Team:HKU HongKong/Project/Future Implications.html

From 2012.igem.org

Future Biobrick 1

Construction of J23119-B0034-pvdQ-B0015:

This biobrick was constructed to test the expression of the pvdQ gene amplified by PCR from genomic DNA of Pseudomonas aeruginosa. It is an uncontrollable, standard biobrick that can later be used as a baseline reference to test whether our future biobricks result in an improvement in pvdQ expression level.

The biobrick is a composite part consisting of the already prevalent Constitutive Promoter [2006 Berkley], along with the newly inserted pvdQ gene. It is also composed of a strong RBS and a transcription double terminator.

Constitutive Promoter (J23119): This promoter biobrick is the consensus sequence and therefore results in the idealized transcription scenario. Since it is a strong promoter and is constitutively expressed, it complies with our preliminary goal of testing transcription and translation of the pvdQ gene in an AHL-independent manner.

The constitutive promoter directs expression in virtually all tissues. Also, it is independent of environmental and developmental (endogenous) factors. This is why it can be utilized across species. The promoter will ensure that pvdQ will be expressed ubiquitously, and pvdQ expression will increase with increase in cell density of E.coli. As the E.coli growth curve is exponential, so will be the curve of pvdQ expression.

We tried to construct this biobrick during our wet lab sessions. However, several problems were encountered, resulting in its unsuccessful creation:

The J23119 promoter could be ligated to B0034 RBS by digesting with EcoRI and PvuI. This digestion was used instead of the conventional EcoRI + SpeI and EcoRI + XbaI of the insert and vector respectively because the fragment size of the EcoRI+ SpeI digested insert is only a little more than a 100 bp. This produces a band of very low concentration as most of the DNA makes up the unwanted backbone, making the insert difficult to gel purify. Digestion with EcoRI and PvuI, conversely, yields a larger fragment that can be subsequently ligated.

Ligating the J23119-B0034 to the pvdQ-B0015 was a problem, however. EcoRI + PvuI digestion could no longer be used because the backbone of the two biobricks are different.

Forward primers were designed to carry out a two-step PCR. The first reaction involved adding the RBS to the pvdQ-B0015 biobrick. The second reaction would add the Prefix and J23119 promoter to the RBS-pvdQ-B0015 biobrick from the first reaction. The standard suffix was used as the reverse primer. The second PCR, however, was unsuccessful perhaps due to the large difference in Tm between the forward and reverse primers.

Future Biobricks 2 & 3

This biobrick will allow constitutive expression of pvdQ at the baseline level. However, pvdQ expression can be enhanced if it is also dependent on AHL-luxR binding to the pLuxR. This system allows a baseline pvdQ expression even in the absence of AHL. Hence, it may confer an advantage over the BBa_K855001 biobrick we constructed by keeping the AHL from accumulating and further minimizing the oscillatory effect.

Another system would be one that is dependent on another factor of cell density but independent of AHL. If pvdQ is dependent on AHL, then when AHL levels decreases, the pvdQ expression level will decrease accordingly. Such a negative feedback system is not desirable to our project because it may lead to the re-accumulation of AHL, re-establishing biofilm formation through quorum sensing. Thus, a system in which pvdQ expression is dependent on P. aeruginosa cell density may alleviate this problem.

Alginate Encapsulation

Immobilizing living cells or other biomolecules in polymeric, alginate gels is a technique of broadening use in biochemical or industrial applications. These entrapped cells can be manipulated to excrete biomolecules of therapeutic use. The alginate network not only keeps cells viable and catalytically operating, but also protects them from biotic and abiotic stress as well as toxic compounds. Alginate is a copious marine biopolymer that can form heat stable, strong gels. These gels polymerize under moderate conditions at room temperature. Additionally, in the process of cross-linking to enclose the bacterial cell, the gel does not swell or shrink greatly. Instead, it maintains a constant shape and boundary. Thereby, this method of encapsulation supports a steady bacterial cell density even after lengthy durations of storage. Encapsulation efficiency is evaluated by the ability of the system to keep the cells physiologically and metabolically competent, maintain a high bacterial cell density, and maximum encapsulation time. This is why the making of the gel must include appropriate additives that stabilize and protect the cells whilst their storage and transport. These supplements provide nutrition to the bacterial cells without causing cell death. The technology must also take into consideration the coordinated release of the biomolecule, and the system’s biodegradability and cost-effectiveness.  This method can be applied to HKU’s iGEM project as the whole cell E.coli can be enclosed in alginate beads. The E.coli contain synthetic mechanisms for the production of pvdQ. A signal peptide on the 5’-end of the pvdQ gene sequence, which secretes the enzyme from the periplasmic space to the external environment, can be added to directly transport the acylase into the environment where it can interact with and degrade C-12 AHL molecules. Even though the bacterial cell remains trapped within the alginate network, the pvdQ protein is small enough to diffuse from the beads. This system allows E.coli to actively produce pvdQ, which can be utilized without being subject to lengthy procedures of protein purification. Pseudomonas aeuroginosa is a soil-inhabiting bacterium. Thereby, E.coli-alginate beads can be inoculated into the soil, and the subsequent release of pvdQ from the immobilized gel matrix can be used to impede P.aeuroinosa biofilm formation. Additionally, enzymes within the human body do not hydrolyze alginate, unlike cellulose and other polymers that may be alternatively used in encapsulation. Hence, these beads can also be directed to sites of P.aeuroginosa colonization in vivo, mimicking the drug delivery mechanisms.

Suggested Protocol for pvdQ Excretion into the Soil:

Preparing the Alginate Solution: -

1). A 2% concentration of the medium viscosity sodium alginate solution is prepared by dissolving the alginate powder in distilled water. 2). Subject the solution to agitation using a magnetic stirrer at room temperature. 3). Autoclave the solution at 121°C for 20 minutes.

Preparing the Additive Nutrient, Humic Acid: -

1). Air dry 200g of peat soil. 2). Mix the air dried soil with 2,000nL solution of 0.1 M NaOH and 0.1 M Ca(OH)2 (1:1) by shaking at 270 rpm for 4 hours. 3). Centrifuge the solution at 12,000xg for 30 minutes. 4). Collect the supernatant and acidify with 6M HCl to pH 1 overnight. 5). Filter the pH-adjusted supernatant through a sintered glass funnel. Dry filterate at 80°C. 6). Prepare 10% stock solution by dissolving humic acid in deionized water. Store in dark till subsequent use. Preparing the Alginate Beads: - 1). Sterilize all the glassware and solutions at 121°C for 20 minutes. 2). Mix 2.5mL of 10% humic acid with 750uL of 30% glycerol. 3). Add the mixture to the 2% sodium alginate to obtain a final volume of 25mL. 4). Centrifuge 250mL of bacterial culture. Discard the supernatant. 5). Wash the cell pellet with saline, 0.85% NaCl. 6). Suspend the saline-containing pellet in 25mL of the alginate-humic acid mixture. Mix thoroughly. 7). The suspension should be added drop b drop using a 26-gauze needle into pre-chilled sterile 1.5% aqueous CaCl2. Should be performed under mild agitation. 8). Allow the beads to harden for 3-6 hours at room temperature.

The beads formed have a spherical shape with a diameter of 2-3mm and a weight of about 7mg. While selecting the additive, several factors must be considered. Firstly, the nutritional supplement must be able to be mixed homogenously with the alginate polymer. Since alginate is polyanionic, the additive must be of a similar charge. Moreover, the molecule should be of a desirable size such that the bacteria can utilize it for survival in its immobilized state. It should also be able to provide the carbon and nitrogen nutrients required by the entrapped cells. Humic acid is an ideal organic compound for bacterial-beads added into the soil because it satisfies these criteria. It is colloidal, producing particles of 5uM as well as those that range between 0.04-0.5uM.

(a). Surface of the intact bead.

(d). Bacteria distributed in the gel matrix of the beads. A indicates the bacteria, and B indicates the humic acid particles.

Secretction Protein system

For the future development of PseudoKill, we need to develop an extracellular secretion system for pvdQ protein for the following reasons. Firstly, presenting system of PseudoKill can only transport the pvdQ protein into the periplasm which means it can only digest the diffused C12-HSL and its efficiency is limited. Secondly, protein aggregation is likely to happen when gene expression is performed at high levels, threatening the physiological condition of PseudoKill itself.

Type I secretion mechanism are used commly for recombinant protein secretion in E.coli B strains and it would be a suitable choice for PseudoKill.

Type I Secretion Mechanism Advantages:

Can transport target protein in one step across the two cellular membranes without a periplasmic intermediate step.

Capable of transporting high-molecular weight components.

Drawbacks:

Additional cleavage step is required for obtaining intact native protein because the signal peptide remains attached to the secreted peptide.

Co-expression of component proteins in the system are required to increase transport capacity but at the same time lead to the competition with the target protein for the transport machinery.

Type II Secretion Mechanism SecB – Dependent Pathway Advantages:

Drawbacks: