Difference between revisions of "Part:BBa K4182011"
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==Usage&Biology==
 
==Usage&Biology==
  
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===Design new suicide circuits for engineering efficiency and biosafety reasons===
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Biosafety is an important consideration when designing engineered bacteria, and MazF has been commonly used in previously work. Here we develop a novel lysis gene (Gene ID: IF654_RS00240) for the design of controlled suicide circuits for engineering efficiency and biosafety reasons, also can be considered as an alternative of suicide protein MazF.
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To facilitate the modularized design of plasmids, we named the EPS synthesis verification plasmid 4, which will be referred to as plasmid 4 in the following paragraphs.
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==Source and Principle==
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Biosafety is an important consideration when designing engineered bacteria. From the beginning, we designed the bacteria on the premise that it would work in the field soil, so we first needed to consider whether our product could be easily controlled for the time of its operation and whether there were potential risks to soil structure, crop growth, and the balance of soil microbiota. So we designed a "suicide system" at the genetic level to ensure that our engineered bacteria would not pose a potential biosecurity risk to the ecological environment.
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The suicidal behavior of bacteria is a common phenomenon in nature, which is a programmed death mechanism of prokaryotes. quorum sensing (QS) is a form of communication between bacterial cells. Cells synthesize and secrete signal molecules. When the concentration of signal molecules in the environment reaches a certain threshold, a series of genes are activated, and the bacterial population synchronously realizes certain functional and behavioral changes. A quorum-sensing suicide gene circuit has been constructed, and the systematic study and precise regulation of this gene circuit are of great significance both in theory and application [1].
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In addition to population-responsive suicide mechanisms, suicide systems with other regulatory modes can also be designed through synthetic biology. Here, we designed a temperature-responsive cleavage system to achieve temperature-controlled cleavage, that is, cleavage of thermoregulated lysis genes (Gene ID: IF654_RS00240) (Figure 1).
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[[File:XJTU-p5-1.png|500px]]
 
[[File:XJTU-p5-1.png|500px]]
  
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Figure 1: Circuit diagram of plasmid 5: Where CI is the C1857 suppression subsystem, Pλ is the promoter, and the temperature control system is in the dashed box.
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Figure 1: The temperature-controlled suicide circuit with a novel lysis gene
  
 
[[File:XJTU-p5-2.png|500px]]
 
[[File:XJTU-p5-2.png|500px]]
  
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Figure 2 shows the principle of the temperature control system [4]. When bacteria are at a low temperature, the c1857 gene expression protein binds to the Pλ promoter, making downstream genes unable to be translated. At 42℃, the protein will be cleaved, leading to the expression of downstream genes.
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Figure 2: The principle of temperature-controlled suicide circuit
  
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In conclusion, we wanted to take advantage of temperature changes as a variable environmental signal, allowing our engineered bacteria to function at lower temperatures and Lysis proteins to lysis the engineered E. coli cells at higher temperatures, resulting in control of the engineered bacteria and release of the product.
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Figure 1 and Figure 2 shows the principle of our temperature-controlled suicide circuit. When bacteria grows at a low temperature(30℃), the CI857 protein binds to the Pλ promoter, and downstream lysis gene are unable to be expressed, allowing the cell growth. While at 42℃, the CI protein will be degraded and lead to the expression of lysis gene and eventually cell death and release of the product.
  
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==Codon improvement and optimization==
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===Codon optimization of lysis gene===
  
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The Lysis gene we used was expressed in Pseudomonas lundensis. To better express the Lysis gene in engineered bacteria, we optimized the codon of the Lysis gene according to the codon preference of Escherichia coli. Figure 5-3 shows the number of codons we optimized to make our codons more in line with Escherichia coli preference. The modified Lysis gene is shown in BBa No.K4182007.  
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The lysis gene is from Enterobacteria phage KleenX174. To better express the lysis gene in engineered bacteria, the codon optimization of the lysis gene was conducted according to the codon preference of Escherichia coli. Figure 3 shows the number of codons we optimized to make our codons more in line with Escherichia coli preference. The modified lysis gene is shown in BBa_K4182007.  
  
 
[[File:XJTU-p5-3.png|500px]]
 
[[File:XJTU-p5-3.png|500px]]
  
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Figure 3: Optimized Sequence Codon in plasmid Ⅴ
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Figure 3: Codon optimization of lysis gene
  
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==Plasmid design and improvement==
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===Construction and optimization of suicide circuit===
  
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Initially, based on the design of the assay protocol, we planned to construct plasmid 5 (FIG. 4) using plasmid pSB1K3 as the skeleton and lysis gene + temperature regulator mode.
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Initially, we planned to construct our suicide circuit (Plasmid 5) using vector backbone pSB1K3 by Golden Gate assembly, and we can obtain several clones. However, it was found that after colony PCR verification, only weak target bands could be observed (Figure 4), and the plasmids extracted from the recombinant DH5α cells was at very low concentration, and sequencing could not be completed.  
  
 
[[File:XJTU-p5-10.png|500px]]
 
[[File:XJTU-p5-10.png|500px]]
  
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Figure 4: Plasmid 5 map based on pSB1K3
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Figure 4: Plasmid 5 map based on pSB1K3 and its verification (he target band is approximately 1600bp)
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However, in our subsequent experiments, it was found that when the plasmids designed in this way were transferred to DH5α cells after Golden Gate cloning for expression, only dark target bands could be observed in colony PCR (Figure 5), and the extraction of plasmids and sequencing could not be completed due to the low concentration
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[[File:XJTU-p5-5.png|500px]]
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Figure 5: PCR results of plasmid 5 colonies based on pSB1K3. The target band is approximately 1600bp long and is marked by blue arrows
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Therefore, we judged that due to the insufficient copy amount of pSB1K3 plasmid, we could not extract the product with a sufficient concentration in the engineered bacteria.
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Therefore, we replaced the vector of plasmid 5 with pSEVA341, which had a higher number of copies, and redesigned the plasmid (Figure 6).
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[[File:XJTU-p5-6.png|500px]]
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We supposed that due to the low copy number of pSB1K3 vector, it is difficult to extract sufficient plasmid from the engineered bacteria for further validation. Therefore, we replaced the backbone to pSEVA341, a higher-copy-number vector and re-constructed the plasmid (Figure 5). As shown in Figure 6, the obvious target bands were observed, and the plasmid correctness was further confirmed by sequencing.
  
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Figure 6: Map of modified plasmid 5
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[[File:XJTU-p5-11.png|500px]]
  
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==Experimental verification==
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Figure 5: the new plasmid 5 with pSEVA341 backbone and its verification
  
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===Colony PCR===
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===The verification of heat triggered cell lysis and suicide===
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The engineered cell harboring plasmid 5 and blank vector respectively, were culture at 30℃ overnight, and  then the temperature was shift to 42℃. The OD600 of each group was detected every 1 h, and the growth curve of these strains were determined as follows.
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The results clearly demonstrated the cell growth was significantly inhibited after heat at 42℃ compared to the strain without lysis gene.  And about 9% of whole cell population was retained after heat. Compared to the commonly used suicide protein MazF (BBa_K302033), the lysis protein in our study is also efficient but shorter and easy to be manipulated, which can be used as an alternative and update for MazF.
  
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After the primer design was completed, the fragments were connected by Golden Gate ligation and transferred into competent DH5α cells, which were cultured in the medium containing chloramphenicol. The primers were designed for colony PCR, and obvious target bands were observed (Figure 7). The sequencing results were correct
 
  
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[[File:XJTU-p5-7.png|500px]]
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[[File:XJTU-p5-12.png|500px]]
  
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Figure 7: PCR results of plasmid 5 colonies after vector change. The target band is marked by a blue arrow
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Figure 6: The cell growth of strains with suicide plasmid 5 and blank vector
  
 
==References==
 
==References==

Revision as of 18:45, 13 October 2022


Temperature regulated suicide circuit

In addition to population-responsive suicide mechanisms, suicide systems with other regulatory modes can also be designed through synthetic biology. Here, we designed a temperature-responsive cleavage system to achieve temperature-controlled cleavage, that is, cleavage of thermoregulated lysis genes (Gene ID: IF654_RS00240)

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1018
    Illegal suffix found in sequence at 1446
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1018
    Illegal SpeI site found at 1447
    Illegal PstI site found at 1461
    Illegal NotI site found at 1024
    Illegal NotI site found at 1454
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1018
    Illegal XhoI site found at 2478
    Illegal XhoI site found at 3504
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1018
    Illegal suffix found in sequence at 1447
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1018
    Illegal XbaI site found at 1033
    Illegal SpeI site found at 1447
    Illegal PstI site found at 1461
  • 1000
    COMPATIBLE WITH RFC[1000]



Profile

Base Pairs

3733

Design Notes

This gene has been optimized for E. coli

Source

Escherichia phage phiX174(from https://www.ncbi.nlm.nih.gov/gene/2546400)

Usage&Biology

Biosafety is an important consideration when designing engineered bacteria, and MazF has been commonly used in previously work. Here we develop a novel lysis gene (Gene ID: IF654_RS00240) for the design of controlled suicide circuits for engineering efficiency and biosafety reasons, also can be considered as an alternative of suicide protein MazF.

XJTU-p5-1.png

Figure 1: The temperature-controlled suicide circuit with a novel lysis gene

XJTU-p5-2.png

Figure 2: The principle of temperature-controlled suicide circuit

Figure 1 and Figure 2 shows the principle of our temperature-controlled suicide circuit. When bacteria grows at a low temperature(30℃), the CI857 protein binds to the Pλ promoter, and downstream lysis gene are unable to be expressed, allowing the cell growth. While at 42℃, the CI protein will be degraded and lead to the expression of lysis gene and eventually cell death and release of the product.

Codon optimization of lysis gene

The lysis gene is from Enterobacteria phage KleenX174. To better express the lysis gene in engineered bacteria, the codon optimization of the lysis gene was conducted according to the codon preference of Escherichia coli. Figure 3 shows the number of codons we optimized to make our codons more in line with Escherichia coli preference. The modified lysis gene is shown in BBa_K4182007.

XJTU-p5-3.png

Figure 3: Codon optimization of lysis gene

Construction and optimization of suicide circuit

Initially, we planned to construct our suicide circuit (Plasmid 5) using vector backbone pSB1K3 by Golden Gate assembly, and we can obtain several clones. However, it was found that after colony PCR verification, only weak target bands could be observed (Figure 4), and the plasmids extracted from the recombinant DH5α cells was at very low concentration, and sequencing could not be completed.

XJTU-p5-10.png

Figure 4: Plasmid 5 map based on pSB1K3 and its verification (he target band is approximately 1600bp)

We supposed that due to the low copy number of pSB1K3 vector, it is difficult to extract sufficient plasmid from the engineered bacteria for further validation. Therefore, we replaced the backbone to pSEVA341, a higher-copy-number vector and re-constructed the plasmid (Figure 5). As shown in Figure 6, the obvious target bands were observed, and the plasmid correctness was further confirmed by sequencing.

XJTU-p5-11.png

Figure 5: the new plasmid 5 with pSEVA341 backbone and its verification

The verification of heat triggered cell lysis and suicide

The engineered cell harboring plasmid 5 and blank vector respectively, were culture at 30℃ overnight, and then the temperature was shift to 42℃. The OD600 of each group was detected every 1 h, and the growth curve of these strains were determined as follows. The results clearly demonstrated the cell growth was significantly inhibited after heat at 42℃ compared to the strain without lysis gene. And about 9% of whole cell population was retained after heat. Compared to the commonly used suicide protein MazF (BBa_K302033), the lysis protein in our study is also efficient but shorter and easy to be manipulated, which can be used as an alternative and update for MazF.


XJTU-p5-12.png

Figure 6: The cell growth of strains with suicide plasmid 5 and blank vector

References

1. Din, M.O., et al., Synchronized cycles of bacterial lysis for in vivo delivery. Nature, 2016. 536(7614): p. 81-85.

2. Saeidi, N., et al., Engineering microbes to sense and eradicate Pseudomonas aeruginosa, a human pathogen. Mol Syst Biol, 2011. 7: p. 521.

3. Restrepo-Pineda, S., et al., Thermoinducible expression system for producing recombinant proteins in Escherichia coli: advances and insights. FEMS Microbiol Rev, 2021. 45(6).

4. Aparicio, T., V. de Lorenzo, and E. Martínez-García, Improved Thermotolerance of Genome-Reduced Pseudomonas putida EM42 Enables Effective Functioning of the PL/cI857 System. Biotechnology Journal, 2019. 14(1): p. 1800483.