1. Keep the vial sealed and on ice until ready to use.
2. Centrifuge at 6,000x g for 1 minute at 4°C.
3. Resuspend pQE-80L-Kan.in 10 uL of IDTE.
4. Resuspend pUC57-Kan containing collagen-like region from Scl2(CO) in 20 uL of nuclease-free water.
5. Keep both tubes on ice and proceed with transformation of competent DH5 alpha cells.

1. Obtain agar plates with KAN antibiotic and label them appropriately.
2. Allow competent E. coli DH5-alpha or BL21 to thaw on ice. Aliquot 25 uL of DH5-alpha into an Eppendorf tube (Initial volume in each tube contains 50). For BL21, use the entire tube.
3. Add 1-5 uL of plasmid into the first tube containing the competent E. coli.
4. Place mixture on ice for 20 minutes.
5. For DH5-alpha, heat-shock the bacteria for 60 seconds using a heat block at 42°C. For BL21, heat-shock the bacteria for exactly 10 seconds using a heat block at 42°C.
6. Immediately place tubes to ice for 2 minutes for DH5-alpha and 5 minutes for BL21.
7. Add 970 uL of SOC.
8. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
9. Centrifuge for 2 minutes at 4000 rpm and discard 900 uL of supernatant to concentrate the bacteria.
10. Carefully resuspended the pellet by pipetting up and down and transferring 50uL of transformed bacteria to the respective agar plate containing the appropriate antibiotic.
11. Spread gently using plastic spreader.
12. Incubated overnight (16 hours) at 37 °C.

1. Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with vigorous shaking.
   • Growth for more than 16 h is not recommended because cells begin to lyse and plasmid DNA yields may be reduced. Use a tube or flask with a volume of at least 4 times the volume of the culture.
2. Harvest the bacterial cells by centrifugation at >8000 rpm (6800 x g) in a conventional table-top microcentrifuge for 3 min at room temperature.
   • The bacterial cells can also be harvested in 15 ml centrifuge tubes at 5400 x g for 10 min at 4°C. Remove all traces of supernatant by inverting the open centrifuge tube until all medium has been drained.

This protocol is designed for the purification of up to 20 μg of high-copy plasmid DNA from 1–5 ml overnight cultures of Escherichia coli in LB medium. Note: All protocols steps at room temperature.

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
   • Ensure that RNase A has been added to Buffer P1. No cell clumps should be visible after resuspension of the pellet.
   • If LyseBlue reagent has been added to Buffer P1, before use vigorously shake the buffer bottle to ensure LyseBlue particles are completely dissolved. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
2. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times
   • Mix gently by inverting the tube. Do not vortex, because this will result in shearing of genomic DNA and contamination of plasmid. If continue inverting the tube until the solution becomes viscous and slightly clear. Do not allow the lysis reaction to proceed for more than 5 min.
   • If LyseBlue has been added to Buffer P1, the cell suspension will turn blue after the addition of Buffer P2. Mixing should result in a homogeneously colored suspension. If the suspension contains localized colorless regions, or if brownish cell clumps are still visible, continue mixing the solution until a homogeneously colored suspension is achieved.
3. Add 350 μl Buffer N3. Mix immediately and thoroughly by inverting the tube 4–6 times.
   • To avoid localized precipitation, mix the solution thoroughly, immediately after addition of Buffer N3. Large culture volumes (e.g., ≥5 ml) may require inverting up to 10 times. The solution should become cloudy.
   • If LyseBlue reagent has been used, the suspension should be mixed until all trace of blue is gone and the suspension is colorless. A homogeneous colorless suspension indicates that the SDS has been effectively precipitated.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge. A compact white pellet will form.
5. Apply 800 μl of the supernatant from step 4 to the QIAprep 2.0 Spin Column by pipetting.
6. Centrifuge for 30–60 s. Discard the flow through.
7. Recommended: Wash the QIAprep 2.0 Spin Column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow through.
   • This step is necessary to remove trace nuclease activity when using endA+ strains, such as the JM series, HB101 and its derivatives, or any wild-type strain, which have high levels of nuclease activity or high carbohydrate content. Host strains, such as XL-1 Blue and DH5α, do not require this additional wash step.
8. Wash QIAprep 2.0 Spin Column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow through, and centrifuge at full speed for an additional 1 min to remove residual wash buffer.
   • Important: Residual wash buffer will not be completely removed unless the flow-through is discarded before this additional centrifugation. Residual ethanol from Buffer PE may inhibit subsequent enzymatic reactions.
10. Place the QIAprep 2.0 Spin Column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) or water to the center of each QIAprep 2.0 Spin Column, let stand for 1 min, and centrifuge for 1 min.
11. Measure DNA concentration using NanoDrop.

1. Set up the reaction as follows:

   Component	50 µl REACTION
   DNA	1 µg
   10X rCutSmart Buffer	5 µl (1X)
   BamHI-HF	1.0 µl (20 units)
   HindIII-HF	1.0 µl (20 units)
   Nuclease-free Water	to 50 µl

2. Incubate at 37°C for 5-15 minutes as both enzymes are Time-Saver qualified.
3. Heat inactivate at 80°C for 20 minutes.

1. Set up the following reaction on ice.

   	2–3 Fragment Assembly*	4–6 Fragment Assembly**	NEBuilder Positive Control✝
   Recommended DNA Molar Ratio	vector:insert = 1:2	vector:insert = 1:1
   Total Amount of Fragments	0.03–0.2 pmols* X μl	0.2–0.5 pmols** X μl	10 μl
   NEBuilder HiFi DNA Assembly Master Mix	10 μl	10 μl	10 μl
   Deionized H2O	10-X μl	10-X μl	0
   Deionized H2O	20 μl✝✝	20 μl✝✝	20 μl

   *Optimized cloning efficiency is 50–100 ng of vector with 2-fold excess of each insert. Use 5-fold molar excess of any insert(s) less than 200 bp. Total volume of unpurified PCR fragments in the assembly reaction should not exceed 20%. To achieve optimal assembly efficiency, design 15-20 bp overlap regions between each fragment.

   **To achieve optimal assembly efficiency, design 20-30 bp overlap regions between each fragment with equimolarity of all fragments (suggested: 0.05 pmol each).

   ✝Control reagents are provided for 5 experiments.

   ✝✝If greater numbers of fragments are assembled, increase the volume of the reaction, and use additional NEBuilder HiFi DNA Assembly Master Mix.

2. Incubate samples in a thermocycler at 50°C for 15 minutes (when 2 or 3 fragments are being assembled) or 60 minutes (when 4–6 fragments are being assembled). Following incubation, store samples on ice or at –20°C for subsequent transformation.
3. Transform NEB 5-alpha or 10-beta Competent E. coli cells with 2 μl of the chilled assembled product, following the transformation protocol.

1. Using a sterile toothpick or inoculating loop, pick a single colony from a selective antibiotic LB agar plate and inoculate 10 ml of LB medium containing 50 μg/ml kanamycin in a 50 ml flask. Grow the cultures at 37°C overnight.
2. Inoculate 150 ml of prewarmed media (with ampicillin) with 4 ml of the overnight cultures and grow at 37°C with vigorous shaking until an OD600 of 0.6 is reached (30–60 min).
3. Take a 0.5 ml sample immediately before induction for noninduced control; pellet cells and resuspend them in 25 μl 5x SDS-PAGE sample buffer. Freeze and store the sample at –20°C until SDS-PAGE analysis.
4. Induce expression by adding IPTG to a final concentration of 1 mM.
5. Incubate the cultures for an additional 6 hour and overnight (16 hour). Collect a second 0.5 ml sample as the induced control; pellet cells and resuspend them in 50 μl 5x SDS-PAGE sample buffer. Freeze and store the sample at –20°C until SDS-PAGE analysis.
6. Harvest the cells by centrifugation at 4000 x g for 20 min.
7. Freeze and store cell pellet overnight at –20°C.

1. Resuspend the cell pellet in 10 ml Denaturing Lysis Buffer.
2. Incubate at room temperature (15–25°C) for 60 min. Mix 2–3 times by gently swirling the cell suspension. Lysis is complete when the suspension is translucent.
3. Centrifuge lysate at 14,000 x g for 30 min at room temperature (15–25°C) to pellet the cellular debris.
4. Retain the cell lysate supernatant. The supernatant contains the recombinant protein.
5. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of the supernatant and store at –20°C for SDS-PAGE analysis.
6. Gently resuspend the resin in a Fast Start Column by inverting it several times.
7. Break the seal at the outlet of the column, open the screw cap, and allow the storage buffer to drain out. It is important that the outlet seal is broken before the screw cap is removed.
8. Apply the cell lysate supernatant from step 3 to the column.
9. Collect the flow-through fraction. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of the flow-through fraction and store at –20°C for SDS-PAGE analysis.
10. Wash the column 2 times with 4 ml of Denaturing Wash Buffer.
11. Collect both wash fractions. Add 5 μl 2x SDS-PAGE sample buffer to a 5 μl aliquot of each wash fraction and store at –20°C for SDS-PAGE analysis.
12. Elute bound 6xHis-tagged protein with two 1 ml aliquots of Denaturing Elution Buffer.
13. Collect each elution fraction in a separate tube. Add 5 μl 2x SDSPAGE sample buffer to a 5 μl aliquot of each elution fraction and store at –20°C for SDS-PAGE analysis.
14. Analyze all fractions by SDS-PAGE.

1. Start overnight seed culture at 37 °C:
2. 50ml LB containing antibiotics (pQE80L-KAN-Scl2.28) KAN and CHL.
3. Samples of seed culture in 50ml LB containing:
   • Kanamycin-KAN (50 ug/mL)
   • Chloramphenicol-CHL (25 ug/mL)

1. Clean the bench and pipettes with alcohol, turn on the fire.
2. Creating 4L of each sample : Grab 1 L of L-Broth in 4 flasks each.
3. Add 500 uL of KAN and CHL each to every flask (1:500 antibiotics:LB ratio). Kan (50 mg/L) and CHL(25 mg/L).
4. Add 10 ml of pre-inoculated bacterial culture to each flask
5. Transfer everything to the shaking incubator (Temperature:37℃ , RPM: 200) for 4 hours. Check if they are stable and properly stuck to the pads.
6. Checking optical density using spectrophotometer:
   • Grab 2 cuvettes (one for blank and one for the sample)
   • Clean the bench and pipettes with alcohol, turn on the fire.
   • Add (less than 5 ml- filling 3/4th cuvette ) blank: plain L-Broth used for inoculation and sample : from the flasks in shaking incubator using the pipette with glass tubes.
   • Switch on and run the Spectrophotometer software
   • Add the blank, on measuring the OD, press on auto-zero
   • Add the sample and measure the OD
   • If OD>=0.3, start inducing. Else continue incubating in the shaker.

Isopropyl-ß-D-thiogalactopyranoside (IPTG) is a chemical analogue of galactose, which cannot be hydrolyzed by the enzyme ß--Galactosidase. Hence, it induces the E. coli lac operon activity by binding and inhibiting the lac repressor without being degraded.

1. Prepare an ice bath to store the IPTG
2. Grab IPTG from the -30 freezer
3. Clean the bench and pipettes with alcohol, turn on the fire.
4. Add 500uL of IPTG to the culture using autoclaved tips in aseptic conditions.
5. Transfer everything to the shaking incubator (Temperature: 37℃ , RPM : 120) for 18 hours.

1. From the growth curve experiment, choose the optimum concentration of proline and lysine for residue-specific incorporation of Hyp and Hyl.
2. Inoculate single colony of proline/lysine auxotroph containing pQE-CLTP into Luria Bertani medium containing 100 μg ml−1 of kanamycin and incubate in a shaker at 37 °C overnight.
3. Inoculate 100 ml of adaptation medium (minimal medium with 40 μg ml−1 of proline) from the overnight grown culture, having 0.2 OD at 0th hour and incubated overnight at 37 °C. 1% of inoculum.
4. Prepare 1-liter minimal medium containing 0.04 mM of proline.
5. Inoculate overnight adaptation culture into the newly prepared 1 liter minimal medium, having 0.2 OD after inoculation.
6. After the culture reaches the stationary phase (OD: 0.9 - 1) 10–80 mM of trans-4 hydroxyproline (Hyp) was added along with 1 mM of IPTG.
7. Then 500 mM of NaCl was added to each medium and incubated for 7 h at 37 °C. The trans-4 hydroxyproline incorporation efficiency was analyzed in whole cell lysate using SDS-PAGE.
   • NaCl assists in intracellular accumulation and the incorporation of Hyp by assisting in transport of Hyp. It is unique to Hyp and may or may not be necessary for Hyl.

1. Cool the bacterial culture on ice before the centrifugation at 8000rpm for 10min at 4°C. Use the 1L centrifuge bottles. Make sure to balance your tubes to prevent centrifuge from damage and ensure safety.
2. Dissolve the cell pellet (1L overnight cell culture) in about 40 ml lysis buffer (0.5g of bacterial pellet/ml of lysis buffer).
   • Meanwhile, equilibrate the nickel column (3 ml resin/bed volume) in the cold room by 30ml lysis buffer.
3. Use homogenizer to dissolve the bacterial pellet in the lysis buffer. You can vortex to further dissolve the bacterial pellet.
4. Sonication in glass beaker. Sonication on for 5 sec, stop for 5 sec. Total sonication time is 5 min on time. 30% power with large tip
5. Centrifuge the cell extract using at 18000 rpm for 45 min at 4 degrees.
6. Load the supernatant to 3 mL column bed volume of Ni-NTA by pumping 3 rpm on the peristaltic pump.
7. Wash the column by (1) 50 ml lysis buffer then (2) 30ml wash Buffer A, by pumping 5 rpm on the peristaltic pump. Collect both washes each in a single 50mL falcon tube.
8. Elute the His-tag protein by elution Buffer B, 1mL fractions each time, 10 fractions in total.
9. Check for protein in the elution fractions using Bradford, 100ul Bradford/3ml protein.
   Keep 10uL of each fraction to be used for SDS-PAGE analysis that will be done later. Pool fractions that have high blue color intensities. If your last fractions still have high blue color, you can collect more elution fractions to make sure all your protein target is out of the column.
10. Run the pooled fractions of CLTP from Ni-NTA column on 16/60 S200 Gel Filtration (GF) column overnight at 1 mL/min. The GF column needs to be priorly equilibrated or a column wash step should be added prior to running the sample.
11. Next day, determine the protein concentration with Bradford assay then run SDS-PAGE analysis to evaluate the sample purity.

GF Buffer

+

1. 50mM \(PO_4\) buffer pH 7.5 (CD analysis) or 20mM Hepes pH 7.5
2. 150 mM NaCl
3. 0.5 mM TCEP

SDS-PAGE: Continuous 12% expected band (∼35 kDa)

1. For handcasting of 12% discontinuous polyacrylamide gels, add the following components to two 50 mL Falcon tubes.

   Resolving gel:

   1. 5.5ml of ddH2O
   2. 5ml of 1.5 M Tris with pH=8.8
   3. 0.2ml of 10% SDS
   4. 8.7ml of 30% Acryl/Bis

   Directly before casting the gel, add the following:

   1. 100μl of 10% APS
   2. 10μl TEMED
   
   

   Stacking gel:

   1. 6.1ml of ddH2O
   2. 2.5ml of 0.5 M Tris-HCl with pH=6.8
   3. 0.1ml of 10% SDS
   4. 8.7ml of 30% Acryl/Bis

   Directly before casting the gel, add the following:

   1. 50μl of APS
   2. 10μl of TEMED
2. As soon as the APS and TEMED, proceed as quickly as possible to the step of casting the gel. Be very careful for any delay will result in gel polymerization in the tubes and you will have to repeat the preparations.
3. Use 1ml pipettes to transfer the gel into the setting and 4 to 5 times should be enough for the resolving gel and 2 times for the stacking. Once you add the resolving gel, add a layer of water or isopropanol. Once, resolving gel is set, add the stacking gel and the comb.
4. Keep the excess in the falcon tubes. Do not discard. This will allow you to monitor the polymerization of the gel.
5. Add 5ul of sample to 5ul of 2X Laemmli sample buffer.
6. Load the sample onto the wells and run the SDS-PAGE for 40 minutes at 200 V.
7. Wash gel 3 times in distilled water for 5 minutes each.
8. Add EZBlue Gel staining reagent and agitate for one hour.
9. Rinse with water for 1 hour to overnight.

Source

1. Prepare a small, overnight culture of the bacteria in LB broth. Grow at 37°C.
2. About 2 h before you are ready to begin the main procedure, use 1.0 mL of the overnight culture to inoculate 100 mL of fresh LB broth. This culture is grown with rapid shaking at 37°C until it reaches roughly 5 x 107 cells/ml. Corresponding to OD between 0.3 and 0.5. Do not allow the cells to reach stationary phase.
3. Take a 5 mL aliquot of each transformation reaction and transfer to sterile plastic centrifuge tubes. Cool on ice for 10 min.
4. Pellet the cells by spinning for 5 min at 5000g. It is necessary for the centrifugation to be performed at 4°C.
5. Pour off the supernatant and resuspend cells in 25 mL of cold 0.1M CaCl2. Leave on ice for at least 20 min.
6. Centrifuge as in Step 3. You should observe a more diffuse pellet than previously. This is an indication of competent cells.
7. Resuspend the cells in 0.2 mL of cold 0.1M CaCl2. And aliquot 50 uL into 1 mL Eppendorf tubes.
8. To each tube add up to 0.1 mg of DNA, made up in a standard DNA storage buffer such as TE to a volume of 100 mL. Leave on ice for 30 min.
9. Heat shock at 42°C using a water bath or heat block.
10. Return to ice fo 3 minutes.
11. Add 970 uL of SOC media.
12. Incubate the tubes on a shaker incubator set to 220 rpm and 37°C for one hour.
13. Centrifuge for 2 minutes at 4000 rpm and discard 900 uL of supernatant to concentrate the bacteria.
14. Carefully resuspended the pellet by pipetting up and down and transferring 50uL of transformed bacteria to the respective agar plate containing the appropriate antibiotic.
15. Spread gently using plastic spreader.
16. Incubated overnight (16 hours) at 37 °C.