Team:IISER Berhampur/Experiments

IISER-BPR IGEM
 Preliminary_Standardization Transformation RE_Digestion Cell_Culture Notebook 

Work Outline


Due to the COVID-19 pandemic, we had to re-strategise our project. Owing to the lockdown, the team could not access the lab till the beginning of October. For the same reason, reagents could not reach us on time either.


However, we decided to utilize this time in planning and revisiting our experiments thoroughly.




Preliminary Standardization of Plasmid


And these made us realise that instead of waiting for the reagents to arrive and then start optimizing protocols and techniques, we should start working on trial experiments to optimize our cloning, transfection and FRET approaches, as soon as we could access the lab.


We were able to do this with the help of a FRET reporter (Bulusu et al., 2017), that was available in the Department of Biological Sciences (DBS), IISER Berhampur.


All the protocols below have now been optimized and we plan to use this for our constructs for the second phase of the project.


Details of our Optimization Plasmid

pCKI – PYRATES


The pCKI vector is a modified pCAGGS (CMV-enhancer, beta-actin promoter, beta-globin polyA) ubiquitous expression vector. This backbone contains the coding sequence for variants of CFP (mTurquoise) and YFP (cp173Venus) and Ampicillin resistance as a selectable marker. The plasmid was kindly gifted to us by Dr. Vinay Bulusu (Assistant Professor, DBS), IISER Berhampur.




Figure 1 Plasmid Map showing the components of the pCKI plasmid with the coding sequences of CFP and YFP variants.


In Silico Restriction Digests

Overview

We performed in silico digests with the circular pCKI sequence using NEBcutter V2.0 (http://nc2.neb.com/NEBcutter2/ ) to obtain virtual gel images and find the non-overlapping open reading frames in the obtained plasmid. We assumed usage of a 1.0% agarose gel, to determine the mobility of known size fragments based on existing experiments in the NEB database. Three possible sets of high fidelity restriction enzymes were used (See Results below). The mobility of the fragments resulting from custom digests are predicted by polynomial interpolation from the standard cubic spline curve for known digests and the expected gel image is generated which can be matched with experimental digests.


Since the pCKI plasmid contains coding sequences of the CFP and YFP variants, we decided to excise out these fragments for additional FRaPPe constructs. In silico digests to release these fluorescent reporters was performed. We aim to use these variants as they are more stable forms than conventional CFP and YFP. mTurquoise is one of the brightest and most bleaching-resistant FRET donor fluorophores with a high quantum yield while cp173 Venus shows a high FRET efficiency. This pair also prevents the formation of bright aggregates of sensors, or speckles which can impede accurate measurement. (Klareenbeek et al., 2015)


This will enable us to use the pCKI plasmid both for optimization of our cloning and transfection protocols as well as for reporter construction. By using these variants in place of the existing sequences in Addgene plasmids (#20148 and #20160) we will modify them to carry an enhanced FRET sensor.



Results

1. Restriction Enzyme Digestion Map for pCKI, all commercially available NEB enzymes.



Figure 2 pCKI RE digest Map


2. Custom Restriction Digests were done using the following sets of enzymes.


  • HindIII and EcoRI Double Digest
  • HindIII and BamHI Double Digest
  • BamHI Single Digest


Figure 3a Circular Digests and ORFs generated




Figure 3b Virtual Gel Images (1 kb Marker, 1% Agarose gel


3. In Silico Digest for excising mTurquiose

Restriction Enzymes: MfeI and AgeI


Length: 711 bp, 26.7 kDa protein, 237 amino acids


4. In Silico Digest for excising cp173Venus

Restriction Enzymes: KpnI and NotI


Length: 735 bp, 27.3 kDa protein, 245 amino acids




Figure 4a Circular Digests and ORFs generated




Figure 4b Virtual Gel Images (1 kb Marker, 1% Agarose gel)



Competent Cell Preparation


We planned to do this in the last week of October. But with the current availability of competent cells in the department, the competent cells were acquired from the stock which was stored at -80°C. DH5α is the available competent cell which was prepared in the month of March.


  • Inoue transformation buffer prepared and filter sterilized using a 0.45 mm Nalgene filter. This was chilled on ice.
  • A single DH5α colony from a streaked plate was inoculated into 25 ml of LB medium in 250 ml flask. This culture flask was incubated at 37 °C overnight, with shaking at 250 RPM.
  • After approximately 8 hours of starter culture growth, three 1 litre flasks containing 250ml of LB were inoculated with differing volumes of the starter culture as follows, 2 ml, 5 ml and 10 ml respectively.
  • The flasks were incubated at 22 °C at 100 RPM, overnight.
  • After overnight incubation, the OD was measured for 1 ml of the culture, measured at OD600.
  • The 10 ml starter culture inoculated flask crossed the OD and hence it was discarded.
  • The 5 ml starter culture inoculated flask reached the OD in about 4 hours, this was immediately placed in ice bucket for 10 minutes.
  • The cells were harvested by centrifugation in the Eppendorf centrifuge at 3000 rpm for 10 minutes at 4 °C.
  • After discarding the supernatant in disinfectant containing disposal flask, the pellet was resuspended in 80 ml of ice cold Inoue transformation buffer. The resuspension was as gentle as possible with minimum pipetting and then mixed by swirling.
  • The cells were harvested by centrifugation at 3000 RPM for 10 minutes at 4 °C.
  • The supernatant was discarded as described above.
  • The cell pellet was carefully resuspended in 20 ml of ice-cold Inoue transformation buffer. To this mix, 1.5 ml of DMSO was added. This was mixed gently, and 400 µl aliquots were made into prechilled 1.5ml tubes.
  • Once the aliquoting was done, the competent cells efficiency was tested by using different controls.
  • The competent cell aliquots were stored at -80 °C.


Bacterial Transformation


In order to carry out the transformation, we started with the preparation of antibiotics followed by the preparation of fresh media and agar plates.


Antibiotic: The antibiotic used for this transformation was ampicillin (Himedia CMS64) which was procured from Dr. Vinay’s research lab and prepared a stock solution with a concentration of 100 mg/mL. The working concentration was made by diluting the stock with Double Distilled Water (DDW) to 100 µg/mL.


LB media preparation: Luria Bertani Broth, Miller (Himedia M1245) media was prepared for a total volume of 200 mL with 5 gm of Miller. With a thorough mixing of solution, media was transferred to a 500 mL air tightened conical flask for autoclaving


LB-Amp Agar plate preparation: For the preparation of four agar plates a composition of Luria Bertani Agar media (Himedia M1151) was prepared with a total volume of 120 mL with 4.8 gm of agar. The autoclaving proceeded for 45 minutes and ampicillin was added to the media after cooling. The media was now enriched with antibiotics and WE proceeded to make plates.


As the plates and media were made, for proceeding to bacterial transformation, 200 µL of DH5α and 1 µL DNA of plasmid pCKI were taken in a 1.5 mL eppendorf tube and mixed, incubated on ice for 30 minutes. This was followed by heat shock at 42°C for 90 seconds and immediately incubated on ice for 5 minutes; this heat shock will allow the integration of DNA to the E.coli competent cells and the final incubation on ice will help to seal this cell part respectively. 300 µL of LB was added and incubated for 40 minutes at 37°C in shaking condition. With the completion of this step the cells were centrifuged, the pellet was resuspended in 100 µL of LB. Now, the cells were plated on LB-Amp plates (see above for the preparation protocol) uniformly using a sterilized spreader and left the plate inverted at 37°C for 12 hours in an incubator with air fan mode on. A strict regime of 12 hours was maintained to avoid the growth of any satellite colonies.


Bacterial Culture for Plasmid Preparation

With the completion of 12 hour incubation, we could observe colonies in the LB-Amp plates. A single colony was picked up using a pipette tip and inserted in a falcon tube with 5 mL LB media added with Ampicillin (5 µL). Replicates were made and left for shaking incubation with an RPM of 100 at 37°C for 12 hours.


Plasmid Preparation


  • The 5ml culture was centrifuged in batches of 1.5 ml, three times in 1.5 ml tubes.
  • The pelleted ~4ml culture was resuspended in 600 µl of TE buffer.
  • The resuspended solution was mixed with 100 µl of 7X lysis buffer and mixed by inverting the tube.
  • The solution changed from opaque to clear blue, which is a mark of complete lysis.
  • Immediately, 350 µl of cold neutralization buffer was added and mixed by inverting, the solution turned yellow in colour.
  • The above sample was centrifuged at 13000 RPM for 3 minutes.
  • The supernatant was carefully transferred to the Zymo spin column and centrifuged for 13000 rpm for 15 seconds.
  • The flow through was discarded.
  • The column was washed with 200 µl of endo-wash buffer. The tube was centrifuged at 13000 rpm for 15 seconds.
  • The column was washed with a 400 µl wash buffer at 13000 rpm for 30 second.
  • The column was transferred from the collection tube to a 1.5 ml microcentrifuge tube.
  • 30 µl elution buffer was added to the column. After incubation at room temperature for 1 minute, the column was centrifuge again at 13000 rpm for 15 second and DNA was eluted.

Concentration estimation by nanodrop (Thermofisher)

2 µl of the plasmid DNA was used for concentration estimation


Replicate 1: 70.6 ng/ µl


Replicate 2: 93.7 ng/ µl


RE Digestion


COMPONENTS (µL) RESTRICTION DIGESTION 1 (EcoRI) RESTRICTION DIGESTION 2 (HindIII) RESTRICTION DIGESTION 3 (EcoRI/HindIII)
PLASMID PCKI 3.5 (from 70 ng/µL conc. ) 3.5 (from 70 ng/µL conc. ) 2.6 (from 95 ng/µL conc.)
&10X BUFFER 2.5 (1X conc.) 2.5 (1X conc.) 2.5 (1X conc.)
ENZYME 1 (EcoRI) 0.5 0.5
ENZYME 2 (HindIII) 0.5 0.5
NUCLEASE FREE WATER 18.5 18.5 18.9
TOTAL (µL) 25 25 25

- Concentration estimation by nano-drop


Incubation conditions: Water bath at 37℃ for 1 hour.


Agarose Gel Electrophoresis


The fragments of the plasmid after single and double digestions were run on an agarose gel to visualise and analyse the DNA.


Protocol


  • 0.8% agarose gel was prepared by dissolving 0.4 g of agarose powder (HiMedia, MB002-100G) in 50 mL TBE buffer (of 1X concentration) in a microwavable flask.
  • 12.5 µL of EtBr (final conc. = 0.5 µg/mL) was added and mixed properly after the solution cooled down to a tolerable temperature.
  • The agarose solution was poured into a casting tray with an 8-well comb in place.
  • The solution was allowed to cool and solidify at room temperature.
  • The gel was moved into the gel box and 1X TBE buffer was poured into the box such that the gel got immersed completely.
  • 25 µL of each sample and 5 µL loading dye were mixed for loading into each well.
  • 20 µL of each sample was loaded onto each well in the sequence as shown in Fig.5. Two ladders were used; Purple 1kb plus ladder (NEB - N055OS) and a ladder of 100bp - 3kb (HiMedia).
  • The gel was run at 120 V for around 1.5 hours.
  • The bands were visualised under UV light in the Gel Doc imaging system (UVP ChemStudio - Analytik Jena).



Figure 5: Restriction digestion of pCKI with EcoRI and HindIII, 0.8% agarose gel, 1X TBE


Results

In the 1st and 6th well, we can observe the ladders, the first one of which is of a higher molecular weight (1kb above) ladder and the other one is a broad-ranging ladder from 100 base pair to 300-kilo base pair. As we used the uncut plasmid having a size of 7 kb, the first ladder is suitable to confirm the size and the second ladder for confirming low molecular weight fragments generated upon RE digestion. Hence, the use of both ladders will help to locate the specific fragments.


The second well contains an uncut (i.e., no RE added) plasmid and as the position of the band is not too far from the well, this possibly depicts the actual size of the plasmid which is ~7kb. Ideally, however, a denser band should have been observed, but in this case as the concentration of plasmid used was 250 ng, so the brightness in this picture in a way, mirrors the concentration of plasmid.


In the third well (digested with the RE EcoRI), and fourth wells (digested with the RE HindIII), a comparison with the in silico digests, reveals that we should obtain one band (i.e., 1 fragment) of size 7 kb since both these enzymes have a single site in the sequence (Refer to Figure 3a). This situation is not reproduced experimentally however and we could observe two bands in both these lanes and fragments were not in the exact expected size range.


In the combination of EcoRI and HindIII, which is a double digest and in reference with the in silico analysis we should have observed 2 bands (6.6 kb + 0.4kb) but, only a single clear band is visible here, which means the double digest shows only linearization suggesting incomplete digestion. The main conclusion is that the Restriction Enzymes digestion needs to be repeated with increased/different enzymes and additional incubation time. Also for the double digest, the compatibility of the buffer for both enzymes remains to be confirmed. Given that these are our optimization experiments, we will use these to troubleshoot and modify the protocols for the experiments using our FRaPPe constructs.


Mammalian Cell Culture

Since our entire project runs around HEK 293 cells, this SOP is to elaborate on the principles and protocols for maintaining HEK 293 cells in culture and preparation for transfection.



HEK Cell Line

The Human Embryonic kidney 293 cells are specific cell line originally derived from human embryonic kidney cells grown in tissue culture.


HEK 293 cells were generated in 1973 by transfection of cultures of normal human embryonic kidney cells with sheared adenovirus 5 DNA in Alex van der Eb’s laboratory in Leiden, the Netherlands.


There is evidence that HEK 293 cells and other human cell lines generated by adenovirus transformation of human embryonic kidney cells have many properties of immature neurons, suggesting that the adenovirus preferentially transformed the neuronal lineage cell of the original kidney culture.


HEK 293 cells have a complex karyotype, exhibiting two or more copies of each chromosome and with a modal chromosome number 64. They are described as hypotriploid, containing less than three times the number of chromosomes of a haploid human gamete. (Chromosomal abnormalities include three copies of X chromosomes and four copies of chromosome 17 and chromosome 22).


Purpose of choosing HEK 293


It is easy to grow and transfect cells and has been widely used for cell biology industry to produce therapeutic proteins and viruses for gene therapy. It is also not a cancerous cell line unlike Hela.


High reproducibility, which makes it preferable over other less-robust and slow-growing cell lines.



Cell Culture Work Flow


ISOLATE

  • Separate fresh tissue of interest.
  • Treat tissue with enzyme(s) (e.g. trypsin, collagenase, protease) and/or mechanically isolate cells.
  • Wash, count and seed cells.

VERIFY

  • Examine cells under a brightfield microscope to access their growth state, attachment to culture vessels/flasks and to check for any signs of infection.
  • Monitor cells for the following days until they reach confluence.
  • Verify isolated cell types by their morphology and expressed biomarkers.

CULTURE

  • Passage cells to propagate cell line.
  • Make master and working cell banks.
  • Immortalize cells if necessary.

INVESTIGATE

  • Plan and execute experiments.
  • Keep monitoring cell state and possible infections using a brightfield microscope.

Basic cell culture equipment

The specific equipment of a cell culture laboratory depends on the type of research conducted. However, all cell culture laboratories have the same common equipment, free from pathogenic microorganisms


  • Wrapped disposable pipettes
  • Waste container
  • Glass pipette (if using)
  • Waste liquid
  • Reagents and media
  • Pipettor
  • Work surface
  • 70% ethanol antiseptic
  • Autoclave

Reagents

  • Medium: 500ml Dulbecco’s modified Eagle Medium (SIGMA, D5796 ; HIMEDIA, TC185), FBS (HYCLONE, SV30160.03), Penicillin Streptomycin solution (HIMEDIA, A0002)
  • Trypsin (SIGMA, 59428C)
  • Cell culture 6 well plate – equivalent to 30 mm plates (TARSONS, 950040)
  • Cryovials (TARSONS, 523182)
  • Cell culture vented flasks with slight inclinatioN (T25 TARSONS, 523182)

Apparatus

5 % CO2 incubator, hemocytometer, inverted microscopes (to observe confluent cells, light source from below)


Procedure

  • The media was discarded from vented flasks (prewarm the media at 37°C).
  • The medium was aspirated from the flask and wash with PBS (2 ml) to remove all traces of media, serum etc. so that trypsin can act.
  • It was gently swirled to mix. Some cells may detach by the force already.
  • Next, 1 ml of trypsin was added and swirled gently.
  • Incubated at 37°C for 2 minutes.
  • One flask was seeded for 1 F, one made into stock, others packaged.
  • Media was to neutralize trypsin (9 ml media + 1 ml trypsin).
  • If thread-like structure appears, which indicates cells are in clumps. (Note: Thread-like structures before step 1 may indicate fungal fungal spore contamination).
  • Mixture was pipetted up and down to homogenize. (No bubble formation)

STEP 1 : Contribute to ROS

  • Aliquots taken for cell count (for seeding for 1F)
  • 3 coverslips were put in each 30mm plate of 6 well-plate, 2.5 ml (approx.) media was added into each plate (maximum can be 3 ml) + 0.5 ml cells in each plate.

STEP 2 : Passaging of cells

  • For passage, solutions were distributed and 4 ml media added into new culture flasks and cells washed with PBS and trypsinized at 37°C.

STEP 3 : Stock preparation

  • Centrifuged after aliquoting -->freezing media + DMSO and serum -->stored at - 80°C for 3-4 weeks

Maintaining HEK 293 cells in culture

HEK 293 cells should be grown in a monolayer, preferably in plastic petri dishes or flasks. Under optimum growth conditions (37°C, 5% co2), 293 cells double after every 36hr. To maintain consistency, one should not passage cells indefinitely. For best results, one may use low passage 293 cells for transfection and titration procedures.


To prevent contamination, work with media and uninfected cells should be done in a vertical laminar flow hood, using sterile technique. Ideally, we should use another hood for all virus work. All virus-contaminated materials, including fluids, must be autoclaved or disinfected with 10% bleach or a chemical disinfectant before disposal.


Antiseptic techniques required while working with cell culture

To have a successful cell culture, it is essential to maintain a contamination free environment (bacteria, fungi, etc.). Aseptic techniques ensure that no microorganisms enter the cell culture set-ups. Cell culture sterility is ensured by a defined set of procedures.


HANDLING REAGENTS/MEDIA WORKPLACE
Slow/careful handling Pre-sterilization of all reagents/equipment Cell culture hood should function properly
Sterilization of all items before starting. No contamination in reagents (expiration date, appearance normal). Frequent de-contamination (hood, fridge etc.)
No touching of sterile items to non-sterilized surfaces Work area should be sterile and tidy.

Electroporation


Electroporation— the use of high-voltage electric shocks to introduce DNA into cells—is a procedure that is gaining popularity for standard gene transfer. It also allows the generation of genetically modified mice. It can be used with most cell types, yields a high frequency of both stable transformation and transient gene expression, and, because it requires fewer steps, can be easier than alternate techniques.


The method uses an electric field that transiently permeabilizes the membrane. During this period the foreign material can enter the cell. This method-


  • Works well with almost all cell types.
  • Ensures high transfection efficiency.
  • Requires optimization as cell permeability can lead to cell damage.
  • Experienced user needed.
  • Quite an expensive method.

Materials


Mammalian cells to be transfected.

Complete medium without and with appropriate selective agents

Electroporation buffer, ice-cold

Linear or supercoiled, purified DNA preparation

Beckman JS-4.2 rotor or equivalent

Electroporation cuvettes (Bio-Rad #165-2088) and power source

Additional reagents and equipment for stable transformation in selective medium and for harvesting transfected cells

Procedure

  • Cells to be transfected are grown to late-log phase in complete medium. Each permanent transfection will usually require 5 × 106 cells to yield a reasonable number of transfectants. Each transient expression may require 1–4 × 107 cells, depending on the promoter.
  • Cells are then harvested by centrifuging 5 min at 640 × g (1500 rpm in a JS-4.2 rotor), 4°C. Adherent cells are first trypsinized and the trypsin inactivated with serum.
  • Cell pellet is resuspended in half its original volume of ice-cold electroporation buffer. The choice of electroporation buffer may depend on the cell line used.
  • Again, cells are harvested by centrifuging for 5 min as in step 2.
  • 1ml aliquots are dispensed into labelled freezing vials and place in a cell freezing container (reduces temperature ~1°C/min) at -80°C overnight.
  • Cells are resuspended at 1 × 107/ml in the electroporation buffer at 0°C for permanent transfection. Higher concentrations of cells (up to 8 × 107) may be used for transient expression.
  • 0.5-ml aliquots of the cell suspension are transferred into desired number of electroporation cuvettes set on ice.
  • DNA added and electroporation of the cells done.
  • DNA added to cell suspension in the cuvettes on ice.
  • DNA/cell suspension is mixed by holding the cuvette on the two “window sides” and flicking the bottom. Incubated for 5 min on ice.
  • The cuvette is placed in the holder in the electroporation apparatus (at room temperature) and shocked one or more times at the desired voltage and capacitance settings. The number of shocks and the voltage and capacitance settings will vary depending on the cell type and should be optimized.
  • After electroporation, the cuvette containing cells and DNA is returned to ice for 10 min.
  • Transfected cells are diluted 20-fold in nonselective complete medium and cuvette rinsed with this same medium to remove all transfected cells.
  • Cells are responded at 1 × 107ml in the electroporation buffer at 0°C for permanent transfection. Higher concentrations of cells (up to 8 × 107) may be used for transient expression.
  • For stable transformation: Cells should be grown for 48 hours (about two generations) in nonselective medium, then transferred to antibiotic-containing medium. Selection conditions will vary with cell type.
  • For transient expression: Cells should be incubated for 50 to 60 hours, then harvested for transient expression assays. Transfected cells can be visualized by standard transient expression assays.

Wet Lab Notebook

Day 1 – 17/10/2020


pCKI plasmid was kindly provided by Dr. Vinay Bulusu.

Agar media and plates were prepared and ampicillin was spread over the plate.

Transformation of the pCKI plasmid and DH5α competent cells was performed.

Transformed cells were plated on LB-ampicillin agar plates along with an additional control plate for the competent cells. These plates were incubated at 37°C for 12 hours.


Day 2 – 18/10/2020


There were few colonies on the plates, which suggested that the transformation process had not gone well. So, we decided to repeat the process.

This could be because of problems with the cells or the culture media.

The LB media and LB-Amp agar plates were prepared freshly. After transformation of the plasmid and competent cells, the cells were plated and incubated at 37°C for 12 hours.

We used two different plasmid concentrations.


Day 3 – 19/10/2020


With the completion of 12 hours incubation, we were successful in witnessing the colonies in both plates. Stored these plates at 4°C to proceed further.

A single colony was added to a falcon tube containing 5 mL of LB Broth media with different concentrations of ampicillin and two replicates were taken for each concentration. A pipette tip was used to scoop out a single colony.

The falcon tubes were placed in a shaking incubator with 100 RPM for 12 hours at 37°C.


Day 4 – 20/10/2020


We were able to observe the dense colour of the media signifying the growth.

Cells were processed for plasmid extraction.

Plasmids that were extracted, were stored at -20 °C


Day 5 - 22/10/20


Restriction digestion of the plasmid was done to confirm the plasmid.

Single enzyme digests and Double enzyme digests using 250ng of plasmid were performed at 37°C for 1 hour.


Day 6 - 23/10/20


Restriction digested plasmids were subjected to electrophoresis on 0.8% agarose gel.

Reagents for cloning and plasmid purification were received from NEB.


Day 7 - 24/10/20


Cell culture media was prepared and incubated in the cell culture incubator for sterility check.


Day 8 - 25/10/20


HEK293 cells were recovered from previously cryopreserved vials.

Plasmid restriction digestion was repeated with 1µg of pCKI


References

1. Bulusu V et al., (2017) Spatiotemporal Analysis of a Glycolytic Activity Gradient Linked to Mouse Embryo Mesoderm Development. Dev Cell. 40(4):331-341.


2. Klarenbeek, J. et al., (2015). Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity. PloS one, 10(4), e0122513. https://doi.org/10.1371/journal.pone.0122513/


©iGEM IISER Berhampur