Bacillus subtilis 168

We are modifying the bacterium Bacillus subtilis 168 in order to achieve desired features in our bacteria. This will be achieved through the design and transfer of a specialized plasmid, containing the targeted genetic material, into Bacillus subtilis 168. Once this genetically modified bacterium is introduced to Orchids, Bacillus subtilis 168 will interact with the orchids in a manner that leads to enhanced resilience against soft rot. Scroll down to navigate the elements we engineered in our plasmid.

aiiA gene

The aiiA gene is one of the most crucial elements in our project as it is known for its ability to produce enzymes that binds with AHL molecules and degrade them.

p43 promotor

Constitutive Promoter 43 continuously drives the expression. Therefore pivotal for ensuring the continuous expression of the aiiA gene in B. subtilis.

AHL molecule (OHHL)

OHHL (3-oxo-hexanoyl homoserine lactone is one of the AHL (acyl-homoserine lactone), and it is the dominant signal molecule responsible for the communication between Dickeya fangzhongdai in the production of plant cell wall degrading enzymes. This communication is known as quorum sensing.

AHL

The diagram depicts the expected scenario: the enzymes produced by the aiiA gene will bind to AHL molecules and subsequently break them down. This will complete the quorum quenching process.

constantly producing AiiA is not energy efficient and may exhaust Bacillus subtilis

we need to save energy

scroll down to see how we manage to save the energy consumption and simultaneously bring more benefits to our project!

what if AiiA is only produced when AHL is presented?

To conserve energy until the appropriate moment for the aiiA gene to be expressed, we've implemented an advanced degron system. This system is constructed using dCas9, CI repressor, and ssrA tag. The degron system serves the purpose of not only suppressing the expression of aiiA but also that of sspB and YsnE.

the regulator:
dCas9_CI_ssrA degron

To match the optimal moment for the aiiA gene to be activated, we implemented a groundbreaking degron system composed of dCas9, a CI repressor, and ssrA tag. This degron system is designed with the specific goal of suppressing gene expression until the appropriate time arrives. This degron system is also applied to other fragments that we’ve implemented on our plasmid.

guide RNA

the guide RNA (gRNA) will help locate the degron system to the correct location (target gene).

dCas9

is a modified form of Cas9 protein that is responsible for finding target genomic DNA sequences, and is also responsible for inhibiting the expression of certain genes until the degron system is degraded

CI Repressor

suppresses gene expression. This component is linked with dCas9 by the GSlinker. In our design, CI repressor works in tandem with the gRNA to guide the dCas9 to its precise destination, ensuring that the target genes remain suppressed until the appropriate moment of activation

ssrA-tag

plays a critical role in the degradation process. Once the ssrA-tagged dCas9 and CI repressor are in place, they become recognizable by ClpXP, a protease complex. This complex is aided by sspB, a protein that can direct ClpXP to the ssrA-tag’s location (as illustrated in the image). The SspB assists the overall system by speeding up the process of recognizing the complex

the illustration of ssrA tag leading the degradation of the degron system when the SspB is presence.

McGinness, Kathleen E., Tania A. Baker, and Robert T. Sauer. "Engineering controllable protein degradation." Molecular cell 22.5 (2006): 701-707.

target gene

the degron system we designed will lay itself on the gene we want to temporarily repress. This includes the aiiA gene, sspB and ysnE.

additional benefits!

ysnE is a gene that produces IAA, a plant growth promoting hormone. With the degron system, we can have IAA at expected time.

degron trigger:
LuxR and sspB

LuxR and sspB fragments are used to detect quorum sensing activities - OHHL in particular. When LuxR sensed the presence of AHL (OHHL), they combine, to triggering aiiA expression. sspB plays an essential role as it can speed up the combination of AHL with LuxR and aid ClpXP in degradation.

luxR

luxR acts as a receptor for AHL molecules. When they are attached, they will bind to the pLux promotor, which allows sspB to continue carrying out the process.

p23106

p23106 is the promotor that allows the constitutional expression of luxR.

Lux promotor

the promoter, responsible for the production of sspB, will only be activated when LuxR binds with AHL.

sspB ClpXP

sspB serves as a crucial facilitator in the interaction between ClpXP and the protein associated with the ssrA-tag. In this particular stage of the degron system, dCas9 undergoes degradation, paving the way for the activation of the remaining three components to commence their respective functions.

our idea is backed by the research done by McGiness as they proved the presence of SspB can facilitate the degrading process of ClpXP

McGinness, Kathleen E., Tania A. Baker, and Robert T. Sauer. "Engineering controllable protein degradation." Molecular cell 22.5 (2006): 701-707.

toxin antitoxin
kill switch.

to avoid any artificial pollution to the environment, we designed a killswitch that can be operated by us to kill Bacillus subtilis after it completes its job. This killswitch system is inspired by previous iGEM team, iGEM17_CCU_Taiwan. We made improvements and modifications so that it is more compatible with our project.

toxin and antitoxin

MazF induces cell death by precisely cleaving mRNA at a specific location, whereas mazE serves as an antitoxin, countering the effects of the toxic protein MazF.

pgrac promotor is responsible for the expression of both mazF and MazE

how do we
manipulate
the killswitch?

the killswitch is only activated when lactose is applied.

To exert additional control over this system, we've integrated a lactose inducer based on the lac operon, controlled by the constitutive promotor: pveg.

This operon allows the translation of the coding sequences only when lactose is present. In situations where lactose is lacking, both genes' translation is halted, resulting in the faster degradation of mazE compared to mazF. As a result, mazF is left unrestricted, ultimately leading to cell death. This design not only ensures the effective functioning of the kill switch but also enables activation within controlled timeframes.

lac operon

this diagram illustrates the condition where the killswitch is not activated

the protein produce by LacI will block the way of mazE, mazF, therefore repressing the expression of the killswitch

this diagram illustrates the condition where lactose is presented. The lactose will bind with the protein LacI produced, then leave the
operator. The killswitch is then activated.

Our plasmid, pHT_WIST168_Bacillus

We use golden gate assembly technique to reconstruct the vector of modified pHT10, named as pHT100. We insert two fragments that are multifunction in terms of expressing the genes and triggering their funcitons, and can be replicate into B. subtilis.