Part:BBa_K4955002
Restriction enzymes selective for chromosomes
Six-base recognition type restriction enzyme with many recognition sites in the ribosomal coding region
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 860
Illegal XhoI site found at 547 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
This system was developed by Takahito Mukai of Rikkyo University's Su'etsugu Laboratory.
Profile
name: restriction enzyme
Base Pairs: 1077 bp
Function (summary): Six-base-recognition restriction enzymes with multiple recognition sites in the ribosomal coding region.
Introduction
Our goal was to provide the public with E. coli that have accumulated antidepressant compounds in their cells right in the cookie. Current regulations on genetically modified organisms focus on cellular proliferation. Therefore, we hypothesized that eliminating the proliferating potential of our E. coli could lower the regulatory hurdle for implementing our cookies. Based on this hypothesis, we set a new goal to develop a system to eliminate the growth potential.
Because we envisioned social implementation, we sought a system that would minimize the incidence of escape. It is known that creating chromosome-free (i.e., non-proliferative) E. coli is possible by using chromosome-selective restriction enzymes and a strict expression regulation system [1]. We have started to develop a system that mimics this.
We demonstrated its function in BL21(DE3) and E. coli Nissle 1917 strains. In iGEM, we were the first team to propose the concept of using restriction enzymes as described above, and we were also the first to achieve chromosome-free E. coli successfully. Our system has strong potential and is highly standardized, and we expect it to be used by various iGEM teams in the future.
Currently, we are trying to reduce the incidence of escape mutants to a socially acceptable level by developing a more robust expression system. Since we aim to commercialize the iGEM project this year and obtain patents for the above-mentioned expression management mechanism and the system for reducing the incidence of escape mutants in the future, the information we can disclose at this stage is limited. However, if we successfully commercialize and obtain patents, we will disclose these systems to iGEM.
Biology & Function
Fig.1 Mechanism of our system
We have implemented a chromosome-free system using a six-base-recognition restriction enzyme with many recognition sites in the ribosomal coding region. We chose this system because of its low escape rate, and as a result, we created a highly standardized system that can be used in all living cells and various situations.
The strengths of the system we have developed are twofold:
1. It is usable in all living cells.
2. The designed plasmid does not degrade even if the chromosome is degraded.
Regarding the first point, the ribosomal coding region is conserved among all organisms. BBa_K4955002 has many restriction enzyme sites in the ribosomal coding region, so it can be applied to all cells. We have demonstrated the function of BBa_K4955002 in BL21(DE3) and E. coli Nissle 1917.
Regarding the second point, BBa_K4955002 is a six-base recognition mega-restriction enzyme. Therefore, it is impossible to have recognition sites in the designed plasmid (if there were, the number would be such that primers could be designed and synonymous codon substitutions could be made). This means the chassis can be made chromosome-free while maintaining the designed functionality. We have shown that the designed function can work in the chromosome-free BL21(DE3) strain. The advantages of this are as follows:
▶ Advantages of no-growth
・It may be able to lower the barriers to social implementation due to the Cartagena Act and other regulations.
The system we have developed may allow to run designed functionality on a chassis that does not proliferate. The type of chassis and functionality designed does not matter. This could be a powerful tool for microbial production, biosensing, drug delivery, cell surface display, and almost any other iGEM project that can be implemented in society.
▶ Advantage as a novel chassis
・Eliminate concerns about interference from the native gene network.
The goal of synthetic biologists is to design organisms to perform their functions in a safe, reliable, and robust manner. In contrast, organisms continue to change through adaptation, evolution, and proliferation. This conflict leads to interference from the native gene network (for example, variability [2] and unpredictable expression [3]). However, chromosome-free cells have great potential to express synthetic gene circuits predictably. This is not only a scientific advance in synthetic biology but also one of the factors that will ensure the high resolution of the product, which is essential for its societal implementation.
▶ Other advantages
・Cell proliferation requires the consumption of large amounts of ATP. Chromosome-freeing allows energy originally used for proliferation to be used for functional circuits [5].
・Chemical and irradiation bacterial inactivation techniques can damage cellular systems; chromosome-free cells are replication-deficient, and because the enzymatic genome removal process does not damage the cellular system, they are safe and effective for use in situations where biological containment is critical.
Measurement
Plasmid
We used the Jungle Express System [4], which uses inexpensive Crystal Violet (CV) as an inducer and has a very strict expression control mechanism as an expression system.
We aimed to introduce BBa_K4955002 into a chassis containing pRK404-BBa_K4955000, which was already confirmed to produce crocetin, to see how the designed plasmid would function under the chromosome-free environment. However, since pRK404-BBa_K4955000 happened to have a recognition site for BBa_K4955002, we redesigned the plasmid with a synonymous codon substitution. The redesigned plasmid will be referred to as pRK404-BBa_K4955000 (ΔRE).
E. coli strain
BBa_K4955002 was introduced by the TSS method into BL21(DE3), and E. coli Nissle 1917 strains heat shocked with pRK404-BBa_K4955000 (ΔRE).
Confirmation of no further proliferation by colony formation
The transformed BL21(DE3) and E. coli Nissle 1917 strains were plated on LB agar medium containing appropriate antibiotics and CV (1 μM) and on LB agar medium containing appropriate antibiotics only. The results showed a clear inhibition of colony formation in both CV-containing plates. This result indicates that we have produced a non-proliferative E. coli strain.
However, some colony formation is observed even in plates containing CV, which is thought to be due to the appearance of a certain percentage of escape mutants.
Confirmation of chromosome-free by fluorescence microscopy
We used DAPI and fluorescence microscopy to confirm the chromosome-free status for further functional verification. Since DAPI is a fluorescent dye that binds to DNA, the presence or absence of chromosomes can be confirmed by the presence or absence of fluorescence.
We successfully confirmed the chromosome-free status of the E. coli strains by sampling the culture medium at several time points after CV induction and observing the results under fluorescence microscopy.
Result of CV induction with OD600=0.2
Protocol
Cultivation
Preculture: Inoculate in 5 mL LB with appropriate antibiotic and cultured in test tubes by shaking (140 rpm sideways, 37°C). After 18-24 hours, the culture is transferred to the main culture.
Main culture: The pre-culture was diluted to the final culture 5 ml, OD600=0.01, and cultured in test tubes by shaking (140 rpm, 37°C). 1 μM final CV was added at OD600=0.2. Test tubes without CV were also cultured simultaneously as a negative control.
After CV induction, samples were collected at 0h, 0.5h, 1h, 2h, and 4h for fluorescence microscopy.
~Fluorescence Microscopy
Sample Preparation
OD measurement was performed before the preparation of plates for observation.
The target Culture was sampled at 500 µl and vortexed for 30 sec with 2.5 µl of DAPI in a refrigerator at 4°C. The DAPI was added on ice.
The light was shaded and allowed to stand at room temperature for 5 min.
Centrifuged at 5000 rpm for 3 min at room temperature and discarded 300 µl of supernatant.
Ten μl of POLY-L-LYSINE stored in a refrigerator at 4°C was applied to a slide on ice, spread over the entire surface using a tip and dried.
Once the POLY-L-LYSINE was completely dry, 5 μl of the sample adjusted as described above was spread, and a cover glass was placed.
The cover glass was pressed against the cover glass using Kimwipes, and any overflowing liquid was wiped off with the Kimwipes.
The gap between the cover glass and the slide was applied with clear nail polish to seal the gap and left at room temperature until completely dry.
~How to use the Keyence fluorescence microscope
The microscope(BZ-X710, Keyence) and PC were started, and BZ-X Viewer was launched.
Selected still image capture.
The objective lens was set to 100x oil immersion lens, the microscope cover was opened, and a drop of oil was placed on the tip of the objective lens.
A slide was set, and the lens tip was aligned with the location to be observed.
When observing, bright field (CH4) was selected, multicolor was enabled, and overlay was set to disabled.
The focus was set so that the fungus was clearly visible.
If the number of visible bacteria was low, the supernatant was discarded after centrifugation at 5000 rpm for 5 min at room temperature in an appropriate volume.
If the amount of bacteria was too large to observe cleanly, it was suspended in TB.
Fluorescence was set to blue (CH3), and the fluorescence exposure time was simultaneously set to CV+- of the sample.
Results
Only the CV-induced sample showed a loss of fluorescence. This indicates that the function of BBA_K495502 chromosome-freed the sample.
Result of CV induction at OD600=8
Our system’s strength is maintaining the functionality of the designed plasmid even when it becomes chromosome-free. When we looked at substance production in BBa_K4955000 and others, we performed IPTG induction at OD600=8. We tried to see if we could confirm material production in chromosome-free E. coli by performing CV induction simultaneously.
After CV induction of the E. coli strain, the culture was measured by HP-LC-MS/MS when the chromosome-free state was confirmed. Crocetin production was confirmed in the culture in which CV induction was performed. However, it is possible that this was due to the detection of crocetin produced by E. coli before the chromosome-free state or by an escape mutant.
Protocol
Preculture: Inoculate in 5 mL LB with appropriate antibiotic and cultured in test tubes by shaking (140 rpm sideways, 37°C). After 18-24 hours, the culture is transferred to the main culture.
Main culture: 1 mL preculture solution and 49 mL TB with appropriate antibiotics were cultured in a 300 mL Erlenmeyer flask with shaking (150 rpm, 25°C). Final 0.1 mM IPTG and 1 μM CV were added when OD600=8-12. A sample without CV was prepared as a negative control. Cells were then incubated for 72 hours.
After CV induction, samples were collected at 0h, 0.5h, 1h, 2h, 4h, and approximately every 12h thereafter, and fluorescence microscopic observation was performed.
The protocol for Fluorescence Microscopy is the same as above except the OD600 values.
Chromosome-free proportions were analyzed using ImageJ. Scripts for ImageJ are available here:https://gitlab.igem.org/2023/software-tools/japan-united/-/blob/main/imagejMethods.ijm?ref_type=heads
Results
CV induction at OD600=8 also confirmed the chromosome-free status of E. coli. ImageJ was used to determine the proportion of E. coli that were chromosome-free. At this time, at least 300 bacteria were analyzed per condition. Compared to the negative control sample without CV induction, the 30-72h sample in the CV-induced sample was chromosome-free.
The fact that chromosome-free status was confirmed even in the samples without CV induction is considered to be a problem with the images analyzed or the analysis tool. The sharp drop in the proportion of E. coli in the chromosome-free state in the 84h CV-induced sample is thought to be due to the proliferation of escape mutants.
HP-LC-MS/MS analysis of a sample taken 72 hours after CV induction, in which the chromosome-free state was confirmed, and a sample taken without CV induction but cultured under the same conditions, confirmed the production of crocetin in both cases. However, it is possible that this was due to the detection of crocetin produced by E. coli before the chromosome-free state or production by an escape mutant. The extraction and HP-LC-MS/MS conditions were the same as the protocol used for BBa_K4955000, which had already successfully detected crocetin.
References
[1]Catherine Fan, Paul A. Davison, Robert Habgooda, Hong Zeng, Christoph M. Decker, Manuela Gesell Salazar,Khemmathin Lueangwattanapong, Helen E. Townley, Aidong Yang, Ian P. Thompson, Hua Ye, Zhanfeng Cui,Frank Schmidt, C. Neil Hunter, and Wei E. Huang.(2020).Chromosome-free bacterial cells are safe andprogrammable platforms for synthetic biology.PNAS.
[2]R. Kwok. (2010). Five hard truths for synthetic biology.Nature 463, 288–290
[3] R. M. Blumenthal, S. A. Gregory, J. S. Cooperider. (1985). Cloning of a restriction-modificationsystem from Proteus vulgaris and its use in analyzing a methylase-sensitive phenotypein Escherichia coli.J. Bacteriol.164, 501–509
[4]Thomas L. Ruegg, Jose H. Pereira, Joseph C. Chen, Andy DeGiovanni, Pavel Novichkov, Vivek K. Mutalik, Giovani P. Tomaleri, Steven W. Singer , Nathan J. Hillson , Blake A. Simmons , Paul D. Adams & Michael P. Thelen
[5]Kasari, M., Kasari, V., Kärmas, M. & Jõers, A. Decoupling Growth and Production by Removing the Origin of Replication from a Bacterial Chromosome. ACS Synth. Biol. 11, 2610–2622 (2022)
| None |