Team:METU HS Ankara/Experiments

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Experiments

Protocols

Transformation Protocol:

1. Throw competent cell on ice
2. Mix ligation product 50 microliter cell+2 microliter plasmid
3. Incubate cells on ice 30 mins
4. Heat shock 55 sec CaCl2, 75 sec RuCl2
5. Add 900 ml LB
6. Incubate 37 celcius for 80 minute
7. Centrifuge at 3000 po m 10 min
8. Discard supernatant
9. Resuspend pellet in 100 ml LB
10. Spread cells and wait 14-16 hours in the incubator

LB Broth Protocol:

1. 10 gr peptone
2. 5 gr yeast extract
3. 10 gr NaCl
4. up to 1000ml dH20 & autoclave
5. LB Broth 25 gr/1L
6. LB Agar 40 gr/1L

LB Agar Protocol:

1. 10 gr peptone
2. 5 gr yeast extract
3. 10 gr NaCl
4. 15 gr agar
5. up to 1000ml dH20 & autoclave
6. use 20 gr for 1lt dH2O

Overnight Culture:

1. Put 5 ml LB into a tube.
2. Put a loop of E. coli aseptic
3. Seal the tube
4. Place the tube digonally into a shaker for 14-18 h ( optimum 16 h) at 37℃ and 200 RPM

Gel Preparation:

• 130 ml TAE Buffer
• 1.3 Agarose

ONC Protocol:

1. Put 5 ml LB into a tube.
2. Put a loop of E. coli dH5 aseptic.
3. Seal the tube using parafilm.
4. Place the tube diagonally into a shaker for 14-18 hours.(optimum 14)

Competent Cell Preparation by Calcium Chloride Protocol:

1. Inoculate 1ml ONC to 100 ml LB in a flask.
2. Incubate the flask for 2-4h at 37 ˚C, check via spectrophotometer at 600nm for 0.300..
3. Divide the solution into two falcon tubes(50 ml).
4. Spin them down at +4 ˚C and 5000g for 5min.
5. Discard supernatant.
6. Resuspend the cells with 10 ml cold CaCl2.
7. Put in ice for 10 minutes.
8. Spin the suspension down at +4 ˚C, 5000g for 5 minutes.
9. Discard supernatant.
10. Add 10 ml cold CaCl2 and resuspend the cells.
11. Put the cells in ice for 30 min.
12. Centrifuge at 5000 rpm for 5 min at +4 ˚C.
13. Discard supernatant.
14. Put 1 ml CaCl2 and dissolve pellet.
15. Put it on ice for 5 min. and keep it at +4 ˚C.

Getting the DNA Parts from the Kit Plate:

1. Add 10 microliter of ddH2O into the wanted well.
2. Wait and get the part by pipetting.
3. Make sure to keep stock from these parts since there is so less of them.
4. In case of having too less sample, you can dilute the stock 10:1 with ddH2O.

Getting the DNA Parts from the Kit Plate:

A. Bacterial culture, harvest, and lysis.

1. Pellet 25 ml (high copy) or 100ml (low copy) overnight LB culture at 6000xg for 15 min at +4˚C.
2. Homogeneously resuspend the bacterial pellet in 4 ml Buffer P1.
3. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4-6 times, and incubate at room temperature for 5 min.
4. Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4-6 times, and incubate on ice for 15 min.

B. Bacterial lysate clearing

1. Centrifuge at ≥20.000 xg for 30 min at +4 ˚C. Re-centrifuge the supernatant at ≥20.000 xg for 15 min at +4 ˚C.

C. Bind, wash, and elute plasmid DNA on QIAGEN-tip.

1. Equilibrate a QIAGEN-tip 100 by applying 4ml Buffer QBT and allow column to empty by gravity flow.
2. Apply the supernatant(step 5) to the QIAGEN-tip and allow it to enter the resin by gravity flow.
3. Wash the QIAGEN-tip with 2x10 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.
4. Elute DNA with 5 ml Buffer QC into clean 2 ml, 15ml or 50ml vessel.

D. Precipitate, wash, and redissolve plasmid DNA

1. Precipitate DNA by adding 3.5 ml room-temperature isopropanol to the eluted DNA and mix. Centrifuge at ≥ 15.000 xg for 30 min at +4℃. Carefully decant supernatant.
2. Wash DNA pelelt with 2ml room-temperature 70% ethanol and centrifuge at ≥ 15.000 xg for 10 min. Carefully decant supernatant.
3. Air-dry pellet for 5-10 min and redissolve DNA in a suitable volume of buffer.

Plasmid Isolation Protocol:

• Resuspend the pelleted cells in 250μl of the Resuspension Solution. Transfer the cell suspension to a microcentrifuge tube. The bacteria should be resuspended completely by vortexing or pipetting up and down until no cell clumps remain.
• Add 250μl of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
• Add 250μl of the Lysis Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
• Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
• Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white precipitate.
• Centrifuge for 1 min. Discard the flow-through and place column back into the same collection tube.
• Add 500μl of the Wash Solution to the GeneJET spin column. Centrifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
• Repeat the wash procedure using 500μl of the Wash Solution.
• Discard the flow-through and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
• Transfer the GneJET spin column into a fresh 1.5 ml microcentrifuge tube. Add 50μl of the Elution Buffer to the center of GeneJET spin column membrane to elute the plasmid DNA. Take care not to contact the membrane with the pipette tip. Incubate for 2 min at room temperature and centrifuge for 2 min.
• Discard the column and store the purified plasmid DNA at -20 ˚C.

80% Glycerol Preparation:

1. Add 80ml 99.7% glycerol to 20ml demineralized water
2. Autoclave

Digestion:

1. Restriction Digests
   • The left part sample is cut out with EcoRI and SpeI.
   • The right part sample is cut out with XbaI and PstI.
   • The linearized plasmid backbone is a linear piece of DNA. It has a few bases beyond the EcoRI and PstI restriction sites. It is cut with EcoRI and PstI.
2. All 3 restriction digests are heated to heat kill all of the restriction enzymes.
3. An equimolar quantity of all 3 restriction digest products are combined in a ligation reaction.
4. The desired result is the left part sample's SpeI overhang ligated with the right part sample's XbaI overhang resulting in a scar that cannot be cut with any of our enzymes.
5. The new composite part sample is ligated into the construction plasmid backbone at the EcoRI and PstI sites.
6. When the ligation is transformed into cells and grown on plates with antibiotic C, only colonies with the correct construction survive.

Single Reaction

1. Add 250ng of DNA to be digested, and adjust with dH20 for a total volume of 16ul.
2. Add 2.5ul of NEBuffer 2.
3. Add 0.5ul of BSA.
4. Add 0.5ul of EcoRI.
5. Add 0.5ul of PstI.

• There should be a total volume of 20ul. Mix well and spin down briefly.
• Incubate the restriction digest at 37C for 30min, and then 80C for 20min to heat kill the enzymes. We incubate in a thermal cycler with a heated lid
• Run a portion of the digest on a gel (8ul, 100ng), to check that both plasmid backbone and part length are accurate.

Double Digestion:

10 μl Plasmid
6 μl MGW (water)
1 μl Buffer 1
1 μl Buffer 2
1 μl Enzyme 1
1 μl Enzyme 2

Single Digestion:

10 μl Plasmid
8 μl MGW (water)
1 μl Buffer 1
1 μl Enzyme 1

Experiments

Growth of cells in culture

The agar stabs contain live bacteria (E. coli) that house your parts. The parts themselves belong to a plasmid which replicates within the E. coli cell.

Before working with our parts, we had to be sure that we have enough copies of them. We did this by growing up cell cultures from the agar stabs: more E. coli cells means more plasmids, which means more copies of our parts. Use the provided agar stabs to streak out the bacteria containing our parts onto agar plates. Then we pick a single colony to grow up a cell culture.

Part A and Part B are both maintained on pSB1AK3 plasmid backbones. The ‘AK’ means that our plasmid backbone is resistant to Ampicillin and Kanamycin antibiotics. When we are growing these parts up, we have to be sure to use media that has these antibiotics, that way we can ensure that only the bacteria containing our parts will grow.

Streaking from agar stabs:

Materials

• 70% ethanol
• Paper towels
• Lab marker/Sharpie
• Agar Stab: Part A - BBa_J04500 (kit)
• Agar Stab: Part B - BBa_J04650 (kit)
• Inoculating loops (kit)
• LB agar plates – Amp/Kan (kit)

Growing up cell cultures:

Materials

• 70% ethanol
• paper towels
• Lab marker/Sharpie
• 14ml culture tubes (kit)
• 10ml of LB broth - Amp/Kan (kit)
• Inoculating loops (kit)
• Agar plate: Part A – BBa_J04500
• Agar plate: Part B – BBa_J04650
• Rotator/Shaker

Production of Competent Cell

Competent cells are what we used to grow up and express your ligation result. These cells are modified bacteria which were selected for their transformation ability: their ability to uptake foreign DNA from their surrounding environment and express it. This makes them useful as cellular models of a system, such as the RFP system we’re making in the 3A Assembly kit protocol.

Transformation efficiency is a measure of how “competent” these competent cells are: highly efficient competent cells will yield many more colonies than less efficient competent cells. DNA is provided at different concentrations so teams can determine how sensitive their cells are.

Preparation of Seed Stocks

To make our own stock of competent cells to use we need to first create seed stocks. Each vial of seed stock can be used to create a separate batch of competent cells, stored in 2.0ml tubes. Transformations are performed using a tube of these competent cells.

Materials

• Agar stab of NEB10 Beta competent cells
• SOB plates
• Inoculating loops
• Paper towels
• Lab marker
• Agar stab of NEB10 Beta cells (kit)
• SOB plates (kit)
• Inculating loops (kit)
• SOB media
• Glycerol
• 2.0 ml screw-cap tubes
• 70 % ethanol

Miniprep

After growing up cell cultures of E. coli containing lots of copies of Part A andPart B. In order to access the plasmid DNA containing your part, we need to lyse the cells (make them burst open), separate the DNA from the cellular material, and purify the sample to make sure you only have the plasmid DNA and not the E. coli's own genomic DNA.

Materials

• 70% ethanol
• Paper towels
• Marker/Sharpie
• Centrifuge/microcentrifuge
• 2 Beakers/waste collection containers
• Bleach
• Buffers: P1, RNAse A, P2, N3, PB, PE (kit)
• 1.7 ml Microcentrifuge tubes (kit)
• Qiagen spin columns (kit)
• Distilled water (kit)
• Cell culture: Part A – BBa_J04500
• Cell culture: Part B – BBa_J04650
• Nanodrop machine (optional)

Restriction Digest

After miniprepping, we had the plasmid DNA containing Part A and Part B. Part A and Part B, we have to cut them out from their pSB1AK3 plasmid backbones. We uses restriction enzymes to cut the parts out. This will leave small overhangs at the end of each part which will act like connectors to assemble Part A and Part B together into the pSB1C3 linearized backbone, which we will need to digest as well.

Materials

• Ice and bucket/container
• (1) 8-tube strip, or (3) 0.6ml thin-walled tubes
• BioBrick Part in BioBrick plasmid (Purified DNA, > 16ng/ul)
• dH2O
• NEB Buffer 2
• BSA
• Restriction Enzymes: EcoRI, SpeI, XbaI, PstI
• Thermal cycler

Materials

• 70% ethanol
• Paper towels
• Ice
• Container for ice
• Lab marker/Sharpie
• pSB1C3 linearized plasmid backbone (25ng/ul) (kit)
• Part A (25ng/ul) (kit)
• Part B (25ng/ul) (kit)
• RFP Control (20ng/ul) (kit)
• 0.6ml tubes (kit)
• NEB buffer 2
• BSA
• NEB enzymes: EcoRI, SpeI, XbaI, PstI
• Thermocycler, or waterbath and thermometer

Ligation

After your restriction digest we have Part A, Part B, and the pSB1C3 linearized plasmid backbone cut. Now we have to assemble all of these components together (Part A + Part B, into pSB1C3). We did this by using the overhangs (connectors) we created by cutting the DNA with restriction enzymes and ligase, which will keep everything glued together.

Materials

• 70% ethanol
• Paper towels
• Lab marker/Sharpie
• 0.6 tubes (kit)
• Distilled water (kit)
• Ice
• Container for ice
• T4 DNA Ligase Reaction Buffer
• T4 DNA Ligase
• Microcentrifuge
• Thermocycler, or waterbath and thermometer
• Restriction Digest: pSB1C3 linearized plasmid backbone
• Restriction Digest: Part A
• Restriction Digest: Part B
• Restriction Digest: RFP Control

Transformation

We had an assembled product through ligation (Part A + Part B, in pSB1C3). To get this new part into E. coli cells, we need to use a process called transformation. So it can uptake our ligated product (the plasmid containing your new part). Once the cells have taken in our plasmid, we had grow them on a chloramphenicol plate (only cells containing the pSB1C3 plasmid backbone will live), and from those cells that live, there will be red colonies that have the correct new part.

Materials

• 70% ethanol
• Paper towels
• Lab marker/Sharpie
• Ice
• Container for ice
• Timer
• NEB10 cells
• 2.0ml microcentrifuge tubes (kit)
• Inoculating loops (kit)
• Ligation: New Part
• Ligation: Control
• DNA: RFP Control (20ng/ul) (kit)
• SOC media (kit)
• LB agar plates - Chloramphenicol (kit)
• Sterile glass beads (kit)
• Waterbath and thermometer

Induction Protocol:

If the protein is membrane-bound , expression in mutant strains C41 , BL21 DE3 and C43(DE3) could improve expression levels.

Expression method

• Picking of a single colony from a freshly streaked plate of the expression host containing the recombinant vector. When the heterologous protein is toxic for the cells , higher expression levels are obtained by using the so-called ‘plating’ method.
• Growing of a starter culture.
• Inoculate with the picked up colony to 50 ml of LB medium containing appropriate antibiotic.
• When a larger starter culture is required , inoculate 4ml of rich media with single colony ; grow for 4-8 hours at 37°C and use this to inoculate the starter culture.

(Don’t let cultures grow at 37°C overnight !)

• It is better to grow overnight cultures at 30°C or lower . Alternatively ,the culture can be incubated at 37°C until OD600 is approx. 1. Then store the culture at 4°C overnight. Following morning , collect the cells by centrifugation , resuspend them in fresh medium and use this to inoculate the main culture.
• Inoculation of the main culture and incubation until OD600 reaches 0,4-L. The optimal OD value depends on the culture method and the medium. For flask cultures using LB-medium on OD600 of 0.6 recommended. To increase growth rate we carry out the cultures at 37°C until the OD for induction is reached. Then the cultures are cooled to the induction temperature in ice-water.

Induction of protein Expression

After induction the cultures are are incubated from 3 hours to overnight depending on the induction temprature. Guide lines are below;

Harvesting of the cell pellet by centrifugation ( 20 min at 6000 g ). Cell pellets are stored at -20°C.

E. coli BL21 (DE3) & E. coli K12 M6 1665 Coculture with CaCo-2 Trial Protocol

E. coli cultures were grown overnight and used in the early stationary phase.
Started from on overnight pre-cultured , stationary phase ( OD > 2)

• 100 μl sample ( E. coli : pre-culture ) +10ml LB
• 37°C , 200 rpm , 16 hours

OD600 measurement → OD600 = 1.0 means we have 8 x 108 cells 1ml , OD600 = 2.0 → 1,6 x 109 cells 1ml ( assumed as stationary phase )

Co-culture

CaCo-2 cells were infected with a bacteria at a multiplicity of infection (MOI) of 1000 in 10 ml of cell culture media lacking antibiotics.

This means for 1 cell- 1000 bacteria.

We are growing 3 x 104 cell / well, therefore 3 x 107 bacteria is required. Overall, 1x 109 from each strain is needed.

1. Determine the amount of well for co-culture.
   i.e: K12- 25 well x (3 x 107 ) = 75 x 107 needed
2. Measure OD600 and find out how many cell/ml you have.
   i.e; We have 12,8 x 108 /ml in culture of bacteria.
3. Decide them to find out the amount to centrifuge.
   i.e; (75 x 107) / (12,8 x 108 ) =0,585≈0,600
4. Centrifuge (600 μl) from each bacterial culture.
5. Remove flow-through and resuspend in 500 μl medium. (Now, medium is used for cancer cell. However, we could create new media. (mix of EMEM&LB)
6. 500 μl media is needed for each well.Then, 500μl x 25 well= 12,5 ml
7. Prepare media (EMEM) as substracted the amount that we have used to resuspend bacterial strains. So, 12 ml + 500 μl media (resuspended bacteria) =12,5 ml
8. 3hr, 6 hr, 9hr overnight 24 well plates were prepared to carry K12, BL21 and cells only.
9. 12,5 ml of K12, BL21 and EMEM medium were distributed equally and placed into incubator. (No shaking! But maybe we need very slow shaking if we use early passage of CaCo-2.
10. After incubation, plates were washed with PBS as 500 μl for each well. (3 times)
11. 1,25 ml MTT is mixed with 11,25 PBS (total 12,5 ml) and distributed each well as 500 μl after washing.
12. After 4 hours incubation with MTT, add 500 μl SDS (as we have described preparation of SDS) to each well and leave overnight.

References:

1. Xuan He, Darya O. Mishchuk, Jigna Shah, Bart C. Weimer & Carolyn M. Slupsky. (2013). Cross-talk between E. coli strains and a human colorectal adenocarcinoma-derived cell line. Retrieved from http://www.nature.com/articles/srep0341

2. EMBL. Protein Expression and Purification Core Facility. Retrieved from: http://www.embl.de/pepcore/pepcore_services/protein_expression/ecoli/

3. iGEM transformation protocol. Retrieved from: http://parts.igem.org/Help:Protocols/Transformation

4. iGEM miniprep protocol. Retrieved from: http://parts.igem.org/Help:Protocols/Miniprep

5. iGEM Competent Cells protocol. Retrieved from: http://parts.igem.org/Help:Protocols/Competent_Cells