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Isolation of microorganisms from marbles

Initially, sterile scalpel is used, which is baptized in ethanol to ensure sterilization. Then the scalpel is burned for 100% sterilization. In addition, aluminum foil from marble is used for the purpose of sterilization, and aluminum foil and scalpel will be sterile.

Next, a small sample (20 μL) of biomass is taken from the marble, and this biomass is added to the eppendorf with a solution called "Riger solution". This solution helps to easily peel off the cells from the marble.

After sample preparation, mix (Vortex) the sample for 10-15 minutes. The sample is then centrifuged for one minute at 6000 revolutions, resulting in isolation of the microorganisms.

Finally, dilute the sample three successive times and place the diluted sample in nutrient plates (100 μL in each plate). These steps allow the cultivation and further study of microorganisms derived from marble. This methodology is an important tool in the research of environmental microbiology and the exploration of biological diversity in various environments.

Bacterial strains and growth conditions

E. coli were routinely grown in lysogeny broth (LB) medium with kanamycin (50 lg ml—1), chloramphenicol (35 lg ml—1) or gentamycin (10 lg ml—1) as needed at 37 °C and 30°C respectively. Solid media additionally contained 1.5% (w/v) agar. 1 mM of 3- methylbenzoate, 3 mM rhamnose and 10 mM arabinose were used as inducers of the Pm, PrhaB and ParaB promoters respectively. M9 minimal medium (6 g l—1 Na2HPO4, 3 g l l—1 KH2PO4, 1.4 g l—1 (NH4)2SO4, 0.5 g l—1 NaCl, 0.2 g l l—1 MgSO4, 2.5 ml l—1 trace elements solution) (Nikel and de Lorenzo, 2014) supple- mented either with 2% of glycerol (E. coli).

Transformation

E. coli DH5a chemical competent cells were prepared and transformed as described by Green and Rogers, 100 ng plasmid was electroporated into 100 µl cell suspension aliquots with a voltage of 2.5 kV, 25 lF capacitance and 200 Ω resistance.

DNA manipulation

General cloning procedures, such as endonuclease restriction digest, ligation and PCR, were performed with enzymes and buffers from New England Biolabs® (NEB; Ipswich, MA, USA) or ThermoScientifcTM (Waltham, MA, USA) according to the respective protocols. Q-5 hot start® polymerase was used for PCR if the resulting frag ment was further used, otherwise for colony PCR, Phire® was the polymerase of choice. PCR purification was performed with the Macherey-Nagel purification kit. All primers were synthesized by IDT.

Biphasic PCR protocol with standard SevaBrick Assembly primers

For the preparation of the parts to be assembled and due to the complexity and length of the engineered primers, the standard PCR protocol had to be modified to a biphasic one: 98 °C, 2 min; Phase 1: (98 °C, 20 s; 50 °C, 20 s; 72 °C, part dependent) 9 10; Phase 2: (98 °C, 20 s; 76 °C, 20 s; 72 °C, part dependent) 9 25; 72 °C, 2 min. The final product is obtained using an initial amplification at low annealing temperature (50 °C) for 10 cycles and a subsequent amplification at an annealing temperature of 76 °C (higher than the standard annealing temperature range) for another 25 cycles. Each pri- mer consists of a short 3’-end annealing sequence, a modular sequence in a non-annealing loop and optionally another standardized 5’-anchor sequence. The short 3’-end sequence requires a low annealing temperature (Phase 1) to be correctly hybridized with the template, while the anchor sequence, which optimizes the annealing fidelity elevates the Tm. High annealing temperature leads to negative results, while the low temperature cycling provides PCR amplified DNA but at a low yield. This low DNA concentration is due to the structural complexity of the long primers at this temperature. Running Phase 1 for 10 cycles provides enough PCR product which carries the additional sequences to be used as the template for Phase 2. At Phase 2, 76 °C is used for 25 additional cycles providing enough DNA to be used downstream in the process. 25 µl reactions followed by agarose gel purification are recommended for optimal results.

sfGFP fluorescence and growth measurements

All the genetic functional elements such as RBS, promoter and origin of replication parts were characterized in P. putida and E. coli. Single colonies were picked in triplicate and grown overnight at 37 °C (E. coli) in 10 ml LB with antibiotics. Cell density was measured with IMPLEN OD600 photometer at 600 nm and cells were diluted into 200 ll of M9 medium with antibiotics, and inducers where needed, in a 96-well Greiner plate to a starting OD600 of 0.1. The plate was incubated at 37 °C for 12 h in a Synergy plate reader (Biotek). OD600 and sfGFP measurements were recorded every 10 min. Fluorescence was determined with the following settings: Ex. 467 nm, Em. 508 nm and the levels were corrected with the fluorescence signal of a blank sample. Relative sfGFP production was quantified after 5 h for E. coli from an average of triplicate data.

Competent Cells

• Bacterial cells of interest (e.g., E. coli)
• Sterile microcentrifuge tubes
• Ice
• Calcium chloride (CaCl2) solution (e.g., 100 mM)
• Electroporation cuvettes (if using electroporation)
• Electroporation apparatus (if using electroporation)
• LB (Luria-Bertani) broth or another appropriate growth medium
• SIncubator at 37°C
• Centrifuge

Procedure

1. Inoculate a single colony of the bacteria that we want to make competent ce into a small volume of sterile LB broth or medium. Incubate the culture at 37°C with agitation until it reaches mid-log phase (OD600 of approximately 0.4 to 0.6). The time this takes will vary depending on the bacterial strain.

2. We transfered the culture to a larger container with more LB broth and allow it to grow until it reaches an OD600 of approximately 0.6 to 0.8.

3.We Placed the culture on ice or in a cold room for 10-15 minutes to chill the cells.

4. We transfered the culture to sterile centrifuge tubes and centrifuge the cells at a low speed (e.g., 3,000 x g) for 10-15 minutes at 4°C.

5. Carefully we removed the supernatant, leaving the cell pellet intact.

6. Resuspend the cell pellet in an appropriate volume of 50 mM calcium chloride (CaCl2) solution. The volume depends on the cell density and the bacterial strain. A common ratio is 100 µl of CaCl2 per 1 ml of the original culture volume.

7. Incubate the resuspended cells on ice for 30 minutes. This step helps to increase the cell membrane permeability.

8. Centrifuge the cells again at the same low speed for 10-15 minutes at 4°C.

9. Carefully we removed the supernatant and resuspend the cell pellet in a small volume of sterile water or a suitable buffer, such as 10% glycerol, which helps protect the cells during freezing.

10. Aliquot the competent cells into small volumes (e.g., 50-100 µl) and flash-freeze them in liquid nitrogen or a dry ice-ethanol bath. Store the aliquots at -80°C until needed.

Upcoming protocols

We ran out of time to finish our project because it would take months to perform the peptide isolation and purification techniques below. However, our team has carried out the experimental plan, and we expect to be given the chance to continue working on the project.

The process of isolating peptides from complex analytical materials, such as human or animal physiological fluids, by utilizing a maltose column normally involves the use of a column containing attached maltose molecules (also known as a maltose affinity column). Peptides attached to the molecular structures of maltose can be recovered selectively using maltose columns. The complicated structure of the glucose branch known as maltose makes it possible to isolate peptides with particular affinities. The following actions are typically taken during the peptide purification procedure utilizing a maltose column: (1) Binding: The maltose column is used to bind the peptide-containing material. Other contaminants flow through the column but peptides that have been pushed to bond to the maltose molecular structures are retained. (2) Wash: To eliminate undesired chemicals and contaminants, the column is washed with specialized washing solutions after binding. (3) Elution: Using particular elution solutions, such as reactive sugars or other solvents, bound peptides to the column are extracted from maltose. (4) Collection: The column's isolated peptides are collected and made available for use in studies, tests, and other procedures. A maltose column is a useful tool for separating peptides with particular affinities, and it is frequently used in biochemistry and biotechnology for the cleaning and purification of peptides.