Our main objective is to get synthetic bacteria containing the two enzymes required for the transformation of tryptophan extracted from by-products to serotonin:
Bacteria with the tryptophan-5-hydroxylase (TPH)
Bacteria with the L-amino acid decarboxylase (TDC)
When these two types of synthetic bacteria express their enzymes of interest and these ones are put together in a test tube with the necessary amount of by-products-coming tryptophan, the TPH enzyme is able to convert it into the intermediate 5-hydroxytryptophan and the TDC enzyme gets it to transform it into 5-hydroxytryptamine, better known as serotonin.
The lab procedure will be divided into two cloning secondary experiments:
The obtaining of this bacterium is achieved by means of the following procedures, based on restriction enzymes and ligase cloning:
Miniprep of the pET28a plasmid
First of all, it is needed to extract the DNA plasmid corresponding to the pET28a vector from bacteria containing it. It is also required to ensure the correct extraction by gel electrophoresis. It corresponds to the 5369 bp band.
Digestion and purification of pET28a
Once the vector is obtained, it must be digested with the corresponding restriction enzymes: EcoRI and HindIII, which will leave sticky ends at both sites of the linearized vector. Then, a gel purification is required to ensure the digestion of the vector.
Zoom of the region where the restriction enzymes cut the plasmid:
PCR of TDC
As the gBlock was ordered just with the coding sequence, it is required to add the restriction sites of EcoRI and HindIII at the ends flanking it. Concretely, EcoRI site must be at the 5’ end and HindIII at the 3’ end.
Digestion and purification of TDC
The next step is the digestion of the TDC fragment flanked by the restriction sites with the corresponding enzymes. Therefore, we will have the TDC gene with the necessary sticky endings to perform the ligation. It must also be purified after the diagnostic gel.
Ligation of TDC and pET28a
Then, the digested and purified TDC and pET28a must be ligated with the T4 ligase.
Transformation of bacteria and plating
The transformation must be performed with the ligation containing the gene of interest. The bacteria selected are E.coli and the methods of transformation are heat shock and electroporation, in order to increase our probabilities of success by developing different ways of transformation. Then, the synthetic bacteria are platted in agar with kanamycin, as the plasmid contains the gene for kanamycin resistance. Therefore, a positive selection will take place because only the transformed bacteria that contain the plasmid will be able to survive and grow in that medium.
Purification of the enzyme
The next step is to purify the enzyme expressed by the bacteria thanks to the histidine tag it owns that will bind to the metal ions immobilized in the resin matrix of purification columns. This is called His-Tag purification, which is based on the affinity of histidines residues for metal ions (added at the C-terminal of the protein because of its previous cloning in pET28a).
The obtaining of this bacterium is achieved by means of LIC cloning. The following procedures are required:
LIC or ligase-independent cloning is a strategy which relies on the 3'-5' exonuclease activity of T4 polymerase. Although the polymerase activity of T4 prevails over its exonuclease activity, this predominance can be reversed by modifying the reaction conditions. For this, it is necessary that the ends of the vector and the insert of interest are complementary and are composed of all the dNTPs except for one of them, which should appear once at the end of these borders. By PCR, we can add to the insert of interest the sequence complementary to the flanking regions around the insertion site in the vector.
If the reaction mix contains all the required elements with only the dNTP complementary to the one that appears uniquely at these borders, the polymerase will use its 3' → 5' exonuclease activity to digest the strand it is working on to the point where it can add the nucleotide from the mix. At that point where it can add a nucleotide because of the presence of a template, the polymerase activity will precede over the exonuclease and incorporate the nucleotide that has been added to the reaction. In this way, large overhangs are generated that will allow binding between the vector and the insert of interest without the need for a complementarity ligase enzyme. For this, these overhangs must be at least 15 nt in length.
Let’s say that the nucleotide of choice is thymine, T. With the sequences flanking the insertion site, primers are designed with which to add this complementarity to both sides of the insert of interest. These sequences are composed of all possible nucleotides and have a single T at the end. At the start of the reaction, the T4 enzyme will begin to digest one of the strands due to the lack of nucleotides in the mix. This enzyme will not recover its polymerase activity until it reaches the T position, where it can insert its complementary base, A, which is the only one containing the reaction cocktail (dATP). This will create single-stranded ends of approximately 15 nt in length in both the insert and the vector that will be complementary to each other and will bind immediately, without the need for ligase.
A variant of the LIC method is the InFusion method, which consists of a commercial kit with an enzyme that has this intrinsic capacity, so that with a commercial mix and correctly designed primers, it acts as a polymerase/exonuclease, generating these cohesive ends necessary to generate the complementarity in both the insert and the vector, and to get them to join without the need for subsequent ligation.
The obtaining of this bacterium is achieved by means of the following procedures:
Miniprep of the pET15-MHL plasmid
The first step is based on the isolation of the pET15-MHL plasmid from the bacteria.
Linearization and purification of pET15-MHL
The plasmid pET15-MHL must be linearized by digestion using BseRI (type II restriction enzyme) and then purified by gel electrophoresis.
PCR of TPH
On the other hand, the insert requires the addition of overlapping ends in order to be able to join the plasmid ends resulting from linearization. These ends consist of complementary 15 nucleotide DNA sequences.
T4 polymerase reaction using 3’ exonuclease activity
First, a reaction must take place between T4 polymerase and the linearized pET15-MHL, in the presence of dGTPs. The same reaction must take place with TPH but in the presence of dCTPs, instead of dGTPs.
Annealing between TPH and pET15-MHL
Then, both parts join together as they present 15 bp overlapping ends. No ligase is required for this step, and this is the reason for the cloning method’s name.
Transformation of bacteria and plating
The resulting construction must be introduced inside the competent bacteria (TOP10) by heat shock. Then, the synthetic bacteria are platted in agar with ampicillin, as the plasmid contains the corresponding resistance gene. Therefore, a positive selection will take place because only the transformed bacteria that contain the plasmid will be able to survive and grow in that medium.
Purification of the enzyme
The next step is to purify the enzyme expressed by the bacteria thanks to the histidine tag it owns that will bind to the metal ions immobilized in the resin matrix of purification columns. The histidine tag is added at the N-terminal of the protein because of its previous cloning in pET15-MHL.
Once we have the two purified enzymes: TPH and TDC, we have the need to obtain our raw material, which is the tryptophan. This must be extracted from by-products, such as broccoli or milk whey.
Then, the in vitro serotonin production will take place. This reaction requires both enzymes and the tryptophan as precursor. The aromatic amino acid will be hydroxylated to 5-hydroxytryptophan (5-HTP) by TPH. The 5-HTP acts as an intermediate as it can be transformed by TDC to 5-hydroxytryptamine, more commonly known as serotonin. Eventually, the serotonin is extracted by using chromatographic techniques, specifically, high performance liquid chromatography (HPLC).
At the end, this serotonin can be nanoencapsulated in non-polluting biological enclosures with the aid of local experts in nanotechnology and biotechnology. This way, our nanocapsules of serotonin could be used for helping in the recovery of mental disorders patients as a potential supplement, thus contributing to the improvement of global mental health and local economy in an eco-friendly way.
And this is the biological basis of our circular economy solution to fight depression!