**2. Strategies for cloning and expression**

### **2.1 Melittin primers**

The preparation of the melittin cDNA primers, both forward and reverse, was carried out first by searching for its sequence in Gen Bank (NCBI), with the accession NC\_007073.3. This sequence contains 100 bp and was published by Suchanek et al. [20]. The sequences of restriction sites for ApaLI and SacII were added to the selected primers. Thus, the final sequences of the primers were the following: primer forward 5′TTTTGGGCCCTTAACAGGAAGGA AGGAAGGAA3′ primer reverse 5′AAAACCGCGGAGATCGATAAATCG GCATCG3′.

#### **2.2 RNA extraction**

Fifty bees were collected in duly sterilized glass bottles and frozen at −30°C for 30 min in order to conserve the genetic material. The PureYield ™ RNA Midiprep System RNA extraction kit was used to extract and purify the total RNA. The quantification of total RNA was carried out by using the Quantus™ fluorometer [21]. The retrotranscription to total cDNA was carried out using the PureYield RNA Midiprep System (Promega), adding 5 μl of the total RNA extraction to the reaction mixture obtaining a final volume of 20 μl per tube.

#### **2.3 PCR amplification**

The PCR mixture was prepared according to the components and the amounts described briefly: a volume (μl) of nuclease-free water 13.25 μl; 5× GoTaq® flexi reaction buffer 5.00 μl; 25 mM MgCl2 2.00 μl; 10 mM PCR nucleotide mix 0.50 μl; 133.1 pM upstream primer 147.9 pM downstream primer 5 u/μl GoTaq® Flexi DNA polymerase 78 ng/μl cDNA obtained a final volume of 25.0 μl.

The mixture was placed in a thermocycler preheated to 94°C to start the denaturation with for 30 seg. Different temperatures were used for annealing (Tm) in order to determine which of them gave a greater number of copies at the end of the PCR (68.0, 68.2, 68.4, 68.6, 68.8, and 69.0°C; named respectively as Tm1, Tm2, Tm3, Tm4, Tm5, and Tm6) for 60 seg. Finally, the elongation temperature was 72°C for 90 seg, all of them for 40 cycles, and the complete PCR lasted 2 h.

The PCR product was run on 1.5% agarose gel electrophoresis, and the exact amount of cDNA obtained on the most visible band was established by the use of Quantus™ fluorometer (Promega).

#### **2.4 Sequencing**

The sample was sent to Macrogen-Korea in order to sequence this amplified fragment by sequencing of new generation. Once the sequence was obtained, it was compared with the melittin accession NC\_007073.3 by searching for DNA homologies using the BLAST v1.4 program in GenBank (http://www.ncbi.nlm.nih.gov/ BLAST/).

#### **2.5 Insertion of the melittin cDNA in the pGEM-T easy vector**

The PCR product was purified using the PCR CleanUp System™ to eliminate primer dimers or other unwanted reaction products in order to improve the ligation efficiency. In order to insert the gene in the vector, 1 μl of the PCR product was taken and mixed with 5 μl of 2× rapid ligation buffer (T4 DNA), 1 μl of pGEM®-T Easy vector (50 ng/μl), 1 μl of T4 DNA ligase (3 μg/μl), and 2 μl of nuclease-free water. These vectors were prepared by cutting with EcoRV and adding a 3'terminal thymidine to both ends. They contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha-peptide coding region of the enzyme beta-galactosidase. Insertional inactivation of the alpha-peptide allows recombinant clones to be directly identified by blue/white screening on indicator plates. The reagents were incubated for 1 h at room temperature. In order to obtain a maximum number of transformants, the reactions were then incubated overnight at 4°C.

#### **2.6 Bacterial transformation**

The commercial strain of *E. coli* JM109 was used, maintained at −30°C. Once thawed, 50 μl of this tube was transferred to 1.5 ml microcentrifuge tube, inserted in the ice, and 2 μl of the ligation product was added. The transformed cells were subjected to ice for 2 min, and 950 μl of Super Optimal Broth with Catabolite Repression (SOC) liquid medium [22] at room temperature was added. This solution was incubated for 1.5 h at 37°C with shaking at 150 rpm. Subsequently, aliquots of 100 μl were placed in different plates with Luria-Bertani (LB) semisolid broth medium [23] with 100 μg/ml of ampicillin, 0.5 mM of IPTG, and 80 μg/ml of X-Gal. The plates were incubated overnight at 37°C to perform the Blue-White Screening for positive bacterial transformed colonies/clones.

#### **3. Results**

#### **3.1 RT-PCR**

It was performed at different annealing temperatures (68.0, 68.2, 68.4, 68.6, 68.8, and 69.0°C). After electrophoresis, it was observed that all the cDNA samples hybridized with the primers obtaining the most visible band at the annealing temperature of 68.4°C. This is, therefore, the hybridization temperature that has resulted in a greater amount of cDNA during PCR. After quantification with the fluorophore, the quantity of cDNA obtained resulted in 78 ng/μl. The PCR product was sequenced prior to cloning by MACROGEN-South Korea.

#### **3.2 BLAST-DNA homology**

Searching of the NCBI GenBank database (http://www.ncbi.nlm.nih.gov/) using the melittin accession (Accession no. NC\_007073.3) resulted in a similarity index around 80%. The genetic transformation of *E. coli* JM109 with the insert in the vector pGEM-T was corroborated by the blue-white screening test. The colonies formed by nonrecombinant cells therefore appeared blue in color while the recombinant ones appeared white and allowed discrimination between transformants containing recombinant plasmids versus those maintaining self-ligated or uncut vector.

The homology is deduced from the excess of similarity recognized from statistical estimates. A common empirical rule is that two sequences are homologous if

they are more than 30% identical over their entire length (much higher identities are seen in short alignments) [24], so it can be firmly stated that both sequences are similar. Due to this, it can be affirmed that *E. coli* has been genetically transformed with the cDNA of western bee melittin. Also, the best annealing temperature has been 68.4°C.
