Engineering Success

The Symbiosis system

Design 
    The symbiosis system is designed to make the three microorganisms thrive in the human gut and live with each other to deliver drugs. The system contains three microorganisms: unengineered vector Clostridium butyricum, and engineered vector Kluyveromyces marxianus and Bacillus subtilis. Some theories hypothesized that the first colonizers of the human gut are aerobic microorganisms. As people grow up and develop, the aerobic microorganisms consume oxygen in the gut, making the gut suitable for the growth of more anaerobic microorganisms. In our system. Bacillus subtilis, generally regarded as an aerobe, can grow under strict anaerobic conditions, such as the human intestines. Kluyveromyces marxianus is a facultative anaerobe, and Clostridium butyricum is an obligate anaerobe. Firstly, the growth of beneficial anaerobic bacteria, so kluyveromyces marxianus will thrive, Bacillus subtilis rapidly consumes oxygen in the intestinal tract, leading to low oxygen conditions in the gut and promoting consumption of more oxygen, providing an ideal habitat for Clostridium butyricum to flourish. Also, Kluyveromyces marxianus can produce prebiotics, a group of nutrients that serve as sustenance for the gut microbiota, further enhancing their growth within the gut.  

Build 
   Clostridium butyricum, Kluyveromyces marxianus, and Bacillus subtilis are co-cultured in YPD medium for one week. 

Test 
   To confirm the system work, we first culture five groups of microorganisms with different combinations: Clostridium butyricum, Kluyveromyces marxianus, Bacillus subtilis, Bacillus subtilis and Clostridium butyricum, Bacillus subtilis and Clostridium butyricum and Bacillus subtilis, and numbers 1 to 5 are given to each tube, respectively. YPD is added near to the rim of the tube, simulating the low-oxygen environment of the human gut. Then, we utilized direct observation and spectrophotometer to examine the efficacy of the symbiosis system. In the culture, C. butyricum would produce hydrogen, so the foam over the liquid culture indicates the presence of C. butyricum. K.marxianus in the culture is engineered to produce astaxanthin, so the red color of the liquid culture indicates the presence of K. marxianus. Firstly, only culture 4 and 5 have foams over the liquid culture, substantiating that b. subtilis consume oxygen to make the environment suitable for C. butyricum’s growth. It is worth noting that culture 5 has more foam over the liquid culture, implying that the presence of k. marxianus enhances the growth of C. butyricum. After one week of culturing, cultures 4 and 5 turn red, showing the presence of engineered k. marxianus. Moreover, culture 4 and 5 are the most turbid among the five, showing the growth of microorganisms in the symbiosis system. These results imply that the co-culture system allows microorganisms to thrive. 

   Secondly, to examine the efficacy of the symbiosis system, we used a spectrophotometer to determine the optical density of five cultures measured at a wavelength of 600 nm in 1 cm light path (ODˊ600). After 24 hours of culturing, culture 1 has low optical density because C. butyricum is a strict anaerobe; culture 3 has low optical density because the B. subtilis strain in the culture (Bacillus subtilis 168) requires much oxygen than is in the tube. The facultative K. marxianus grows as expected. Culture 4 and 5 have the highest ODˊ600. Furthermore, culture 5 turns red after a week.

   Combined direct observation and the data obtained from spectrophotometer, in the culture, B subtilis and K. marxianus consuming oxygen allows for the subsequent thrive of C. butyricum, and at the same the other microorganisms can still grow. The symbiosis system, a promising co-culture system for live biotherapeutic products that localize in the human gut, allows the three microorganisms to live and grow together.

Secretion system

K. marxianus-Astaxnthin 

Design 
   The design of the astaxanthin biosynthesis pathway incorporates numerous essential enzymes that catalyze the production of astaxanthin from acetyl-CoA. The design complies with yeast’s monocistronic system and utilizes constitutive promoter Adh1 and terminator Adh1 to facilitate gene expression in yeast. In the pathway, genes chyB, crtE, crtI, CrtYB, and bkt express catalytic enzymes, beta-carotene 3-hydroxylase, geranylgeranyl diphosphate synthase, phytoene dehydrogenase, Bifunctional lycopene cyclase/phytoene synthase, and β-carotene ketolase, that facilitate the production of astaxanthin. One hygromycin-resistance gene hph is included in the construct for selection. The construct is integrated into the pklac2 expression vector, an expression vector capable of both replication in E. coli and stable integration into the genome of the yeast, especially kluyveromyces lactis.  

Build 
   The gene fragments are synthesized from Twist Bioscience and assembled through Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO). In the PGASO system, each cassette has a homologous recombination site that allows homologous recombination between two different cassettes to take place. Then, we do the colony PCR test to check if the gene fragments are assembled correctly.   

Test 
   The kluyveromyces marxianus turns red after being engineered, indicating the accumulation of intracellular astaxanthin and engineering success. Yeasts reproduce by budding. There may be some buds that have not yet broken off from cell bodies. Some of the buds might not contain the desired genes but can still survive after antibiotics selection because the engineered cell bodies have antibiotic resistance. We observed that cell sizes are not uniform and some of them are not red, implying that some of the cells in the culture do not contain the desired genes. Given the readily observable phenotype of the engineered Kluyveromyces marxianus, we employed a three-generation streak selection method to further purify the cell culture. 

Zeroth generation 
   The zeroth generation of the engineered k. marxianus is the first culture we obtain after transformation. In this culture, we observed that cell sizes are not uniform and some of them are not red, indicating that some of the cells in the culture do not contain the desired genes.  

First generation 
   4 agar plates are divided into 64 streaks in total. We pick 64 red colonies from the zeroth generation of the engineered K. marxianus and culture them in a separate streak on 4 agar plates. The cells turn much more red in this generation but can still be further purified. 

Second generation 
   4 agar plates are divided into 64 streaks in total. We pick 64 red colonies from the first generation of the engineered K. marxianus and culture them in a separate streak on 4 agar plates. The cells turn much more red in this generation but can still be further purified. 

Third generation 
   2 agar plates are divided into 32 streaks in total. We pick 32 red colonies from the first generation of the engineered K. marxianus and culture them in a separate streak on 2 agar plates. In this generation, the phenotype of the engineered cells are improved, which brings an end to the streak selection 

HPLC test 
   Lastly, We conducted an HPLC test on culture 4, 5, 9, 12, 14, 56 to quantify the astaxanthin produced by the cells. Among them culture 56 shows the best result, with 0.11 g astaxanthin per gram yeast powder.

K. marxianus- NMN

Design 
   The NMN biosynthesis pathway design incorporates a multitude of essential enzymes responsible for catalyzing the conversion of aspartate and glucose into NMN. This design adheres to yeast's monocistronic system, employing the constitutive promoter Adh1 along with the terminators Adh1 and Prm9 to facilitate gene expression within yeast cells. Within this pathway, genes NadA, NadB, NadE are responsible for expressing catalytic enzymes that are essential in the production of NMN.  

To enable the functionality of this genetic construct, it is integrated into the pklac2 expression vector. This vector possesses the ability to both replicate in E. coli and stably integrate into the yeast genome, particularly in the case of kluyveromyces lactis. 

Build 
   The gene fragments are synthesized from Twist Bioscience and assembled through Promoter-based Gene Assembly and Simultaneous Overexpression (PGASO). In the PGASO system, each cassette has a homologous recombination site that allows homologous recombination between two different cassettes to take place. Synthetic fragment NadA is derived from , NadB, NadE. Then, we do the colony PCR test to check if the gene fragments are assembled correctly.

Test 
   We conducted the HPLC test to confirm that the engineered K. marxianus can produce NMN. The HPLC test shows that the engineered K. marxianus is able to produce NMN, but we think that the production of NMN can be further enhanced so we do the redesign. 

Redesign 
   The NMN biosynthesis pathway design incorporates a multitude of essential enzymes responsible for catalyzing the conversion of aspartate and glucose into astaxanthin. This design adheres to yeast's monocistronic system, employing the constitutive promoter Adh1 along with the terminators Adh1 and Prm9 to facilitate gene expression within yeast cells. Within this pathway, genes NadA, NadB, NadE, NadV, and PrsA are responsible for expressing catalytic enzymes that are instrumental in the production of NMN. Additionally, NMN intracellular accumulation is proven to decrease the rate of NMN biosynthesis. PnuC serves as the transporter responsible for pumping NMN out of the cell. A plasma-membrane associating sequence (PMassc) follows after the PnuC coding sequence. This sequence will translocate PnuC to the cell membrane if yeast. 
   To enable the functionality of this genetic construct, it is integrated into the pklac2 expression vector. This vector possesses the ability to both replicate in E. coli and stably integrate into the yeast genome, particularly in the case of kluyveromyces lactis. 

Build

Test 
   First of all, we have to prove the PMassc sequence derived from s.cerevisiae work in kluyveromyces marxianus. We fuse reporter GFP with the construct to see if the transporter is delivered to the cell membrane. Fortunately, under a microscope, we observe fluorescence on the cell membrane of kluyveromyces marxianus, proving the functionality of the PMassc sequence in K. marxianus.

Due to time constraints, we have not finished testing for this construct. 

B. subtilis-HMB

Design 
   The design of the HMB biosynthesis pathway incorporates numerous essential enzymes that catalyze the production of HMB from leucine. In the pathway, RNA polymerase binds to promoter veg, initiating transcription. Genes HPB and LAAD express catalytic enzymes 4-hydroxyphenylpyruvate dioxygenase and soluble l-amino acid deaminase that facilitate the production of astaxanthin. The construct is integrated into the pet28a expression vector.  

Build
   The gene fragment is synthesized by Twist Bioscience (ABreal). The synthetic hpd gene is derived from Pseudomonas entomophila. The synthetic Laad gene is derived from Proteus vulgaris. The gene fragment and pet28a plasmid are digested with BamHI enzyme and EcoRI enzyme and ligate with each other. Then, we do the colony PCR test to check if the gene fragment is integrated into the plasmid 
Test 
   We used the HPLC test to quantify the amount of HMB produced by the cell culture and show success in engineering Bacillus subtilis. The total amount of HMB produced by the engineered Bacillus subtilis is 3.97 g/L. 

Kill Switch system

B subtilis-Kill switch

Design 
To tackle the issue of potential microorganism release, we've devised a safety mechanism known as a kill switch. This kill switch relies on Hydrogen Sulfide, a commonly found gas in the human digestive system, as its indicator. Our design draws inspiration from the TA (Toxin-Antitoxin) system to regulate the controlled termination of our organisms. 

Given that we are developing a Life Biotherapeutic Product (LBP), it's imperative that the kill switch remains inactive within the medicine capsule while it's being ingested. Otherwise, the product would lose its effectiveness. To achieve this, we employ DRE recombinase to govern the activation of the kill switch. 

Here's how it works: Initially, when the microorganism is inside the medicine capsule, where there is an absence of hydrogen sulfide, it remains viable because the kill switch remains deactivated. Subsequently, after entering the gut, the organism continues to survive. Even though the kill switch has been triggered, the Antitoxin within the system counteracts the Toxin, allowing the engineered organisms to persist.

However, in the event of a genetically modified organism escaping during elimination, our genetic organism is programmed to self-terminate to prevent any potential harm to the environment. For further details, please refer to the kill switch page on our website. The entire process and the gene map is illustrated below:

Build 
Due to time constraints, we weren’t able to complete the engineering cycle of this particular construct. 

K. marxianus-Kill switch

Design 
While the fundamental concept of the kill switch remains consistent for Bacillus subtilis, we've made specific adaptations tailored to Kluyveromyces marxianus. Our primary adjustment involves replacing the recombinase with CRE, and we've carefully customized the promoters and terminators to align with the unique genetic characteristics of Kluyveromyces marxianus, which differ from those of Bacillus subtilis. Additionally, we've divided the composite kill switch into four distinct segments, each controlled by its own unique promoter. This modification is essential because Kluyveromyces marxianus operates according to a monocistronic pattern, in contrast to the polycistronic pattern observed in Bacillus subtilis.

In addressing the issue of promoter compatibility – specifically, the inability of prokaryotic promoters to function in eukaryotes – we've integrated two promoters into a single entity, combining Promoter ADH1 with Promoter sqr. The ADH1 promoter acts as the driver for RNA polymerase, while the sqr promoter plays a regulatory role by facilitating the binding of the sqrR repressor. It's important to note that this composite promoter is currently a hypothesis and requires further experimental validation to ensure its functionality and compatibility within the Kluyveromyces marxianus system.

Build 
Due to time constraints, we weren’t able to complete the engineering cycle of this particular construct.