Team:IISER Berhampur/Experiment

iGEM IISER_Berhampur
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GLP(Good Lab Practices)

After gaining lab access the first thing we did was a training session on Good Lab Practices (GLP). Here we learned to ensure and promote safety, consistency, high quality, and reliability in the process of laboratory testing. In this session, we learned the Importance of maintaining a logbook and also how each instrument functions, and how to use it safely. We also learned the importance of maintaining the notebook in the laboratory which will help us in troubleshooting if we made any mistake in some step of a protocol. Notebook snapshots have been uploaded with experiments performed.




Biosafety

After a GLP session, we had a biosafety training where we learned the roles and responsibilities of persons conducting research with recombinant DNA and other biohazardous agents and also learned about the practices, safety equipment, and facility design required to ensure safety in laboratories. We also applied and received approval from IBSC which is a committee overlooking biosafety aspects in IISERs. Authorization to use laboratory instruments was also granted to us.





Experiments We Performed

Media and buffers:-

  1. 250mM KCl solution: 100ml - 1.86g of KCl was dissolved in double-distilled water and volume was made up to 100ml.
  2. SOB medium: 500ml - The recipe contained 10g of tryptone, 0.25g of NaCl, 2.5g of yeast extract, and 5ml of 250mM KCl solution. The total volume of solution was made up to 500ml with double, distilled water. The media was stored at 4 degrees until use.
  3. 5M KOH solution: 50ml - 14g of KOH pellets were dissolved in double distilled water and volume was made up to 50ml.
  4. PIPES buffer: (0.5M pH 6.7) 100ml - 15.1g of PIPES was dissolved in double-distilled water. The pH was adjusted to 6.7 using a 5M KOH solution. The final volume was made up to 100ml with double distilled water.
  5. Inoue buffer: 1000ml - The recipe contained 55mM(10.88g) of MnCl2.4H2O, 15mM(2.2g) of CaCl2.2H2O, 250mM(18.65g) of KCl and 10mM(20ml) of PIPES buffer. The total volume of solution was made up to 1000ml with double distilled water. The buffer was filtered through a 0.45 micron Nalgene filter. It was stored at 4 degrees until use. All other buffers and solutions were stored at room temperature.

Procedure

  • Plate colony growth - 100ul of competent cells were taken from existing vials and plated on an LB Agar Petri plate. The plate was left in an incubator at 37 degrees, overnight.
  • Primary inoculum - A grown colony was taken from the Petri plate with a microtip and put into a 20ml LB medium culture. The tube was left in an incubator at 37℃ for 6-8 hours.
  • Secondary culture - 10ml of primary inoculum was added to 250ml of the SOB medium in a culture flask. 1.25ml of 2M MgCl2 solution was added to the culture flask. The flask was incubated at 18 degrees, 184 rcf rotation, overnight.
  • The OD was measured at 600 nm after roughly 12-14 hours and was found to be 0.595.
  • The culture was centrifuged at 5514 rcf, 4 degrees for 10 minutes to pellet down the cells. The supernatant was discarded.
  • The pellet was resuspended in an 80ml ice-cold Inoue buffer.
  • Again, the resuspended mix was centrifuged at 5514 rcf, 4 degrees for 10 minutes.
  • The supernatant was discarded and the pellet was resuspended in a 20ml ice-cold Inoue buffer.
  • 1.5ml DMSO solution was added into the resuspension and incubated in ice for 10 minutes.
  • The solution mixture was distributed into microcentrifuge tubes in 500ul aliquots and the tubes were snap-frozen in liquid nitrogen.

Table 3.1: List of competent cells in use

Competent cells Purpose of use
NEB Stable Used for stable cloning methods.
BL21 DE3 Used for stable protein expression since they lack certain cellular proteases like Lon and Omp T. They also work well with inducible promoter constructs.

NOTES : Click on the buttons to scroll through the notes


buffers preparation:-

Ethanol was added to Monarch Plasmid Wash Buffer 2 prior to use (4 volumes of ≥ 95% ethanol per volume of Monarch Plasmid Wash Buffer 2).
We added 24 ml of ethanol to 6 ml of Monarch Plasmid Wash Buffer 2



Protocol (Protocol is taken from Monarch® Plasmid Miniprep Kit):

All centrifugation steps were carried out at 16,000 x g (~13,000 RPM).

If precipitate has formed in Lysis Buffer (B2), incubate at 30–37°C, inverting periodically to dissolve.

Store Plasmid Neutralization Buffer (B3) at 4°C after opening, as it contains RNase A.

  1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatants.
  2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
  3. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert the tube immediately and gently 5–6 times until the color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.

    Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert the tube until the color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
  4. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
  5. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
  6. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein, and endotoxin. (Add a 5-minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
  7. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.

    Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.

    Add ≥ 30 μl DNA Elution Buffer to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.

Use nanodrop for DNA quantification tools to find out the concentration of nucleic acid present.

NOTES : Click on the buttons to scroll through the notes


Protocol (Protocol is taken from Agarose Gel Electrophoresis):

https://dx.doi.org/10.17504/protocols.io.78rhrv6

This protocol is for making 0.8% agarose gel and the run of an agarose gel electrophoresis on a DNA sample.

  1. Melt 0.8% times 0.4gm of agarose in 50 ml of 1X TAE buffer in a microwave oven until the liquid is fully transparent.
  2. Add 1 μl (10mg/ml) of Ethidium bromide (EtBr) in the melted agarose.
  3. Pour the melted agarose in the gel cast with the comb set. make sure the "comb" is well placed and the solution is balanced.
  4. Wait 30 minutes until the gel solidifies (faster in the 4°C fridges).
  5. Remove the combs carefully and cover the gel with 1X TAE buffer
  6. Load the samples in the wells:
    • 5 μl of 1 kb DNA ladder
    • Mix 5 μl of DNA with 1 μl of 6X times loading buffer
  7. Run the gel at 100 volts for 40 minutes. Remove the gel from the chamber.
  8. Visualize the DNA fragments using a UV transilluminator.

NOTES : Click on the buttons to scroll through the notes

Locate the DNA on the kit plate.

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross-contamination between the wells.
  2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye.


NOTES : Click on the buttons to scroll through the notes

Protocol

  1. Thaw the competent cells on ice.
  2. Add 5 µl of Plasmid DNA to 500 µl of the competent cells. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.
  3. Place the mixture on ice for 30 minutes. Do not mix.
  4. Heat shock at 42°C for 30 seconds*. Do not mix.
  5. Add 500 µl of room temperature media* to the tube.
  6. Keep the tube in the incubator at 37°C and 250rpm for 60 minutes.
  7. Keep selection plates ready with specific antibiotics.
  8. Spread 50–100 µl of the cells onto the plates.
  9. Incubate overnight at 37°C.

Please note: For the duration and temperature of the heat shock step as well as for the media to be used during the recovery period, should be followed which are provided by the competent cells’ manufacturer.

NOTES : Click on the buttons to scroll through the notes

Protocol

  1. Take one clear plate for absorbance and one opaque plate for fluorescence.
  2. Put 50μl of PBS in the first row from well 1 to 12.
  3. Add 50μl of venus protein to the well 12.
  4. Mix properly (50μl of PBS+50μl of venus) and then take 50μl from well 12 and add to well 11.
  5. Repeat the same process till well 2 and discard the 50μl taken from well 2.
  6. Do not add venus in the 1st well. Keep it as control.
  7. Add 50μl of PBS in each well (1 to 12) to make a total volume of 100μl.
  8. Check the absorbance and fluorescence using the Plate reader.

NOTES : Click on the buttons to scroll through the notes

Click here for results of the experiments



Future Experiments Planned

WarmStart Colorimetric LAMP 2X Master Mix Typical LAMP Protocol (M1800).

Reaction Setup: For simplicity in setting up reactions, we recommend making stocks of the LAMP primers at a usable concentration. For example, we suggest a 10X Primer Mix containing all 6 LAMP primers.

A 10X LAMP Primer Mix contains:

PRIMER 10X CONCENTRATION 1X CONCENTRATION
STOCK FINAL
FIP 16 μM 1.6 μM
BIP 16 μM 1.6 μM
F3 2 μM 0.2 μM
B3 2 μM 0.2 μM
LOOP F 4 μM 0.4 μM
LOOP B 4 μM 0.4 μM

Note: Make primer stock in molecular biology grade H2O rather than TE or other buffers in order to avoid carryover of additional buffer to the LAMP reaction. Prepare primer stocks in nuclease-free water and store them at –20°C for up to 2 years.

  1. Thaw all components to be used at room temperature and place on ice. Salt may appear in the bottom of the tube so vortex briefly or invert tubes several times to mix thoroughly. Centrifuge to collect material and place on ice.
  2. Prepare reaction mix as described below using Colorimetric LAMP Master Mix, LAMP primers, and nuclease-free water. Volumes are listed per 25 μl LAMP reaction, but other volumes (10, 20, 50 μl, etc.) are all effective if desired, just adjust volumes accordingly. The sample is assumed here to be 1 μl, but for higher sample volumes add as needed and reduce the volume of H2O to compensate. For non-template reactions add an equivalent volume of H2O or sample storage buffer.
    3. DNA TARGET RNA TARGET DETECTION NO- TEMPLATE CONTROL(NTC)
    WarmStart Colorimetric LAMP 2X Master Mix 12.5 µl 12.5 µl 12.5 µl
    LAMP Primer Mix (10X) 2.5 µl 2.5 µl 2.5 µl
    Target DNA 1 µl - -
    Target RNA - 1 µl -
    dH2O 9 µl 9 µl 10 µl
    Total Volume 25 µl 25 µl 25 µl
  3. Vortex reaction mix and centrifuge to collect material.
  4. Pipet 24 μl per reaction into desired reaction vessels and add 1 µl of the sample. Mix by vortexing or by pipetting if using a plate or similar vessel, centrifuge to collect if necessary. Check that the reaction solution has a bright pink color, which indicates the initial high pH required for a successful pH-LAMP reaction.
  5. Seal reaction vessels.
  6. Remove tubes or vessels from incubation and examine by eye. Positive reactions will have turned yellow while negative controls should remain pink. If color change is not robust, e.g. an orange color is visible, return reactions to 65°C for an additional 10 minutes. Reactions can be examined earlier if desired, and high copy or input reactions can exhibit a full-colour change in as little as 10–15 minutes. Color will be visible directly on removal from incubation temperature but can be intensified by allowing the reaction to cool to room temperature.
  7. Keep selection plates ready with specific antibiotics.
  8. The result can be photographed or scanned to record the colorimetric results, or simply kept at room temperature in the reaction vessel.

Electronic LAMP: virtual Loop–mediated isothermal AMPlification

Requirements:-

  1. PERL interpreter
  2. PERL IPC::System::Simple module
  3. Tre library
  4. Perl/Tk module (GUI only)

These things should be installed beforehand from
https://www.nybg.org/files/scientists/dlittle/eLAMP.html

If you cannot install Tre from there use these commands:-
https://zoomadmin.com/HowToInstall/UbuntuPackage/tre-agrep

How to go to the root and install-

>
  1. sudo su
  2. type password
  3. sudo apt-get update -y
  4. sudo apt-get install -y tre-agrep

Open the README file, read it, set parameters according to the doc and run the eLAMP by below command.


Command:-

perl eLAMP.pl -f in–file.fasta -p in–file.csv [-A ###] [-a ###] [-s ##] [-l ##] [-I #] [-M #] [-O #] [-i ###] [-m ###] [-o ###] [-r] [-c]

Example code:- eLAMP.pl -f x.fst -p x.csv -A 300 -a 100 -s 1 -l 20 -r -I 3 -M 3 -O 3 -i 80 -m 80 -o 80 -c > x-out.csv


Extraction from E.coli.
Expression and purification of an Un1Cas12f1 (Cas14a1) protein (Our plasmid – addgene 112502).

Un1Cas12f1 (Cas14a1) protein was expressed in E. coli BL21(DE3) strain from the pLBH531 MBP-Cas14a1 plasmid (Addgene plasmid #112500). Un1Cas12f1D326A and Un1Cas12f1D510A expression plasmids were engineered from pLBH531 using Phusion Site-Directed Mutagenesis Kit (Thermo Fisher Scientific).

  1. Escherichia coli cells were grown in LB broth supplemented with ampicillin (100 g/ml) at 37◦C. After culturing to an OD600 of 0.5, the temperature was decreased to 16◦C and expression induced with 0.5 mM IPTG - Isopropyl ß-D-1-thiogalactopyranoside.
  2. After 16 h cells were pelleted, re-suspended in loading buffer (20 mM Tris–HCl, pH 8.0 at 25◦C, 1.5 M NaCl, 5 mM 2-mercaptoethanol, 10 mM imidazole, 2 mM PMSF, 5% (v/v) glycerol) and disrupted by sonication.
  3. Cell debris was removed by centrifugation.
  4. The supernatant was loaded on the Ni2+-charged HiTrap chelating HP column (GE Healthcare) and eluted with a linear gradient of increasing imidazole concentration (from 10 to 500 mM) in 20 mM Tris–HCl, pH 8.0 at 25◦C, 0.5 M NaCl, 5 mM 2-mercaptoethanol buffer.
  5. The fractions containing Un1Cas12f1 variants were pooled and subsequently loaded on HiTrap heparin HP column (GE Healthcare) for elution using a linear gradient of increasing NaCl concentration (from 0.1 to 1.5 M).
  6. The fractions containing the protein of interest were pooled and the 10×His-MBP-tag was cleaved by overnight incubation with TEV protease at 4◦C. To remove the cleaved 10×His-MBP-tag and TEV protease, reaction mixtures were loaded onto a HiTrap heparin HP 5 column (GE Healthcare) for elution using a linear gradient of increasing NaCl concentration (from 0.1 to 1.5 M). Next, the elution from the HiTrap heparin column was loaded on a MBPTrap column (GE Healthcare) and the Un1Cas12f1 proteins were collected in the flow-through.
  7. The collected fractions with Un1Cas12f1 were then dialyzed against 20 mM Tris–HCl, pH 8.0 at 25◦C, 500 mM NaCl, 2 mM DTT and 50% (v/v) glycerol and stored at –20◦C.

NOTES : Click on the buttons to scroll through the notes



References

[1] MoMonarch® Plasmid Miniprep Kit: https://international.neb.com/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010

[2] Agarose Gel Electrophoresis: https://dx.doi.org/10.17504/protocols.io.78rhrv6

[3] DNA distribution kit: http://parts.igem.org/Help:2021_DNA_Distribution

[4] WarmStart® Colorimetric LAMP 2X Master Mix (DNA & RNA): https://international.neb.com/products/m1800-warmstart-colorimetric-lamp-2x-master-mix-dna-rna#Product%20Information

[5] Electric LAMP: Virtual Loop-Mediated Isothermal AMPlification: https://doi.org/10.5402/2012/696758

[6] Harrington LB, Burstein D, Chen JS, Paez-Espino D, Ma E, Witte IP, Cofsky JC, Kyrpides NC, Banfield JF, Doudna JA. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science. 2018 Nov 16;362(6416):839-842. doi: https://pubmed.ncbi.nlm.nih.gov/30337455/

[7] PAM recognition by miniature CRISPR–Cas12f nucleases triggers programmable double-stranded DNA target cleavage https://academic.oup.com/nar/article/48/9/5016/5815819