Design and Results
Overview
We’ve achieved results mainly in four segments: wet lab, modelling, docking, and hardware.
Wet Lab
Our plans at the wet lab consisted mainly of three parts. First, the expression of our enzyme using E. coli as a chassis. Secondly, expressing our enzyme at Lactobacillus acidophilus as a probiotic. And last but not least, testing our enzymes on C. elegans, a nematode model organism. As demonstrated in the image below, the tasks were divided between the two universities of the team being UFMG in charge of the expression and characterization of both chitinases in E. coli and the test of nematicidal effect. And UFV responsible for characterizing various signal peptides designed and quantifying the production of the chitinases.
Expression of Endo and Exochitinase in E. coli
Although our objective is to express endo and exochitinase in Lactobacillus, at first, we developed a composite part for expression in E. coli, as it is a well-studied and characterized model organism.
Design
For this, our team designed a composite part to express and secrete CfcI exochitinase from Aspergillus niger and PCCHI44 endochitinase from Pochonia chlamydosporia in E. coli. Both constructs consist of a strong constitutive promoter, RBS, NSP4 signal peptide, CDS, histidine tag (6x) and terminator.
We requested both composite parts of the IDT Grant flanked with restriction enzyme site EcoRI and XBal in the prefix and Spel and PstI in the suffix to facilitate our future digestion.
Cloning
We added 3' adenine overhangs with Taq polymerase and proceeded to TA cloning, inserting the construct in pCR™2.1-TOPO™ cloning vector from ThermoFisher (the vector comes with thymine overhangs). Then, we transformed DH5alpha E.coli with the resultant plasmid.
Digestion
The bacteria grew and we performed Miniprep to obtain more quantity of the plasmid. After that, we performed two digestions (in the Miniprep and in the pSB1C3): first with EcoRI and then with PstI. In the image below, we show a 1% agarose gel, where we confirmed the digestion with EcoRI and extracted the resultant bands from the gel. The stains in the gel are caused by RNA contamination, because we forgot to add RNAse during Miniprep.
Then, we digested the resultant samples with PstI. Then, we precipitated the post-digestion DNA samples.
Ligation
After that, we ligated the backbone + the insert with the T4 DNA Ligase. We left the reaction in the Thermocycler 16°C overnight. In the subsequent day, we transformed BL21 E. coli .
Confirmative PCR
We also successfully performed PCR to confirm the insertion of our insert using Vf and Vr primers that flank the gene regions.
Expression
In search of investigating the expression of our enzyme. We cultured 1 liter of each enzyme in LB, and separated its supernatant and pellet. Afterward, we concentrated our supernatant, as our enzymes should be secreted in theory. We then sonicated the pellet, separating the soluble and insoluble fractions to cover all our possibilities. Then, we made a new polyacrylamide gel.
Unfortunately, it was not possible to add the pellet and insoluble fraction samples, because they did not enter the gel, because of their viscosity. We can see that the solution fraction has demonstrated a drag through the gel, probably caused by proteins native to E. coli. Sadly we didn't see the band corresponding to our enzymes in any of the samples, even in the supernatant as expected.
We assume that if our proteins are being secreted, there is a small amount, which cannot be visualized on the agarose gel. We assume that there is an expression of the proteins, in view of the altered visual appearance of the transformation plating (Figs 5 and 6). To confirm, we would have liked to have performed a Western Blotting test, but it was not possible due to the short time.
Expression of Endo and Exochitinase in L. acidophilus
Our main idea was to express and secrete both enzymes in Lactobacillus acidophilus and characterize signal peptides quantifying exported GFP. So we designed constructions totally optimized to produce and send the proteins to the extracellular compartment.
When our constructions arrived, we tried to clone them into pGEM®-T Easy vector, but it wasn't working out (Sep 5 to 9 - Week 1). Meanwhile, in our other lab space (UFMG), the group successfully cloned the constructions optimized for E. coli into pCR™2.1-TOPO™ vector and then into pSB1C3. So they sent us a sample and we could transform the E. coli optimized constructions of Exo and Endochitinases in L. acidophilus
The transformed bacteria grew in 30ug/uL and in 50 ug/uL of Chloramphenicol
But when we did a plasmid extraction and a PCR, the samples showed highly unspecific bands, it was probably an experimental mistake, but the deadline couldn’t allow us to repeat the experiment. However, because the bacteria is growing on MRS broth supplemented with 50 ug/mL of CLO, we believe that the chitinases were transformed.
Along with the PCR we were also trying to turn chitin, which is highly insoluble, in colloidal chitin to add it to agar plates and check if Lactobacillus was secreting the enzymes. The colloidal chitin obtained by the protocol wasn’t as soluble as we expected but we could put it in a plate with the bacteria. The results suggested an initial halo formation that could be due to the degradation of chitin, but further tests need to be done.
Signal peptides for L. acidophilus
For the signal peptides, we assembled multiple constructions using the same promoter, RBS, GFP, and terminator, with the variation of the signal peptide, the full collection can be found here. We also did a codon optimization of the signal peptide SacB form Bacillus subtilis to improve the secretion of extracellular proteins.
But unfortunately, because of the failed cloning into pGEM®-T Easy, and the lack of TOPO in our lab, we could not proceed with the signal peptides testing in enough time before the competition.
Troubleshooting
Since our wet lab team faced many problems, we decided to make a troubleshooting session that can be helpful for future iGEMer's that may come across similar situations
Docking
Our molecular docking simulations show that the chosen chitinases would bind correctly with a good binding affinity score to a chitin polymer (octamer).
A. niger CfcI bound to chitin shows a free energy score (ΔG(Kcal/mol)) of -11.6, pointing to a favorable reaction with chitin binding to the protein pocket.
P. chlamydosporia Pchi44 docking results showed a free energy score of -10.6, also pointing to a favorable reaction.
Modelling
Through our mathematical model, we obtained different parameters from the literature and used them to predict the chitinase production by Lactobacillus acidophilus inside the human intestine. To do that, we created a function that represents the relation between probiotic factor and time. After integrating this function and joining it with other parameters, we obtained the chitinase production by our probiotic chassi inside the human intestine. Dividing the resultant value by the intestinal volume, we achieved the concentration of the chitinase inside the small intestine, according to time and to the probiotic dose. We performed this calculation both with endochitinase and exochitinase, and using the maximum and minimum values of our variables, obtaining our results as maximum and minimum concentration as well.
Additional information can be found HERE.
Hardware
The development was an open-source modular low-cost benchtop bioreactor. It stands autoclave (except for pH sensor which is chemically sterilized using a hypochlorite solution solution). It is able to read pH, temperature, and proxy Optical Density through the reading of the media color changes. Users can also control light exposure (it can be used as a photo-bioreactor) and activate peristaltic pumps to compensate for pH changes or to keep the growth phase steady. A heating pad is also an option to keep it warm, if not using it as a photo-bioreactor.
