Team:IISER-Pune-India/Applied Design

Applied Design

Re-engineering the Cell through an Engineered Phage Delivery Device

For the expression of the Hijack, Detect and Terminate (HDT) genetic circuit it is necessary to dispose of a system to deliver effectively DNA into appropriate bacterial hosts in heterogeneous samples. The expression of synthetic circuits in bacterial hosts can be done through conventional protocols of transformation. Nevertheless, a delivery system based on these protocols is not suitable on the conditions for which this HDT system has been conceived, since samples are heterogeneous and may have extremely low concentrations of target host. Also they are not suitable for its massive use in hospitals and field applications in a labour- and cost-effective manner. Therefore, we propose here a portable 3D printed device that enables the infection of the target sample with engineered synthetic bacteriophages harbouring the synthetic HDT device and allow for the delivery of DNA in heterogeneous samples with low amount of target microorganisms such as M. tuberculosis with high specificity.

A portable 3D printed device

The following Figure 1 and Figure 2 provides an scheme and 3D-models, respectively, of the portable 3D printed device that will be used for the loading and detection of samples of the target microorganism ( here. M. Tuberculosis). In these Figures, it can be seen that the device consists of a Falcon-like tube with a special security system based on the lockage of the cap to the tube in an irreversible way; so that once the potentially dangerous samples are loaded the tube cannot be opened back again. This locking system is composed by a circular protuberance in the upper side of the tube (see Figure 1, B) and a complementary hollow ring in the cap that allows for the lockage of the cap once it has broken the membrane through its piercing stick (see Figure 1, D). This hollow ring can be better appreciated in the Figure 2.

Figure 1. Scheme of the 3D printed device for the delivery of the HDT genetic construct with the use of bacteriophages. (A) Soft-impermeable membrane that allows protecting the Bacteriophage compartment where the bacteriophages will be stored in the tube of the section (B). (B) It is a Falcon-like tube that is characterised by having attached the impermeable membrane at 3 cm from the bottom side. The mouth of the tube (top side) also has a protuberance that in used for blocking the cap once it breaks the membrane. (C) It is the No-Return locked Cap with the Piercing stick. The Cap has a complementary same hollow ring that, once installed upon the Falcon-like tube it gets locked (no-return) but still allows to be rotated for the optimal breakage of the membrane. (D) It is the complete scheme of the device when the Cap has been locked and the membrane broken. The measurements are made in centimetres

Figure 2. Three-Dimensional visualization of the 3D-printed prototype for the delivery of the HDT genetic construct with the use of synthetic bacteriophages (A) Closed tube with the Piercing stick perforating the membrane. (B) Visualization of the no-return lockage system of the Cap attached to the Piercing stick. (C) Empty tube containing the attached membrane. (D) Cap attached to the Piercing stick.

As it can be seen in the Figures 1 and 2, this 3D-portable device is designed to protect under the membrane a charge of lyophilised/freeze dried, jellified or liquefied mycobacteriophage (see pink pellet in Figure 1, B and D).

In the Figure 3 below, a complete procedure that goes from the production of Mycobacteriophages, to the detection of target samples is described. As it has been exposed in the PDF file attached at the bottom of this page, through the bacteriophage designs it is possible to amplify the TM4-like phAE159-ΔLys mycobacteriophages harbouring the HDT synthetic DNA construct in Mycobacterium smegmatis that contain the Lysis cassette expressed in trans. Upon infection, a high titre progeny of mycobacteriophages can be isolated for storage in a lyophilised, jellified or liquefied form. This allows for the long-term storage of the viral particles containing the synthetic DNA construct in a very cost-effective manner and permits the construction and distribution of 3D printed kits in a massive manner. These kits will be composed of two components:

  1. (1)The Falcon-like tube containing the mycobacteriophages which are protected by the impermeable membrane (see Figure 1, part B; Figure 2, part C; Figure 3, part 5)
  2. (2)The Cap attached to the Piercing stick (see Figure 1, part C; Figure 2, part D)

This sample of mycobacteriophages can be transferred to the Falcon-like tube (Figure 3, part 5) and further protected with the impermeable membrane until the target sample with microorganisms is loaded. The target sample will consist of a biopsy from a particular patient that may be infected with the target microorganism (here M. tuberculosis). This biopsy will have to be appropriately mixed with a solution containing the necessary nutrients to boost mycobacterial growth. Once the target sample is loaded into the tube, the cap containing the piercing stick will be used to break the membrane protecting the mycobacteriophages and also as a locking system that will avoid a possible and spreading of the microorganisms. Once the system is blocked and mixed, infection will occur and if will proceed as stated in the designs for the Mycobacteriophage (see PDF file below). The main advantage of using bacteriophage as the delivery systems is their high specificity among a community of microorganisms. This allows for the efficient transformation of Mycobacteria and its Detection through the HDT synthetic DNA that otherwise could not be achieved due to the heterogeneity of the samples, and the necessity to dispose of a system that is able to infect/transform a small concentration of target microorganism.

The infected tubes will be stored at 30º or 37º for induction, or not, of the viral machinery. The induction of the viral machinery will allow for the multicopy expression of the synthetic DNA all together with the viral genes, the non induction (37º) will allow the expression of the synthetic device present within Mycobacteria due to Mycobacteriophage delivery. Nevertheless, in the latter a very low or no copy number can be expected. Once the HDT device is expressed within the target host the Hijack module will be in charge of speeding up the growth rate of the slow growing Mycobacterium tuberculosis at the same time that the Detection chromophores are expressed (shown in purple dots in the Figure 3). When cell the density will increase, this chromophores will enable the detection of the growing Mycobacteria without any special detection system, just by exposing the tube to the naked eye. Finally, when the cells will grow they will be eliminated by means of the Termination module in order to ensure that there is no possibility of further contamination after the visual detection has been granted positive. This termination will proceed according to the devices presented in the project description.

Figure 3. (1) Conditionally replicating phAE159-∆Lys-HDT Mycobacteriophages (red viruses) containing the HDT device infect the fast producing strain Mycobacterium smegmatis that contains the complementing plasmids (Lysis complementing strain with LysA, LysB and Holin genes). (2) Replication and assembly of the synthetic Mycobacteriophage phAE159-∆Lys-HDT. (3) High titre release of new synthetic mycobacteriophages containing the synthetic device can isolated and Lyophilised or Jellified or dissolved in liquid. (4) A pellet or concentrated liquid full of Mycobacteriophages can be obtained from the previous process. (5) The Mycobacteriophages are deposited into the Falcon-like tube and then the tubes are protected by an impermeable membrane (yellow). (6) A heterogeneous target sample with appropriate nutritive media for bacterial growth (blue circle) is loaded in a liquid and remains in the upper side of the tube. This liquid sample contains target microbes (orange) and non-target microbes, tissue and other particles (blue). (7) After loading the target sample, the tube is closed using the special Non-Return Cap (see Figure 1 and 2) with the locking system and the Piercing stick. The closure of the cap provokes the breakage of the membrane and allows for the mixture of the target sample with the previously stored Mycobacteriophages. (8) In the mixture, re-activated bacteriophages infect only the target M. tuberculosis with high specificity. (9) The expression of the HDT genetic circuit can be expressed in a sustained way at 30ºC or 37ºC since the host does not contain the Lysis cassette (see designs in the PDF file below). (10) Upon infection, the target microbe will speed up its growth through the Hijack module at the same time that will display the chromophore-protein encoded in the Detection module of the HDT construct (see purple dots representing the chromophore). The increasing cell densities will make it visible to the naked eye. After excessive growth, the Termination module will lyse the infected cells (not shown).

Below, a PDF containing two different designs of bacteriophages for infection in E. coli and Mycobacteria are provided. The cited DNA sequences in the PDF below are represented in the attached images at the bottom of this page. These images are printed from the DNA constructs created with the SnapGene® software.

Engineering bacteriophages as a conditionally replicating delivery system for Escherichia coli and Mycobacterium spp.

Amplicon_LysA,LysB
pHAE159_Lysis cassette!
pMyBADC_Kan
TM4_from_U_pittspburgh_(featured)_Map

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