Transformation Protocol

All the previously assembled BioBricks were transformed into DH5α competent cells acquired from New England Biolabs (NEB ®). In order to do so, NEB ®’s transformation protocol (3) was used: 50 μl of competent cells were added to microtubes; then, 5 μl of each previously assembled device (DNA concentration between 200 and 300 pg/ml, as determined by spectrophotometry) were pipetted into the tube, which was placed on ice for 30 minutes. Afterwards, the samples were submitted to 30 seconds of a 42°C heat shock; after which they were placed on ice for another 5 minutes. After incubation on ice, 950 μl of SOC medium were added to each mixture.
The tubes were placed at 37°C and 250 rpm for 60 minutes. Finally, 200 μl of each sample were plated into warm, solid LB media with 0.1% v/v of antibiotic (kanamycin 15 mg/ml for device 1, and chloramphenicol 35 mg/ml for devices 2, and 3).
After 12 hours of incubation at 37°C, a single colony was isolated from each plate and cultured overnight in liquid LB media with the previously stated concentration of antibiotic. A part of these liquid cultures was used for plasmid extraction and isolation; while the rest of them was used to perform relative fluorescence measurements. Both extraction and fluorescence measurement protocols are described later.

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Miniprep Protocol

Common miniprep plasmid-DNA extraction was performed. To do so, an isolated colony obtained from the transformation step was transferred from the Petri dish into an Erlenmeyer flask containing LB medium with the selection antibiotic (0.1% v/v). The flask was incubated overnight at 37°C and 250 rpm; 10 ml of the resulting culture were centrifuged at 13500 rpm for 30 seconds, so that biomass could be separated. The supernatant was discarded and cells were resuspended in 350 μl of STET buffer. The mixture was then transferred to a 1.5 ml microtube, where 5 μl of lysozyme (10 mg/ml) were added. The mixture was incubated during 3 minutes, after which the tube was transferred to a boiling water bath for 2 minutes in order to inactivate the enzyme.
Afterwards, the sample was centrifuged at 13500 rpm for 10 minutes. The bacterial pellet was taken out of the liquid using a sterile micropipette, and 10 μl of RNase A were added. The mixture was incubated for 10 minutes at room temperature. Then, 20 μl of sodium acetate (3M), and 250 μl of isopropanol were added. The mixture was gently stirred and incubated for 10 minutes at room temperature. Afterwards, it was centrifuged for 10 minutes at 12400 rpm; the supernatant was discarded, and the pellet washed 2 times with 1 ml of ethanol 70% v/v. Finally, the DNA was resuspended in 100 μl of distilled water and quantified by spectrophotometry.

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Assembly Protocol

Promoters from New device 1 and 2 (contained in a psB1C3 plasmid),were digested using the restriction enzymes SpeI and EcoRI. Reagents were added to a 0.5 ml PCR tube in the following order: 12.5 μl of water for molecular biology, 4 μl of NEB® Buffer 2.1, 0.5 μl of BSA, 20 μl of DNA (BioBrick BBa_K823005 in psB3K3 backbone), 1.5 μl of SpeI enzyme, and 1.5 μl of EcoRI enzyme. The content of the tube was gently mixed, and placed at a Thermoblock at 37°C for 75 minutes. After incubation, the tube was placed at a water bath at 80°C for 20 minutes so that the enzyme could be inactivated. Finally, the digestion product was stored at -20°C.

The GFP cassette, BBa_E0240 (contained in a psB1C3 plasmid) was also obtained by digestion, now using XbaI, and PstI. The same procedure was used, but now 20 μl of DNA (BioBrick BBa_K823005), 1.5 μl of XbaI enzyme, and 1.5 μl of PstI enzyme were added to the tube. The digestion product was also stored at -20°C.

This same protocol was followed to obtain the desired backbone (psB1C3) by digesting a RFP-containing psB1C3 plasmid, using the restriction enzymes EcoRI and PstI; the product was stored at -20°C.

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PCR for gene isolation.

Obtention of Biobricks by PCR

Every part was obtained from plasmid pcDNA 3.1 (-)/Myc-His A.

Primers Design

Table 1 annotations.
A) Extra bases to allow cuts (salmon). B) Extra base to give space (light blue). C) Extra base to avoid methylation sites (royal blue). D) Extra base to avoid methylation site or start codon (green). E) EcoRI Site (light blue background). F) NotI Site (green background). G) XbaI Site (red background). H) Pst1 Site (yellow background). I) SpeI Site (purple background). J) When it comes to coding sequences like NeoR, extra bases need to be added to ensure there is a correct space between ribosome and ATG (yellow font color with black background). K) TTA TTA (salmon font color with black background) Works as a double stop codon.

PCR non specific protocol

Table 1.This protocol is the New England Biolabs Taq2X Master Mix protocol.

The reaction must be gently mixed, if necessary a quick spin can collect all liquid ti the bottom.

Routine conditions for an average PCR are reported as follows:

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Gene isolation testing via digestion

All of the parts were digested with XhoI.

Incubate at 37°C for 1:15 hours.

Inactivate at 80°C for 20 minutes.

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Recombinant Protein Expression Protocol

Materials

Reagents

• 20 mM KH2PO4 and 10 mM KCl (pH 8) solution
• 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution
• Biomass recovered from the 40 mL medium
• Fresh Laemmli Buffer
• Lysozyme (20 mg/mL)

Equipment

• Centrifuge
• Micropipettes
• Conical tubes
• Centrifugal tubes

Procedure

1. Centrifuge 40 mL medium 5000 rpm, 30 min. Remove supernatant.
2. Resuspend pellet in 4 mL of a cold 300 mM KCl (pH 8), 20 mM Imidazole (pH 8) and 20 mM KH2PO4 solution.
3. Add 70uL of Lysozyme.
4. Incubate in shaker 200 rpm the biomass-lysozyme solution at 37° C for 60 minutes.
5. Centrifuge cell lysate at 14,000 rpm for 10 minutes. If the supernatant is not crystalline, the sample has to be centrifuged again. Store soluble phase at 4°C in a conical tube, covered with aluminum for chromatography.
6. Take 20 µL of the supernatant and mix with 20 µL of Laemmli buffer for SDS PAGE analysis.
7. The insoluble phase obtained from the previous centrifugation has to be washed twice with a 1% SDS solution and 20 mM KH2PO4 and 10 mM KCl (pH 8) solution. Then, mix insoluble phase with 20ul Laemmli buffer for SDS PAGE analysis.
8. Run a SDS PAGE with iGEM ITESM CEM’s protocol.

*Do not cease to contemplate that all samples should be perfectly mixed with the indicated reagents (Laemmli Buffer) before they are boiled for 5 minutes. In the case of the inclusion bodies, the boiling should be carried out for 10 minutes.

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