IGEM:IMPERIAL/2009/Encapsulation/cell death

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For more on cell death see here

Contents

Summary

Table 1. Summary of different Cell death mechanisms

Name Genes Proteins Level of action Efficiency Chassis References Pros Cons Feasible?
Restriction enzymes N/A Large cutters (eg.: Ecor571, AsuHP1), they recognise a larger number of nucleotides at one time, thus allowing to be more specific with the targeting. Could be used to delete the plasmid selectively Very effective, maybe possibly too effective, causing damage in the environment, and the proximal cells E. Coli Endo-exonucleases: Enzymes involved in DNA repair and cell death?;Cell death upon epigenetic genome methylation: a novel function of methyl-specific deoxyribonucleases Specificity, Can be chosen for optimal temperature, easily available Might be toxic to the surrounding environment if over expressed Yes
Restriction enzymes N/A Small cutters (eg.: EcoRI, Hsp92), they recognise a smaller number of nucleotides at one time, thus less specific with the targeting and cutting the genome at many different places. Could be used to delete the plasmid selectively Very effective, maybe possibly too effective, causing damage in the environment, and the proximal cells E. Coli References Specificity, Can be chosen for optimal temperature, easily available Might be toxic to the surrounding environment if over-expressed Yes
ccdB ccdB killing geneB ccdB protein Cell-wide killing mechanism Very effective, possibly too effective, would need to be very tightly regulated by a non-leaky promoter E. Coli E. Coli wiki; KULeuven08 Very effective with cells, specific to prokaryotes and E. Coli strains in particular Too effective, would need to be very well regulated with a promoter -> does such a promoter exist? Not likely
Recombinases XerC and XerD XerC-XerD dimer Recombines/excises sequences in between Dif sites Not very fast nor very effective (40% after 48h) E. Coli and B. Subtilis XerDif Paper Elegant solution could be easily engineered by adding Dif sequences Not feasible because it takes too much time No
σe factor-mediated cell death RpoE σe transcription factor Activates a set of genes/operons responsible for programmed cell death ? E. Coli σe factor-mediated cell death Known gene, transcription factor Will act on many different loci in the genome, some of which could be proteases that could degrade our product: use protease-deficient strains More work needed
Toxin/antitoxin mediated cell death mazE, mazF Glucose-6- phosphatase dehydrogenase for production of extracellular death factor (EDF, aka quorum sensing peptide) mazE and mazF counteract each other and lead to the production of toxins/antitoxins to trigger cell death Widely used E. Coli [1] Known gene, transcription factor Since we are targetting the stomach it may not be advisable to release toxins into it, even if they are not harmful Not for the stomach

Methods

  • Title: From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction–modification gene complex [2]
    • Abstract: Genetically programmed cell deaths play important roles in unicellular prokaryotes. In postsegregational killing, loss of a gene complex from a cell leads to its descendants’ deaths. With type II restriction–modification gene complexes, such death is triggered by restriction endonuclease's attacks on under-methylated chromosomes. Here, we examined how the Escherichia coli transcriptome changes after loss of PaeR7I gene complex. At earlier time points, activation of SOS genes and E-regulon was noticeable. With time, more SOS genes, stress-response genes (including S-regulon, osmotic-, oxidative- and periplasmic-stress genes), biofilm-related genes, and many hitherto uncharacterized genes were induced, and genes for energy metabolism, motility and outer membrane biogenesis were repressed. As expected from the activation of E-regulon, the death was accompanied by cell lysis and release of cellular proteins. Expression of several E-regulon genes indeed led to cell lysis. We hypothesize that some signal was transduced, among multiple genes involved, from the damaged genome to the cell surface and led to its disintegration. These results are discussed in comparison with other forms of programmed deaths in bacteria and eukaryotes.
    • Problem: We dont want to use restriction enzymes and it involves cutting out a gene complex. *Nuri Purswani 10:31, 22 July 2009 (EDT):
    • Other:


  • Title: Bacterial Programmed Cell Death and Multicellular Behavior in Bacteria [3]
    • Abstract: Traditionally, programmed cell death (PCD) is associated with eukaryotic multicellular organisms. However, recently, PCD systems have also been observed in bacteria. Here we review recent research on two kinds of genetic programs that promote bacterial cell death. The first is mediated by mazEF, a toxin–antitoxin module found in the chromosomes of many kinds of bacteria, and mainly studied in Escherichia coli. The second program is found in Bacillus subtilis, in which the skf and sdp operons mediate the death of a subpopulation of sporulating bacterial cells. We relate these two bacterial PCD systems to the ways in which bacterial populations resemble multicellular organisms.
    • Problem: The mazEF system involves having toxins in the stomach.
    • Other: B.subtilis skf/sdp system is an interesting possibility to explore. *Nuri Purswani 10:31, 22 July 2009 (EDT):


  • Title: Enterohemorrhagic Escherichia coli Induces Apoptosis Which Augments Bacterial Binding and Phosphatidylethanolamine Exposure on the Plasma Membrane Outer Leaflet [4]
    • Abstract: Enterohemorrhagic Escherichia coli (EHEC) is a gastrointestinal pathogen that causes watery diarrhea and hemorrhagic colitis and can lead to serious and even fatal complications such as hemolytic uremic syndrome. We investigated the ability of EHEC to kill host cells using three human epithelial cell lines. Analysis of phosphatidylserine expression, internucleosomal cleavage of host cell DNA and morphological changes detected by electron microscopy changes revealed evidence of apoptotic cell death. The rates and extents of cell death were similar for both verotoxin-producing and nonproducing strains of EHEC as well as for a related gastrointestinal pathogen, enteropathogenic E. coli (EPEC). The induction of apoptosis by bacterial attachment was independent of verotoxin production and greater than that produced by a similar treatment with verotoxin alone. Expression of phosphatidylethanolamine, previously reported to bind EHEC and EPEC, was also increased on apoptotic cells but with little correlation to phosphatidylserine expression. Phosphatidylethanolamine levels but not phosphatidylserine levels on dying cells correlated with EHEC binding. Cells treated with phosphatidylethanolamine-containing liposomes also showed increased EHEC binding. These results suggest that bacterial induction of apoptosis offers an advantage for bacterial attachment by augmenting outer leaflet levels of the phosphatidylethanolamine receptor.
    • Problem: I believe this is talking about how E. coli cells induce apoptosis in host cells; not in themselves. ~ Tom Adie 15:49, 23 July 2009 (EDT)
    • Other: *Nuri Purswani 10:31, 22 July 2009 (EDT):


  • Title: The Extra-cellular Death Factor (EDF): Physiological and genetic factors influencing its production and response in

Escherichia coli (paper |here)

    • Abstract: Gene pairs specifying for a toxin and its antitoxin (TA) are called toxinantitoxin modules and are found on the chromosomes of many bacteria. The most studied of these modules is Escherichia coli mazEF in which mazF encodes a stable toxin, MazF, and mazE encodes a labile antitoxin, MazE, which prevents the lethal effect of MazF. In a previous report from this laboratory it was shown that mazEF-mediated cell death is a population phenomenon requiring a quorum sensing peptide that we call the Extracellular Death Factor (EDF). EDF is the linear penta-peptide NNWNN (31). Here we further confirmed that EDF is a signal molecule in a mixed population. In addition, we characterized some physiological conditions and genes required for EDF production and response. Furthermore, stress response and the gene specifying for MazEF, Zwf (Glucose-6-phosphate dehydrogenase) and the protease ClpXP are critical in EDF production. Significant strain difference in EDF production and response explain variation in the induction of mazEF-mediated cell death
  • David Roche 11:01, 22 July 2009 (EDT):


  • Title: MazF-Mediated Cell Death in Escherichia coli: a Point of No Return [1]
    • Abstract: mazEF is a stress-induced toxin-antitoxin module, located on the chromosome of Escherichia coli, that we have previously described to be responsible for programmed cell death in E. coli. mazF specifies a stable toxin, and mazE specifies a labile antitoxin. Recently, it was reported that inhibition of translation and cell growth by ectopic overexpression of the toxin MazF can be reversed by the action of the antitoxin MazE ectopically overexpressed at a later time. Based on these results, it was suggested that rather than inducing cell death, mazF induces a state of reversible bacteriostasis (K. Pederson, S. K. Christensen, and K. Gerdes, Mol. Microbiol. 45:501-510, 2002). Using a similar ectopic overexpression system, we show here that overexpression of MazE could reverse MazF lethality only over a short window of time. The size of that window depended on the nature of the medium in which MazF was overexpressed. Thus, we found "a point of no return," which occurred sooner in minimal M9 medium than it did in the rich Luria-Bertani medium. We also describe a state in which the effect of MazF on translation could be separated from its effect on cell death: MazE overproduction could completely reverse the inhibitory effect of MazF on translation, while not affecting the bacteriocidic effect of MazF at all. Our results reported here support our view that the mazEF module mediates cell death and is part of a programmed cell death network. **Problem: See table
  • Nuri Purswani 04:30, 23 July 2009 (EDT):


  • Title: The restriction–modification genes of Escherichia coli K-12 may not be selfish: They do not resist loss and are readily replaced by alleles conferring different specificities [5]
    • Abstract: Type II restriction and modification (R-M) genes have been described as selfish because they have been shown to impose selection for the maintenance of the plasmid that encodes them. In our experiments, the type I R-M system EcoKI does not behave in the same way. The genes specifying EcoKI are, however, normally residents of the chromosome and therefore our analyses were extended to monitor the deletion of chromosomal genes rather than loss of plasmid vector. If EcoKI were to behave in the same way as the plasmid-encoded type II R-M systems, the loss of the relevant chromosomal genes by mutation or recombination should lead to cell death because the cell would become deficient in modification enzyme and the bacterial chromosome would be vulnerable to the restriction endonuclease. Our data contradict this prediction; they reveal that functional type I R-M genes in the chromosome are readily replaced by mutant alleles and by alleles encoding a type I R-M system of different specificity. The acquisition of allelic genes conferring a new sequence specificity, but not the loss of the resident genes, is dependent on the product of an unlinked gene, one predicted [Prakash-Cheng, A., Chung, S. S. & Ryu, J. (1993) Mol. Gen. Genet. 241, 491–496] to be relevant to control of expression of the genes that encode EcoKI. Our evidence suggests that not all R-M systems are evolving as “selfish” units; rather, the diversity and distribution of the family of type I enzymes we have investigated require an alternative selective pressure.
  • Nuri Purswani 04:42, 23 July 2009 (EDT):

Biobricks


Helpful papers: [6], [7]

  • Requirements:
  1. Use a 4-cutter (enzyme that cleaves short sequences) with tight regulation
  2. Flank genes with cleavage sites; include cleavage sites within the genes and other components of the construct
  3. Use a bistable switch to control basal levels of restriction enzyme gene expression??
  4. Use REs that produce blunt ends to reduce likelihood of re-ligation


  • Potential restriction enzymes:


Name Blunt (B)/
Staggered (S) Ends
Cleavage Site Source Already Available in Lab? Cuts PAH gene ? Cuts Cellulase gene? Methylase available?
AatII S GACGT---C
C---TGCAG
Yes No No No
AflII S C---TTAAG
GAATT---C
Yes Yes, 1 No No
AgeI S GACGT---C
C---TGCAG
Yes No No No
ApaI S GGGCC---C
C---CCGGG
Yes No Yes, 2 Yes
AseI S AT---TAAT
TAAT---TA
Yes Yes, 3 No No
Asp7181 S G---GTACC
CCATG---G
Yes No No Yes
AvrII S C---CTAGG
GGATC---C
Yes No No No
BglII S A---GATCT
TCTAG---A
Yes Yes, 1 No No
BsmI S GAATGCN---N
CTTAC---GNN
Yes Yes, 1 Yes, 3 No
BspEI S T---CCGGA
AGGCC---T
Yes No Yes, 2 Yes, Dam
BspHI S T---CATGA
AGTAC---T
Yes No Yes, 1 Yes, Dam
BssSI S C---TCGTG
GAGCA---C
Yes Yes, 1 No No
BstBI S TT---CGAA
AAGC---TT
Yes No No No
BstZI7I B GTA---TAC
CAT---ATG
Yes Yes, 1 Yes, 1 No
ClaI S AT---CGAT
TAGC---TA
Yes No No Yes, Dam
DpnI B GA---TC
CT---AG
Yes Yes, 6 Yes, 9 No
DraI B TTT---AAA
AAA---TTT
Yes Yes, 1 Yes, 1 No
DrdI S GACNNNN---NNGTC
CTGNN---NNNNCAG
Yes Yes, 2 No No
EagI S C---GGCCG
GCCGG---C
Yes No Yes, 2 No
FspI B TGC---GCA
ACG---CGT
Yes No Yes, 1 No
HinfI S G---ANTC
CTNA---G
Yes Yes, 10 Yes, 1 No
HincII B GTY---RAC
CAR---YTG
Yes Yes, 2 No No
KpnI S GGTAC---C
C---CATGG
Yes No No Yes
MaeI S C---TAG
GAT---C
Yes Yes, 2 No No
MluI S A---CGCGT
TGCGC---A
Yes No No No
NarI S GG---CGCC
CCGC---GG
Yes No Yes, 2 No
NcoI S C---CATGG
GGTAC---C
Yes Yes, 2 No No
NdeI S CA---TATG
GTAT---AC
Yes Yes, 1 No No
NheI S G---CTAGC
CGAT---G
Yes No No No
NruI B TCG---CGA
AGC---GCT
Yes No No Yes, Dam
PacI S TTAAT---TAA
AAT---TAATT
Yes Yes, 1 No No
PvuI S CGAT---CG
GC---TAGC
Yes No No No
PvuII B CAG---CTG
GTC---GAC
Yes Yes, 3 Yes, 1 No
RsaI B GT---AC
CA---TG
Yes Yes, 8 No No
SacI S GAGCT---C
C---TCGAG
Yes No No No
SacII S CCGC---GG
GG---CGCC
Yes No Yes, 2 No
SalI S G---TCGAC
CAGCT---G
Yes No No No
ScaI B AGT---ACT
TCA---TGA
Yes Yes, 2 No No
SexAI S A---CCWGGT
TGGWCC---A
No No No Yes
SfoI B GGC---GCC
CCG---CGG
Yes No Yes, 2 Yes
SmaI B CCC---GGG
GGG---CCC
Yes Yes, 1 Yes, 2 ?
SpeI S A---CTAGT
TGATC---A
Yes No No ?
SphI S GCATG---C
C---GTACG
Yes No Yes, 2 ?
SwaI B ATTTT---AAAT
TAAA---TTTA
Yes No No ?
SspI B AAT---ATT
TTA---TAA
Yes No No ?
StuI B AGG---CCT
TCC---GGA
Yes No No ?
XmaI S C---CCGGG
GGGCC---C
Yes Yes, 1 Yes, 2 ?
XmnI B GAANN---NNTTC
CTTNN---NNAAG
Yes No Yes, 1 ?
PovII B CAG---CTG
GTC---GAC
? No Can't find enzyme Can't find enzyme ?
HaeIII B GG---CC
CC---GG
Roche No Yes, 10 Yes, 10 ?
AluI B AG---CT
TC---GA
Roche No Yes, 14 Yes, 9 ?

Papers

  1. Amitai S, Yassin Y, and Engelberg-Kulka H. . pmid:15576778. PubMed HubMed [celldeath4]
  2. Asakura Y and Kobayashi I. . pmid:19304752. PubMed HubMed [celldeath1]
  3. Engelberg-Kulka H, Amitai S, Kolodkin-Gal I, and Hazan R. . pmid:17069462. PubMed HubMed [celldeath2]
  4. Barnett Foster D, Abul-Milh M, Huesca M, and Lingwood CA. . pmid:10816451. PubMed HubMed [celldeath3]
  5. The restriction–modification genes of Escherichia coli K-12 may not be selfish: They do not resist loss and are readily replaced by alleles conferring different specificities,Mary O’Neill, Angela Chen, and Noreen E. Murray

    [celldeath5]

  6. Graves RC, Abernathy JR, Disney JA, Stamm JW, and Bohannan HM. . pmid:1920265. PubMed HubMed [RE1]
  7. Rackham O and Chin JW. . pmid:16408021. PubMed HubMed [RE2]
All Medline abstracts: PubMed HubMed
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