Team:OUC-China
Overview
How can we build a platform that ensures high production? By ensuring the system has the ability to recognize specific molecules and eliminate engineered bacteria that don’t have high production, our system can produce, identify, transport efficiently. We will introduce the results of these functions in the following aspects: number of copies, quorum system, kill switch and transport RNA.
Number of copies
We knocked out the phosphofructokinase gene essential for the growth of the engineered bacteria and planned to introduce it at the downstream of the production gene.We successfully construct production engineered bacteria.
Knock out PFK gene
To verify that we knocked out the phosphofructokinase gene in Aureobasidium melanogenum P16, we extracted the genome of P16 and P16 with PFK gene knocked out then amplified PFK gene.As shown in figure1, lane 2 has the exact length of PFK gene but lane 1 dose not,which indicated we successfully knocked out PFK gene.
We also incubated Aureobasidium melanogenum on YNB solid medium for two days, using glucose (Figure2.A) and lactic acid as carbon source(Figure2.B). 1, 2 and 3 are three phosphofructokinase knocked out strains . From left to right, the inoculated liquid decreased gradually.The results told us strains with PFK gene knocked out could hardly grow on the medium using glucose as carbon source but could grow on the medium as carbon source,which matches well with our expectation.
The activity of strain knocked out PFK
According to the results of measuring the growth curves of strains without PFK gene knockout and three strains with PFK gene knockout in YPD liquid and YPGL liquid medium, it can be seen that the three strains with PFK gene knockout can hardly grow in the medium with glucose as the main carbon source, while in the medium with lactic acid and glycerol as the carbon source, there is only a difference before and after a logarithmic period compared with the strains without PFK gene knocked out, At 60h, the same stable period was reached, which showed that the three strains with PFK gene knocked out had roughly the same production activity as the strains without PFK gene knocked out.
Quorum sensing
n our platform when the signal molecule IP reaches a certain concentration, it activates the SSRE promoter and increases the transcription of production gene. We transformed our fragments into the genome of our engineering bacteria and measured the intensity of SSRE promoter.
Genome transformation
We inserted the gene circuit of the quorum sensing system to the genome of Aureobasidium melanogenum pHPTX13-loxp, which was given to us from our PI. To verify if the DNA segments were inserted into the genome correctly，we amplified the target gene after extracting the genome of each strain. We selected 583bp specific fragment of AtCRE1 gene (Lane 1 ) and the whole length of PTP2+GFP (Lane 4) to test transformation. The electrophoresis gel show that the gene fragments were properly inserted into the genome.
SSRE promoter
At first, we measured the background expression intensity of SSRE promoter while measuring the growth curve of the transformed strain.
For IP dose–response experiments, all strains were precultured for 8h with terminal OD600 of 0.4, then treated with IP for 0，0.5，1，1.25，1.5h at 28℃, 175 rpm, and the fluorescence intensity of the strains was measured. Different concentrations of IP can increase the expression of SSRE promoter. After rough statistical analysis, the expression intensity of SSRE promoter was significantly correlated with IP concentration, showing that SSRE promoter can fulfill our assumption to regulate the production and growth. In order to obtain more SSRE promoters with different dynamic regulation ranges, we obtained various of subtypes of SSRE promoters through modeling and analysis
Other findings
The results of the background expression intensity of SSRE promoter showed that the expression intensity of SSRE decreased at the 6th hour. The results of repeated experiments are the same. Therefore, we suspect that there may be a protein with similar function to AtlPT4 in the genome of Aureobasidium melanogenum.
Kill switch
To make sure our platform always maintains high production, our team used a ribozyme riboswitch to identify the product molecule. And through further engineering, we used the aptamer to construct a kill switch that only lets the engineered bacteria live when the product molecule is produced in high intensity.
SELEX
Our primary part is to screen RNA aptamers that could bind specifically to our product. We decided to perform SELEX 12 times, but we failed in the early rounds and there was no band on the electrophoresis gel. We figured out that the RT-PCR step didn’t work as we expected. So, we changed our experimental protocol, we divided reverse transcription and amplification into two steps during PCR, and we got aptamers that combined specifically and strongly with mannitol.
Dissociation Constant(Kd) of RNA Aptamer
Firstly,we simulated the relationship between the expression of Yope and the dissociation constant of RNA aptamer,which showed that if we want to express YopE while the production is low,we must get the aptamer with low Kd value.
Our team simulated RNA aptamer with high specificity for γ- aminobutyric acid(GABA) by modeling method. We then synthesized the target RNA aptamer sequence with fluorescence 3’ FAM. Therefore, the dissociation constant(Kd) of the aptamer was determined by fluorescence spectroscopy. We incubated the aptamer with a range of concentrations of GABA for two hours at room temperature, respectively. Next, we measured the fluorescence intensity at 525nm of each group and used the following formula to characterize the Kd value.
Here, F0 and F represent the fluorescence intensity of the fluorescent groups in the presence and absence of GABA.
GABA-1 is the aptamer we designed in silicon.But it have worse GABA-binding ability compared to GABA-2.Although the result of Kd value of GABA-1 aptamer is not very good,we decreased it by learning the experiment data to design another aptamer called GABA-2.It has a stronger binding ability,showing we could adapt this sequence to ribozyme riboswitch.
Toxic protein
At the beginning of the project design, we chose YopE as the suicide protein of our engineering bacteria. Injection of YopE into eukaryotic cells induces depolymerization of actin stress fibres. After communicating with other teams, we found that using YopE as the suicide protein in our design not only posed a security threat to the platform's R&D personnel, but also posed a greater security problem to the staff of the microbial factory. After communicating with the DUT-China team to test the sacB toxic protein in their project, we decided to use sacB as the final suicide protein of our platform because of its good killing effect and better biosafety.
As shown in figure 10(on the left), we selected 183bp specific fragment of SacB gene and genome PCR to test transformation. Lane 1 is genome PCR of Aureobasidium melanogenum P16 transformed with empty genome transformation plasmid. Lane 2 is genome PCR of Aureobasidium melanogenum P16 transformed with genome transformation plasmid including SacB gene ,which shows we introduced SacB gene successfully.
As shown in figure 10(on the right), area 1 is Aureobasidium melanogenum with empty genome transformation plasmid; area2 is Aureobasidium melanogenum with SacB gene transformed into the genome;area3 is Aureobasidium melanogenum with SacB gene transformed into the genome and cultured with 5mM sucrose, showing SacB has a great ability to kill Aureobasidium melanogenum.
Because Aureobasidium melanogenum has a strong sugar production capacity, SacB protein has a certain toxicity to it when fructose is not added to the culture medium. In the future, we will optimize the fermentation conditions of Aureobasidium melanogenum and add appropriate sucrose to the culture medium, so that the SacB protein expressed by Aureobasidium melanogenum has an ideal killing effect on it after the low yield triggers the kill switch.
Transport RNA
In order to transport the products out of the engineering bacteria in time and break through the limitation of limited types of protein transporters, OUC-China decided to apply the aptamer to the transmembrane transport of products. This RNA molecule with transporter function consists of three strands of two different RNA. Among them, one RNA is responsible for targeting the cell membrane structure, and one RNA is responsible for specifically binding products. In order to simply test the transport performance of the transduced RNA, OUC-China decided to use the extracellular sealing membrane structure to incubate the transduced RNA and product molecules, and to test the transport performance of the transduced RNA by detecting the molecular residue of the product in the supernatant of the membrane structure.
Due to the epidemic situation, our partner NJU-China did not have enough time to use their exosomes to detect the performance of RNA transport. We believe that we can enrich our wikis and parts after verifying the performance of RNA transport after the competition, which will inspire the following iGEM teams to flexibly apply RNA in different aspects.
