One of the primary usages of this lactate biosensor system is production of anticancer therapeutic agents. The system can be applied to detect lactate bioproduction in the food industry and pharmaceutical fields. Moreover, ALPaGA has a potential to be used in cancer detection since elevated lactate production is one of the hallmarks of solid cancer development. This promoter can also be incorporated into various biotechnology processes. ^[1]

References

[1]Pérez-Tomás R, Pérez-Guillén I. Lactate in the Tumor Microenvironment: An Essential Molecule in Cancer Progression and Treatment. Cancers (Basel). 2020 Nov 3;12(11):3244. doi: 10.3390/cancers12113244. PMID: 33153193; PMCID: PMC7693872.
[2]de la Cruz-López KG, Castro-Muñoz LJ, Reyes-Hernández DO, García-Carrancá A, Manzo-Merino J. Lactate in the Regulation of Tumor Microenvironment and Therapeutic Approaches. Front Oncol. 2019 Nov 1;9:1143. doi: 10.3389/fonc.2019.01143. PMID: 31737570; PMCID: PMC6839026.
[3]Aguilera L.; Campos E.; Giménez R.; Badía J.; Aguilar J.; Baldoma L. Dual Role of LldR in Regulation of th.e lldPRD Operon, Involved in l -Lactate Metabolism in Escherichia coli. J. Bacteriol. 2008, 190, 2997–3005. 10.1128/jb.02013-07>
[4]Bentley W. E.; Mirjalili N.; Andersen D. C.; Davis R. H.; Kompala D. S. Plasmid-encoded protein: The principal factor in the ″metabolic burden″ associated with recombinant bacteria. Biotechnol. Bioeng. 1990, 35, 668–681. 10.1002/bit.260350704.
[5] Iuchi, S., Aristarkhov, A., Dong, J. M., Taylor, J. S., & Lin, E. C. (1994). Effects of nitrate respiration on expression of the Arc-controlled operons encoding succinate dehydrogenase and flavin-linked L-lactate dehydrogenase. Journal of bacteriology, 176(6), 1695–1701. https://doi.org/10.1128/jb.176.6.1695-1701.19
[7]Zúñiga, A., Camacho, M. D., Chang, H., Fristot, E., Mayonove, P., Hani, E., & Bonnet, J. (2021). Engineered l-Lactate Responding Promoter System Operating in Glucose-Rich and Anoxic Environments. ACS Synthetic Biology, 10(12), 3527–3536. https://doi.org/10.1021/acssynbio.1c00456
[8]Spangler, R., Zhang, S. P., Krueger, J., & Zubay, G. (1985). Colicin synthesis and cell death. Journal of bacteriology, 163(1), 167–173. https://doi.org/10.1128/jb.163.1.167-173.1985 https://pubmed.ncbi.nlm.nih.gov/3891723/
[9]Clark, D. P., Pazdernik, N. J., & McGehee, M. R. (2019). Chapter 16–regulation of transcription in Prokaryotes. Molecular Biology (Third Edition), Academic Cell, 522-59.
[10]Sannigrahi, A., & Chattopadhyay, K. (2022). Pore formation by pore forming membrane proteins towards infections. Advances in Protein Chemistry and Structural Biology, 128, 79-111.
[11]Gupta, Y., Kohli, D. V., & Jain, S. K. (2008). Vitamin B 12-mediated transport: a potential tool for tumor targeting of antineoplastic drugs and imaging agents. Critical Reviews™ in Therapeutic Drug Carrier Systems, 25(4). https://www.dl.begellhouse.com/journals/3667c4ae6e8fd136,2f9ffbb97317f5ec,2ab5e3416bb865a1.html
[12}Fathizadeh, Hadis & Saffari, Mahmood & Esmaeili, Davoud & Moniri, R. & J., Amini. (2021). Anticancer Effect of Enterocin A-Colicin E1 Fusion Peptide on the Gastric Cancer Cell. Probiotics and Antimicrobial Proteins. 13. 10.1007/s12602-021-09770-y. https://www.researchgate.net/publication/352426578_Anticancer_Effect_of_Enterocin_A-Colicin_E1_Fusion_Peptide_on_the_Gastric_Cancer_Cell
[13]Kaur, S., Kaur, S. (2015). Bacteriocins as Potential Anticancer Agents. Cancer Molecular Targets and Theraupetics. Volume 6. https://doi.org/10.3389/fphar.2015.00272 https://www.frontiersin.org/articles/10.3389/fphar.2015.00272/full