iGEM Unesp Brazil

Results and Demonstrate

Sense and control

We have built a fully functional regulatory circuit that we named Glucose Responsive System, or pGRS. The circuit was designed to function as a NIMPLY logic gate (Figure 1). It was cloned into plasmid pBs1C and introduced into the genome of Bacillus subtilis 168.

**Figure 1.** In this circuit, glucose acts as input for the production of sRNA and TetR. The sRNA acts by blocking the translation of the mRNA of our gene reporter and TetR acts by repressing the production of T7 RNA polymerase, required to produce our protein reporter (mCherry). In the absence of glucose, Pgal is active and thus both sRNA and TetR are produced, blocking the translation and transcription of mCherry. When there is glucose, Pgal is inactive and thus the sRNA and TetR are not produced, leading to the production of mCherry.

In order to test the circuit, we started by growing the B. subtilis carrying plasmid pGRS in LB and minimal medium supplemented with different carbon sources at 1%: glucose and fructose, that were expected to activate the gene expression and galactose and mannitol, that were expected to deactivate. Although glucose and fructose worked as expected, we saw that mannitol also activated the gene expression. This is because once mannitol enters the cell and is phosphorylated, D-mannitol-1-phosphate is converted to fructose-6-phosphate and then to fructose-1,6-bisphosphate, which is the main activator of CCR in gram-positive bacteria [1]. For galactose, we noticed slow growth both in LB and minimal medium and thus, too low or no protein (mCherry) production was measured.

We then started to look for other carbon sources that would not activate CCR, deactivate our system and yet result in a satisfactory growth. We tested sorbitol, xylose, glycerol, arabic gum and lactose. Using LB supplemented with 1% lactose we finally got a satisfactory growth and deactivation of gene expression. Therefore, we continued our tests with lactose and fructose, as cell growth was higher on fructose than on glucose. Moreover, it is metabolized through the same pathway as glucose, activating CCR. Thus, the circuit should maintain its function both in glucose and fructose equally. All cultures were incubated at 30°C, as we found out that growth at 37°C affects mCherry production.

We grew B. subtilis carrying plasmid pGRS in LB media supplemented with 2% fructose and lactose overnight. The cultures were centrifuged, the cell pellet was resuspended in NaCl 0,9%, and we measured the absorbance at 600 nm and fluorescence of mCherry. Figure 2 shows results for the positive control (B. subtilis producing mCherry constitutively) and the test strain (B. subtilis carrying the synthetic circuit). B. subtilis 168 was used as negative control and its intrinsic cell fluorescence was considered as blank.

**Figure 2.** Relative fluorescence of positive control and test strain in NaCl 0,9%. The test strain exhibits strong fluorescence at the same intensity as the positive control when grew in fructose and no fluorescence when grew in lactose, confirming that fructose activates the gene expression in our system and lactose deactivates it. The experiment was made in duplicate. (*): error bars not shown due experimental problems in one of replicates. pGRS: Plasmid Glucose-Responsive System.

Furthermore, we tried to grow our device carrying B. subtilis in minimal medium supplemented with different concentrations of carbon source to eliminate the possibility of unknown sugars in LB medium interfering with the device’s response. Unfortunately, B. subtilis grows very poorly in minimal medium supplemented with lactose.

As demonstrated in Figure 2, our Glucose Responsive System works as expected. Gene expression is ON when glucose/fructose is present, and it is completely OFF in the absence of it. This is a very important result to show the specificity of our circuit and to demonstrate that we accomplished our goal of avoiding leakages with this design.

Production

Production of penetratin fused proteins

Penetratin-Insulin

In order to validate the production of the Penetratin-Insulin peptide by Bacillus subtilis K07, we cultivate strain 210 in shaker flasks in LB medium. B. subtilis strain 210 carries a his-tagged version of the peptide Penetratin-Insulin. As controls, we cultivated B. subtilis strain 102, which carries an empty plasmid, and the strain 179, which carries an untagged version of Penetratin-Insulin. Samples were withdrawn from each flask after 9h, 12h and 24h of growth. Then, all collected samples were submitted to SDS-PAGE analysis (Figure 1). Surprisingly, the his-tag improved Insulin synthesis greatly.

**Figure 1.** SDS-PAGE of the cell lysates from cells collected after 9, 12 and 24 hours of B. subtilis K07 growth. Strain 102 carries an empty plasmid, strain 179 carries the gene penetratin-insulin under control of the srfA promoter, and strain 210 carries his-tagged penetratin-insulin gene under control of the srfA promoter.

Our results show that B. subtilis produces great amounts of his-tagged Penetratin-Insulin, and that no apparent degradation of insulin occurs in 24h of culture.

Penetratin-GFP

The E. coli BL21 transformed with the pBS1C plasmid carrying the penetratin-GFP gene under control of the T7 promoter. The engineered strain was cultivated in LB medium until OD 0.6 and induced with IPTG 1 mM. Cultures were incubated for further 4h. Cells were harvested by centrifugation, and cell lysates prepared by sonication. Penetratin-GFP was purified using magnetic beads. Both crude and purified protein solutions were analyzed by SDS-PAGE (Figure 2).
We did the SDS-page before and after the purification of the cell lisate.

After purification, we still had some contaminants in the protein solution. Nonetheless, it was possible to identify the 29.9 kDa band corresponding to the his-tagged Penetratin-GFP.

Penetratin permeation through the intestinal epithelium (in vitro assay)

In 2017, our team demonstrated that our genetically engineered probiotic could survive in the simulated microbiome of a diabetic person for up to 16 hours.
This year we wanted to see whether the cell penetrating peptide Penetratin, which is fused to the insulin part we constructed, could promote permeation of a cargo protein through the intestinal epithelium. Therefore, we performed an in vitro permeability test in caco-2 cell monolayer. Caco-2 cells are human colon carcinoma cells that slowly differentiate into a monolayer of intestinal epithelial cells when cultured in a permeable support (HUBATSCH et al., 2007).
We cultured caco-2 cells on a porous plate filter in a transwell system (Figure 3). After 21 days of growth, cell differentiation was observed forming a monolayer of caco-2 cells (Figure 4).

**Figure 3.** In vitro permeability test in caco-2 cell monolayer. Caco-2 cells were cultured in DMEM medium in a transwell plate for differentiation and further permeability test. GFP (control) and Penetratin-GFP were added to the apical compartment of the transwell plate.

**Figure 4.** Image of differentiated caco-2 cells after 21 days of culture.

At the 21st day, we added about the same amount of GFP (control) or Penetratin-GFP to the apical compartment of the transwell plate (3 wells for each treatment). The plate was incubated for further 4h (Figure 3). In order to determine whether the cell layer was viable, we measured the trans-epithelial electrical resistance (TERR). Inserts presenting EQ values at least 260 ± 65 Ohm x cm2 are considered ideal for testing (HUBATSCH et al., 2007). Our monolayers presented EQ values between 253 and 350 Ohm x cm².
Samples from apical and basal compartments were taken for fluorescence analysis at beginning and after 4h of assay. Only the GFP fused to penetratin was able to cross the differentiated caco-2 monolayer (Figure 5). From the total penetratin-GFP added to the apical compartment at time 0, about 71% crossed the monolayer after 4h.

**Figure 5.** GFP fluorescence measured in the apical and basal compartments of the transwell plate. Fluorescence found in the basal compartment indicates that penetratin promoted permeation of GFP through the caco-2 monolayer.

We extrapolate that if penetratin can aid GFP (27 kDa) crossing the caco-2 monolayer, it should also work for the much smaller single-chain insulin (7 kDa).

References

Hubatsch, I. et al. Determination of drug permeability and prediction of drug absorption in Caco-2 monolayers. Nature Protocols 2(9), 2111-2119, 2007.

Biocontainment

We could not build the final version of our kill switch. Our system is a very complex circuit and we had a lot of problems to build it, so we couldn't finish it and test it. However, we were able to construct the sequence Physpank-NCas9-pMag-nMag in pBs1C, missing just the other half of split Cas9.

Team:Unesp Brazil/Demonstrate