Notebook

In the beginning, we planned three approaches to investigate activation of cryptic genes and how to increase the production of the metabolites under its control.

First, Streptomyces genus is known by its ability to produce numerous secondary metabolites of biotechnological interest, specially for the pharmaceutical industry. The synthesis of these molecules depends on a gene cluster controlled by a specific transcriptional regulator named SARP (Streptomyces Antibiotic Regulatory Protein), which is activated under determined conditions, such as incubation in presence of stressors agents. Unfortunally, despite this genus of bacteria have many gene cluster encoding for putative molecules, most of them can’t be activated, once we don’t know the condition inducing for SARP production. Nevertheless, these proteins are positive transcriptional regulators, which makes it possible to genetically engeener Streptomyces to produce the SARP of interest and, consequently, transcribe the proteins that synthethise the molecule of interest. For this part of the project, we planned to clone the SARP that controls the synthesis of cosmomycin, a antimicrobial and antitumor molecule produced by Streptomyces olindensis, a brazilian isolate, and investigate if it starts to be synthethised in commun culture medium. Our construction would be created by inserting SARP gene in pSET152Ec constitutive vector, digesting with NotI and EcoRI restriction enzymes and ligated with T4 ligase. We designed the primers with the following nucleotide sequences:

Oligos were synthethized by IDT (Integrated DNA Technology). Curiously, our primers were delivered twice to somewhere in Florida before sending them to us. Once we received it, PCR was prepared using the high fidelity Phusion polymerase from Thermo Scientific with the following protocol:

Streptomyces colonies were boiled at 95°C for 10 min and used as DNA template for the first two reactions, which yielded significant low amounts of amplicon, impossible to use for digestion and ligation reactions

For the next reaction, instead of the whole colony, we used genomic DNA extracted from Streptomyces using the Wizard genomic DNA purification kit from Promega. We have a lot of trouble with this protocol, since the mycelium formed during the bacterial growth makes the lysis and DNA purification difficult. After three trials, we got enough DNA for PCR, the reaction was prepared with the same protocol as described above, but with a gradient of temperatures ranging from 57 to 68°C (57, 59, 62, 65, 68°C). As showed bellow, the best reaction occured at 68°C where the unspecific band above the desired one wasn’t present. Inserts were purifed from agarose gel using Thermo Scientific GeneGET Gel extraction kit and stored at -20°C until the digestion reaction.

For the next step, we attempted to obtain a higher amounts of pSET152Ec transforming chemocompetent DH5α bacteria by heat shock, incubating overnight at 37°C in 2xTY medium supplemented with 50 μg/mL apramycin, using vector and antibiotic that was given to us. For our surprise, there was a background population of bacteria growing in the agar, which made us believe the antibiotic wasn't effectively selecting the transformants. We tried again to transform it now using 100 μg/mL apramycin, but again the same phenotype was seen. Preparing a serial dilution of the antibiotic in microplates, we determined that the ideal concentration would be 200 μg/mL, that in fact selected the positive transformants without the background growth. Two colonies were picked and inoculated in 2xTY medium with 200 μg/mL apramycin and incubated overnight at 37°C under agitation. In the next morning, the plasmid extraction was done and, unexpectedly, it formed a intense 2.000 bp band in the agarose gel, that migrates to 4.000 bp after digestion with NotI enzyme.

Our vector should have about 5.900 bp, sugesting it was not the expected plasmid. Bacteria was transformed and DNA extracted again, revealing the same result. A new aliquot was given to us, the protocol was repeated again and now the plasmid extraction yielded a construct with 4.000 bp that migrates in the gel forming a band between 7.000 and 8.000 bp after digestion with NotI enzyme. The size diverges a bit with the expected one, but we thought in the possibility that something was already cloned in this plasmid, so we could substitute the insert with our SARP amplicon when digesting with the two described enzymes. Plasmid and insert digestions was prepared with NotI and EcoRI enzymes, and ligation with T4 ligase. DH5α was transformed with 10 ng of DNA from ligation, selected by apramycin, and using a control reaction containing only the plasmid digested and ligated, without insert. For our surprise, the control and the reaction had the same number of colonies, indicating the reaction didn't was not successful. We prepared a new digestion, ligation and transformation, but the same result was obtained. To check if the plasmid was being digested as expected, 3 digestion reactions were prepared using both enzymes (NotI and EcoRI) or each enzyme separately. We found that the plasmid was linearized by NotI enzyme, bot not dor EcoRI. Plasmid was purified again but the digestion reveald the same pattern, making it impossible to conclude the cloning.

For the second part, we wanted to explore the possibility of increasing cosmomycin production through the elevated expression of resistance genes. CosI and cosJ genes encoding for the autotransporter responsible for cosmomycin exportion would be cloned in pSET152Ec using the same strategy descibred for SARP. Also, a construction with cosI, cosJ, cosP and cosU would be prepared, the 4 genes related with cosmomycin resistance. Since we couldn't correctly digest the vector, we were not able to clone it also.

The third part was focused on improving the malonate uptake, increasing the intracellular substrate for cosmomycin production and consequently increasing its production. We designed pIJ6021 plasmid transforming it in a conjugative plasmid by adding the sequences recongnized for mobility. Then, once we didn't found a di-carboxylate carrier protein in the genome of Streptomyces olindensis (only a tri-carboxylate carrier protein related to citrate uptake), we inserted the gene of a di/tri-carboxylate carrier protein from Streptomyces hygroscopicus and a malonyl-CoA enzyme from Rhizobium leguminosarum. For this part, instead of cloning, we planned to synthesize the whole plasmid, but we were informed that the wouldn’t be possible to create the whole plasmid. For this reason, we devided the plasmid in two minor fragments that would be ligated using SLIC. unfortunately, the companies couldn’t synthesize this plasmid due to the high G-C content. We asked if it would be possible at least to produce a synthetic sequence of only the two proteins, but the G-C content was too high even for this.

Our construct can be found on: Primer and Plasmid Constructions