Experiments
Experimental Overview
To give our engineered bacteria antifungal activity against FOC TR4, we devised a chitinase secretion system, and designed a Chi18h8 secretion construct for said system. In order to ensure adequate biocontainment for our engineered bacteria, we designed a Toxin-Antitoxin kill switch system and three constructs to achieve that function - a CcdA-producing construct(BBa_K4137012), CcdB-producing construct(BBa_K4137013), and a mleR-producing construct(BBa_K4137014). These constructs act as modular parts that can be assembled in various combinations to determine systemic behaviors.
The chitinase secretion system uses an unspecific effector that has the potential activity against non-FOC fungus, so the kill switch system will help us rein in the unintended environmental consequences of our low effector specificity. For both systems, we’ve performed multiple cloning cycles to construct our desired plasmids, verified protein expression in BL21 E. coli, and analyzed the results via SDS-PAGE.
For further investigations on whether our designed constructs perform their expected function, we plan to conduct several assays. For confirmation on the functionality of chitinase and extracellular secretion, we ran chitinase secretion and yeast co-culture assays. For identification of the correct co-production of both the toxin and antitoxin in bacteria, we’ll perform CcdA/CcdB expression assay. Induction expression will also be carried to confirm that the transcriptional activator pmleS (BBa_K4137005) operates correctly in conjunction with mleR to induce production of CcdA. To relate the amount of malate with bacterial survival and growth and test the effectiveness of our kill switch, we’ll perform the drop assay to see bacteria survival outcomes in various environmental malate concentrations.
Construct Designs & Cloning
Chitinase secretion construct
The specific chitinase we selected is Chi18h8 – through literature review, many authors have concluded it as having potential applications in antifungal applications for crop disease biocontrol. Most crucially, Chi18h8 was shown to have antagonistic activity against Fusarium oxysporum in Hjort et al.’s analysis.
For our design, we used a T7 promoter and T7 terminator to ensure high transcription levels of our gene of interest by E. coli. Along with the strong promoter and terminator, we included a strong ribosome binding site (RBS) to further optimize the constitutive protein production rate.
Since our goal is to have an extracellular secretion of Chi18h8 using the Sec secretion system, we fused Chi18h8 with a yebF secretion tag, which facilitates the movement of chitinase through the periplasm and into the extracellular medium. The YebF tag is a N-terminal peptide tag that is used to permit extracellular secretion of a protein of interest through protein fusion; the tag is cleaved off when the protein is secreted into the extracellular medium. We’re testing this in E. coli as a proof of concept for chitinase secretion before moving on to handling B. subtilis. We’ve also inserted a flexible glycine-serine linker (GS) between the two genes to stabilize our fusion protein.
To verify the successful production and secretion of the fusion protein of Chi18h8 and yebF tag, we inserted a 6xHis tag at the end of our construct design to help us isolate the protein in downstream protein purification assays.
Purification of the protein will allow us to experiment with various concentrations to determine what amount of chitinase is required to achieve sufficient inhibition of FOC; we can then use this information downstream to tweak the construct designs to optimize FOC inhibition.
CcdA-CcdB malate-induced toxin-antitoxin kill switch construct(s)
The aim of the kill switch system is to constrain the activity of our engineered probiotic B. subtilis to the permissive environment that is the banana root rhizosphere so that it wouldn’t spread and cause unpredictable harm; the rhizosphere supplies sufficient malate to the bacteria through root exudates.
The kill-switch is based on the toxin-antitoxin pair CcdB/CcdA (BBa_K4137000, BBa_K4137001). As a DNA gyrase inhibitor, CcdB targets and traps DNA gyrase(GyrA), causing cell death; this toxicity is sequestered by antitoxin CcdA, which blocks the activity of CcdB by forming a CcdA-CcdB complex, preventing CcdB from binding into GyrA.
In order to control the survival of our bacteria and ensure that it would stay only in areas where malate is present in sufficient quantity, we used the mleR transcriptional activator and its respective regulon to control the expression of CcdA. The mleR regulon requires mleR to achieve maximal expression; without malate, mleR is not activated, and thus transcription via the mleR regulon is not induced, causing low CcdA production. If our bacteria moves beyond the banana root rhizosphere, the subsequent deficiency in CcdA will eventually lead to cell death due to the constitutive production of CcdB.
a. mleR production construct
Here, we also used a T7 promoter, T7 terminator, and strong RBS to ensure a high, constitutive level of mleR production; when mleR binds with malate, it is activated and thus able to perform its function of transcriptional activation.
b. ccdA-ccdB toxin antitoxin constructs
The promoter for CcdA was sourced from mleS, a protein governed by mleR in the mle operon. This regulatory site, nicknamed pmleS, promotes transcription only when bound by activated mleR; by using it as the CcdA-production construct’s promoter region, we make CcdA production inducible with malate. This allows us to achieve malate-controlled production of the antitoxin and thus enforce the permissiveness of the rhizosphere.
On the other hand, ccdB toxin is constitutively synthesized using the pLac promoter. The constitutive expression here enforces the nonpermissiveness of malate-absent environments. We added purification tags to our protein sequences for downstream purification and quantification purposes.
By designing our constructs with biobrick restriction sites bracketing the genetic components, we’re able to modularize each construct to allow for a variety of plasmid combinations; this is explained further in Cloning Plans. This design allows us to carry out different functional tests and verify our theory separately. For example, we could test the toxin-antitoxin theory by cloning ccdA and ccdB together in the same plasmid to prove whether both proteins bind and inhibit the toxicity of ccdB. Moreover, we can compare the results of ccdA production with and without the presence of mleR gene, which would confirm our theory of how mleR regulates the expression of ccdA.
Cloning Plans
In order to physically create our engineered systems, we will do multiple cloning cycles to create plasmids with different combinations of our designed constructs; per biobrick assembly standard, we will be using the backbone pSB1C3. However, because of delays in receiving the distribution kit, we extracted pSB1C3 from a previous iGEM team’s plasmids (pSB1C3-RFP (BBa_J04450) and pSB1C3-pSTE12-18) by removing RFP and pSTE12-18.
To assemble the constructs into our desired plasmids, we will be using the EcoRI(E), XbaI(X), and SpeI(S) restriction sites in conjunction with standard cloning protocol to allow for multiple construct insertions and thus complex combinations of our designed constructs.
To insert one of our constructs into pSB1C3, we’ll cut the E and S sites for both the insert and the vector. To add a second insert into pSB1C3, we’ll cut the E and X sites, as this allows us to preserve our previously inserted construct while attaching a new section in. Using this method, we’ll construct three plasmids with singular inserts (pSB1C3::CcdA, pSB1C3::CcdB, pSB1C3::mleR) and two plasmids with dual inserts (pSB1C3::CcdB::CcdA, pSB1C3::CcdA::mleR) for the experimentation on the Toxin-Antitoxin system.
For cloning results, visit the Engineering page.
Protein Expression
Expression of ccdA-ccdB Antitoxin System
To implement our bacteria into field conditions, we mustn't neglect the need for strong biocontainment. Our intention for the kill-switch system is to prevent the unexpected effects that the bacteria may cause, and to limit the bacteria’s influence within the banana plantation. As such, we must extensively test the constructs of this system via expression to ensure that they perform their intended function precisely.
The goal of experimentation for this system is then to characterize the CcdA, CcdB, and mleR constructs to determine the holistic behavior of the rudimentary kill-switch using protein expression; from this round of characterization we can revise the construct designs to choose different promoters, RBS’s, and terminators to fine-tune the system to behave as we’d like, i.e. CcdB basal expression is higher than CcdA, mleR induction being sufficient to overcome CcdB, etc.
To examine whether our constructs built during the cloning cycles perform its intended function, we’ll verify protein expression via SDS-PAGE. If time and equipment permits, we will perform purification and quantification to collect data of protein production over time, which will aid Drylab in constructing the Toxin-Antitoxin model by providing data points for kinetic constant calculations.
For results, visit the Engineering page.
Chitinase Production
The core of our experiment is to confront FOC by degrading the cell wall with the use of Chitinase which will be secreted by our designed bacteria. In order for us to confirm that chitinase is verily secreted, we’ll transform our designed chitinase construct into DH5𝜶 E. Coli cells and grow them overnight. Part of the overnight culture would be used as the starter culture and its concentration would be verified consistently until it reaches log phase. Same as the toxin-antitoxin system, we’ll harvest and lyse the cell and use SDS-Page to verify the size of the secreted protein.
Testing secretion is not enough to prove our theory, hence we’ll investigate through assays to examine our constructs' effect on FOC. The Bradford Assay would be essential for us to do quantitative tests in the future with the respective on chitinase concentration versus FOC activity. We can also use it to determine the relation on how the secretion of chitinase is affected when we change temperature and other environmental conditions.
In addition to the level of secretion, we also wanted to determine the effectiveness of our transformed bacteria. The Co-culture Assay serves this purpose in which we can examine FOC activity by comparing how it grows when surrounded by the designed bacteria. It can also be used to show differences in the measure of time. We expect to see that the co-culture plate containing our bacteria would be able to inhibit fungal activity longer than plates where Chitinase were added instead.
For results, visit the Engineering page.
Functional Assays
Chitinase Secretion
Since our bacteria is meant to combat FOC, a co-culture assay seems the natural way to go for a proof of concept. To demonstrate and prove the substantial inhibitory effects of our engineered bacteria against FOC is the first and most crucial step to proving the viability of our project; though the secreted chitinase Chi18h8 is proven to have said inhibitory effects against FOC, whether or not this property persists when implemented in B. subtilis remains to be seen. Before that, we must first verify protein production in E. Coli BL21.
Protein production verification for BL21 would be done by running the Bradford assay and SDS-PAGE on the supernatant of the BL21 pSB1c3::Chi18h8 LB incubation mixture and lysed BL21 cell contents.
Division of protein production verification into these two categories allows us to verify extracellular secretion of chitinase and also quantify the inefficiencies of secretion; we could then use this data to improve our protein designs to improve secretion by reducing inclusion bodies.
For results, visit the Proof of Concept page.
Co-culture/Chitinase Activity Assay
We plan on utilizing a co-culture antagonism assay to verify the function of the secreted chitinase. We will use an experimental group of yeast co-cultured with our bacteria to verify that the fusion protein encoded by BBa_K4137009 maintains the same functionalities as chi18h8; though yeast is a unicellular fungus, it nevertheless possesses the chitin cell wall targeted by chitinase. Thus, we will look for significant increases in growth inhibition in the yeast-BL21 pSB1c3::chi18h8 group as compared to the yeast-BL21 pSB1c3 control group as proof of correct function.
For results, visit the Proof of Concept page.
CcdA Induction Expression
Due to our choice of malate concentration as the environmental factor for biocontainment, we performed intensive literature research to find transcriptional regulators that reacted to malate concentration. From a preliminary round of plausible candidate selection, we found CcpA, a malate-dependent transcriptional repressor, and mleR, a malate-dependent transcriptional activator. We couldn’t find relevant gene sequences for CcpA, so we went with mleR and adapted our Toxin-Antitoxin system to this activator. To find the regulatory region that mleR was responsible for, we looked for relevant literature on mleR’s function as a malolactic enzyme regulator and found the mleS promoter(pmleS), which should theoretically be regulated by mleR.
Though pmleS and mleR come from the same organism, because the literature source for the mleR transcriptional activator and the regulatory region supposedly recognized by mleR are different, we plan to perform protein expression of CcdA alongside mleR in various malate induction concentration trial groups to ensure that our transcriptional activator truly performs the function that it theoretically possesses. Through additional research for the mleR regulon and subsequent alignment analysis performed with Benchling, we verified the existence of a highly similar sequence in pmleS - a promising sign for our system; further research yielded a paper for an unrelated field using pmleS to achieve significant induction of luciferase production. As for the induction expression test, a control group of pSB1c3::CcdA-6xHis will be used to determine the basal production of the protein.
Beyond simply ensuring function, we plan to further characterize pmleS, as we see it to be a promoter with significant potential value in agriculture-related applications of synthetic biology - an inducible promoter responsive to a chemical present in root exudates has myriads of possible use cases, and will certainly aid the pursuit of future iGEM teams for sustainable agriculture protected from disease.
For results, visit the Proof of Concept.
References
Hjort, K., Presti, I., Elväng, A., Marinelli, F., & Sjöling, S. (2013). Bacterial chitinase with phytopathogen control capacity from suppressive soil revealed by functional metagenomics. Applied Microbiology and Biotechnology, 98(6), 2819–2828. Link to Source