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Background


In module 1, the E. coli produces an enzyme of our choice, either PAH or cellulase.


Then in module 2, the bacterium will encapsulate itself in colonic acid and produces trehalose to increase the stability of PAH or cellulase during storage.


Finally in module 3, the bacterium will be killed. This is necessary to make the pill non-toxic or pathogenic, and hence safe for human consumption. The self killing mechanism is activated by an increase in temperatures from 28°C to 42°C.





In our system, the lambda cI promoter is initially repressed by the protein cI that is produced constitutively under the strong promoter J23114. At 28°C, functional protein cI will bind strongly to the lambda cI promoter to repress it. Restriction enzymes DpnII and TaqI will not be produced.


When there is an increase in temperature (from 28°C to 42°C [1][2]), a change in conformation of the cI protein occurs, which makes it harder for protein cI will no longer be able to bind to the lambda cI promoter and repress it. In other words, there will be a de-repression of lambda cI promoter, and it will be activated, to produce restriction enzymes DpnII and TaqI. Restriction enzymes will then digest the genetic material of the E. coli cells and cut the DNA, leading to cell death.


The Dam methylase, which is produced constitutively by a weak promoter J23103, has been added to prevent cell death for redundant basal levels of production of restriction enzymes. It ensures that cell death is negligible due to the restriction enzyme. When switched on, E. coli can produce as much PAH or cellulase as possible, with ample time for encapsulation, before we trigger cell death manually.


Dam methylase works by transferring a methyl group to the N6 position of the adenine residues in the GATC[3]. As a result, restriction enzymes cannot recognise their intended cleavage site. This means that DNA will not be cleaved in the presence of Dam methylase and E. coli cells will not die. As it competes with restriction enzymes for DNA, at low restriction enzymes concentrations, Dam methylase will be able to methylate DNA and protect it. However, as the concentration of restriction enzymes increase at higher temperatures, the amount of constitutively produced Dam methylase will no longer be sufficient to protect against cell death, and cell killing occurs.

Goals

With this model, we want to check the validity of our assumptions, and obtain the necessary parameters for data fitting. We also want to have a qualitative understanding of our system, making it a whitebox to us. We can achieve this by:


  • Exploring how temperature and cI protein concentration correlates to the concentration of restriction enzymes.
  • Hence, determining the relationship between temperature and cI protein concentration and live cell population.
  • Varying the strength of lambda cI promoter.


From this, we can characterise impact various factors can have on cell death. Furthermore, we can correlate our assays to the model, to make it more realistic. This can be done by:

  • Fitting data obtained from the live and dead cells assay (hyperlink), to the model for total cell population, and dead cell population (Cell death will be modelled neglecting Dam methylase protection.)


From this, we can monitor the rate of killing and perform data analysis to obtain the necessary parameters. Hence we have modelled for the output of M3.



  1. Jechlinger W, Glocker J, Haidinger W, Matis A, Szostak MP, and Lubitz W. Modulation of gene expression by promoter mutants of the lambdacI857/pRM/pR system. J Biotechnol. 2005 Mar 2;116(1):11-20. DOI:10.1016/j.jbiotec.2004.10.002 | PubMed ID:15652426 | HubMed [1]
  2. Jana NK, Roy S, Bhattacharyya B, and Mandal NC. Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor. Protein Eng. 1999 Mar;12(3):225-33. DOI:10.1093/protein/12.3.225 | PubMed ID:10235623 | HubMed [2]

All Medline abstracts: PubMed | HubMed

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