Team:Hamburg/Experiments

Alginate Encapsulation

To encapsulate our diagnostic bacteria we utilize a microfluidic encapsulation chip. The first task performed by the encapsulation chip is the pinch-off of the solution of alginic acid and bacteria by an oil, the shear fluid, creating an emulsion. In the second and last step the polymerization initiating CaCl2 solution is injected into the pinched-off droplets. By the time the droplets exit the chip they are polymerized. The size of the produced alginate encapsulated bacteria droplets is tuneable by using different flow rates of sunflower oil (shear fluid). The following videos show the dependence of the droplet size from the shear flow rates. To precisely test the influence of the flow rate on the droplet size we have used a syringe pump system. To produce big droplets, we have used a shear fluid flow rate of 15.000 µL/h and an alginate bacteria solution flow rate of 4.000 µL/h. Smaller droplets can be produced with a shear fluid flow rate of 8.000 µL/h and an alginate bacteria solution flow rate of 4.000 µL/h.

Protocols

Biolab Protocols

Agarose gel

AG Ignatova

  • 0.5 g Agarose
  • 50 mL TAE-buffer (gel room)
  1. Heat in microwave until agarose is dissolved
  2. Transfer into gel slide, tip pipet into EtBr, then into gel
  3. Cool for 30 minutes

Backbone dephosphorylation

AG Ignatova

10 μL pSB1C3 (cut)
1 μL FastAP
4 μL FastAP Buffer

→ 37 °C for 30 minutes

→ purify with PCR clean up kit

Chloramphenicol Stock

AG Ignatova

34 mg/mL preparation of Choramphenicol Stock (3 in EtOH)

  1. dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol
  2. filter through a 0.22 μL filter to sterilize
  3. use at 1:1000 dilution in LB or LB-agar
  4. mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics
  5. store at -20 °C

Competent Cells

Zhang Gong

Buffers and solutions:

  • Ca/glycerol buffer: 60mM CaCl2, 10mM PIPES, 150mL glycerol, Fill water up to 1L, pH=7.0
    Filter sterilization. Avoid autoclaving.

Protocol:
  1. Grow the Cells at 37°C till OD=0.2-0.4
  2. Chill the culture on ice for 5min.
  3. Cllect cells by centrifugation, 6000rpm 10min 4°C
  4. Gently resuspend the cells from 500ml LB in 40ml cold Ca/glycerol buffer on ice.
  5. Incubate the cells on ice for 5~30min.
  6. Repeat step 3 and 4.
  7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The longer, the better)
  8. Collect cells by centrifugation, 6000rpm 10min 4°C
  9. Resuspend cells in 6ml Ca/glycerol buffer.
  10. Aliquote 150µl/tube. Freeze in liquid N2 and store at -80°C.

GeneJET PCR Purification Kit

#K0701, #K0702

Thermo SCIENTIFIC Product Information

PURIFICATION PROTOCOLS

Note

  • Read IMPORTANT NOTES on p.3 before starting.
  • All purification steps ahould be carried out at room temperature.
  • All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).

Protocol A. DNA purification using centrifuge

Step Procedure
1 Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
2
for DNA ≥500 bp 		Optional: if the DNA fragment is ≥ 500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
Note. If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
3 Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.
Note. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane.
Close the bag with GeneJET Purification Columns tightly after each use!
4 Add a 700 µL of Wash Buffer (diluted with the ethanol as described on p.3) to the GeneJET purification column. Centrifuge for 30-60 s.
Discard the flow-through and place the purification column back into the collection tube.
5 Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
Note. This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.
6 Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included).
Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.
Note.
  • For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended.
  • If DNA fragment is > 10 kb, prewarm Elution Buffer to 65 °C before applying to column.
  • If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.
7 Discard the GeneJET purification column and store the purified DNA at -20 °C.

GeneJET Plasmid Miniprep Kit

#K0502, #K0503

Thermo SCIENTIFIC Product Information

Growth of Bacterial Cultures

  • Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask with a volume of least 4 times the culture volume.
  • Havest the bacterial culture by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room temperature. Decant the supernetant and remove all remaining medium.
Do not overload the column:

For high-copy-number plasmids (see Table 1), do not process more than 5 mL of bacterial culture. If more than 5 mL of such a culture are processed, the GeneJET spin column capacity (20 μL of dsDNA) will be exceeded and no increase in plasmid yield will be obtained.
For low-copy-number plasmids (see Table 1), it may be necessary to process larger volumes of bacterial culture (up to 10 mL) to recover a sufficient quantity of DNA.

Table 1. Copy numbers of various vectors

High-copy
300-700 copies per cell 		Low-copy
10-50 copies per cell 		Very low-copy
up to 5 copies per cell
pUC vectors
pBluescript vectors
pGEM vectors
pTZ vectors
pJET vectors 		pBR322 and derivatives
pACYC and derivatives 		pSC101 and derivatives

PURIFICATION PROTOCOLS

Note

  • Read IMPORTANT NOTES on p.3 before starting.
  • All purification steps should be carried out at room temperature.
  • All centrifugations should be carried out in a table-top microcentrifuge at >12000 x g (10000-14000 rpm, depending on the rotor type).

Use 1-5 mL of E. coli culture in LB media for purification of higt-copy plasmids.
For low-copy plasmids use up to 10 mL of culture.

Protocol A. Plasmid DNA purification using centrifuges

Step Procedure
1 Resuspend the pelleted cells in 250 μL 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.
Note. Ensure RNase A has been added to the Resuspension Solution (as describe on p.3)
2 Add 250 μL of the Lysis Solution and mix thoroughly by inverting the tube 4-6 times until the solution becomes viscous and slightly clear.
Note. Do not vortex to avoid shearing of chromosomal DNA. Do not incubate for more than 5 min to avoid denaturation of supercoiled plasmid DNA.
3 Add 350 μL of the Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times.
Note. It is important to mix thoroughly and gently after the addition of the Neutralization Solution to avoid localized precipitation of bacterial cell debris.
The neutralized bacterial lysate should become cloudy.
4 Centrifuge for 5 min to pellet cell debris and chromosomal DNA.
5 Transfer the supernatant to the supplied GeneJET spin column by decanting or pipetting. Avoid disturbing or transferring the white prepipitate.
Note. Close the bag with GeneJET Spin Columns tightly after each use!
6 Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube.
Note. Do not add bleach to the flow-through, see p.8 for Safety Information.
7
for EndA+ strains only 		Optional: use this preliminary washing step only if EndA+ strains which have high level of nuclease activity are used.
Wash the GeneJET spin column by adding 500 μL of Wash Solution I (#R1611, diluted)
8 Add 500 μL of Wash Solution (diluted with ethanol prior to first use as described on p.3) to the GeneJET spin column. Centriifuge for 30-60 seconds and discard the flow-through. Place the column back into the same collection tube.
9 Repeat the wash procedure (step 8) using 500 μL of the Wash Solution.
10 Discard the flow-trough and centrifuge for an additional 1 min to remove residual Wash Solution. This step is essential to avoid residual ethanol in plasmid preps.
11 Transfer the GeneJET spin column into a fresh 1.5 mL microcentrifuge tube (not included). Add 50 μL 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.
Note. An additional elution step (optional) with Elution Buffer or water will recover residual DNA from the membrane and increase the overall yield by 10-20%
For elution of plasmids or cosmids >20 kb, prewarm Elution Buffer to 70 °C before applying to silica membrane.
12 Discard the column and store the purified plasmid DNA at -20 °C.

Glycerol stocks

AG Ignatova

from liquid culture

  • 500 μL (50% Glycerol, 50% H2O)
  • 500 μL Cells
  • Store at -80 °C

LB-Agar Plates (+ AB)

AG Ignatova

  1. Heat 250 mL LB-Agar
  2. Cool down to ca. 50°C
  3. Add 250 μL Chloramphenicol/ 12.5 μL Kanamycin (50 μg/mL)/ 25μL Ampicillin (80 μg/mL)
  4. Preparation of 20 plates under sterile conditions
  5. Store at 4 °C, mark as LB CAmp/Kan/Amp Plates iGEM, dd.mm.yyyy

Ligation

AG Ignatova

1 μL pSB-1C3 vector (23 ng)
1 μL/1.5 μL GroEl-Insert (3.5 ng) / miRNA-Insert (3.3 ng)
2 μL T4 Buffer
1 μL T4 Ligase
5 μL ddH20 (6 μL for control)

15 min at room temperature, then 10 min at 65 °C

Media

Tejas

Content

LB-Medium
M9Y Medium
M9 minimal medium

LB Medium

Yeast extrakt 5 g/L
Trypton 10 g/L
NaCl 10 g/L
RO-water add 1000 mL
pH 7.5

autoclave: 20 min 121 °C


M9Y Medium (with yeast extract)

Na2HPO4 6 g/L
KH2PO4 3 g/L
NaCl 0.5 g/L
NH4Cl 1 g/L
Yeast extract 2.5 g/L
RO-water ad 1000 mL
pH 7.4
autoclave: 20 min 121 °C

After autocalving add following substances:
1 M MgSO4 2 mL
20% Glucose 15 mL (end conc. 3 g/L)
0.1 M CaCl2 1 mL

Glucose and MgSO4 autoclave separately, CaCl2 to be steril filtered.


M9 minimal medium

M9 salts (part 1) 10x:
Na2HPO4 68 g
KH2PO4 30 g
NaCl 5 g (do not add for osmotic stress experiment)
NH4Cl 10 g
Add water to 1 L. Adjust pH=7.4. Autoclave
M9 (part 2) 10x:
MgSO4 10 mM
Glucose 30 g
Add water to 1 L. Filter sterilization.

CaCl2 100 mM stock solution. Filter sterilization

AA-Met mixture 250 mg/L each, pH=7.4. Filter sterilization


To use:

10x M9 salts (part 1) 1 mL
10x M9 (part 2) 1 mL
5x AA 2 mL for stress experiment don’t add!
CaCl2 100 mM 0.1 mL
Water up to 10 mL

PCR protocols

Zhang Gong

Content

  • PCR protocols
  • Fragment amplification with Herculase
  • Fragment amplification with Pfu
  • mutagenesis

Fragment amplification with Herculase

Template 1 μL
Primers (100˜200 μM) 1 μL each
10x Herc buffer 5 μL
Water 39.5 μL
dNTPs (10 mM each) 1.5 μL
Herculase 0.5˜1 μL
50 μL
95 °C 2 min
95 °C 30 sec 10 cycles
55 °C 45˜60 sec
72 °C 1 min/kb
95 °C 30 sec 20 cycles
55 °C 45˜60 sec
72 °C 1 min/kb + 10 sec/cycle
4 °C -

Fragment amplification with Pfu

Tamplate 1 μL
Primers (100˜200 μM) 1 μL each
10x Pfu buffer 5 μL
Water 39.5 μL
dNTPs (10 mM each) 1.5 μL
Pfu polymerase 1 μL
50 μL
95 °C 2 min
95 °C 30 sec 30 cycles
55 °C 45˜60 sec
72 °C 2 min/kb + 1 min
4 °C -

Mutagenesis

Tamplate (1:5 dilution) 1 μL
Primers (10 μM) 1 μL each
10x Pfu buffer 5 μL
Water 39.5 μL
dNTPs (10 mM each) 1.5 μL
Pfu polymerase 1 μL
50 μL
95 °C 2 min
95 °C 30 sec 18 cycles
55 °C 45˜60 sec
68 °C 2 min/kb + 1 min
4 °C -
DpnI 1 μl, 37 °C 1 h. Then transform 5 μL into DH5α.

Note:

  1. If you get too less colonies, you may think to increase the cycles to 20, but no more! Otherwise you might get mistakes in the final sequence.
  2. You can try both Pfu and Herculase to perform muatgenesis. Pfu will give less yield but more fidelity, so you should preferably use Pfu. If you cannot succeed with Pfu then you can consider trying Herculase. Just keep the same protocol!

Pick colony

AG Ignatova

  • Inoculate each picked colony into 5 mL of liquid LB-(CAmp/ respective antibioticum)-medium, respectively
  • Tubes in the 37 °C incubation room, on rotator!

Preparation for sequencing

AG Ignatova

  • Primer concentration: 100 μM → Concentration needed: 5 μM (dilute at 1:20)
  • take 50 nm of sample mix with 5 μL of (5 μM primer, only forward)

Restriction

AG Ignatova

20 μL pSB-1C3 (500 ng)
1 μL EcoRI
1 μL PstI
3 μL FD Buffer
5 μL ddH2O

20 minutes at 37 °C, following clean up with kit

SOC medium

Dagmar Stang

Bacto-Tryptone 2.000g 
Bacto-Hefe-Extract 0.500 g
NaCl 0.050 g
KCl 0.019 g
MgCl2 * (H2O)6 0.203 g (without (H2O)6 → 0.095 g)
Glucose 0.036 g
pH 7.0 with pH-Meter
→ 10 mL (fridge)

TB PIPES Buffer

AG Ignatova

for 100 mL:

KCl 1.865 g
CaCl x 2 H2O 0.220 g
0.5 M PIPES 2 mL
pH 6.7
MgCl2 x 2 H20 0.889 g
ddH2O → 100 mL

Transformation Efficiency 'Competent Cell Test Kit'

iGEM HQ

http://parts.igem.org/Help:Transformation_Efficiency_Kit

  1. spin down DNA (0.5, 5, 10, 20 & 50 pg/μL RFP Construct BBa_J04450, pSB1C3) from Competent Cell Test Kit (30 sec, 10.000 rpm)
  2. thaw competent cells on ice, label one 2 mL tube for each concentration + pre-chill
  3. 1 μL of DNA in each tube, respectively
  4. add 50 μL of competent cells to each tube, incubate for 30 min on ice
  5. pre-heat (42 °C), heat shock for 1 min
  6. incubate for 5 min on ice
  7. add 200 μL SOC media, incubate for 2 h at 37 °C
  8. pipet 20 μL on each plate (triplets), respectively
  9. incubate overnight at 37 °C
  10. count colonies, calculate transformation efficiency

Nanolab Protocols

Fabrication of Alginate Capsules

iGEM-Team TU Eindhoven

Buffers and solutions:

  • 100mM CaCl2/BaCl2 solution, 0.9% NaCl solution, 2.5% alginic acid solution

Protocol:

Preparation of 0.9% NaCl Solution

  1. Dissolve 0.9g NaCl in 100ml MiliQ water
  2. Mix/vortex so that all NaCl goes into solution
  3. Filter into a glass flask using a syringe with a 0.22µm filter for sterilization

Preparation of 2.5% Alginate Solution

  1. Dissolve 2.5g alginic acid slowly in 100ml NaCl solution. During the entire dissolving process a mixing magnet should mixing the solution carefully
  2. Autoclave the alginic acid solution at 121°C for 30min

Preparation of 100mM CaCl2/BaCl2 solution

  1. Dissolve 1.1098g of CaCl2/BaCl2 in 100ml MiliQ water
  2. Mix so that all CaCl2/BaCl2 goes into solution
  3. Filter into a glass flask using a syringe with 0.22µm filter for sterilization

Production of Alginate Beads

  1. Mix 1ml bacteria in 0.9% NaCl solution with 4ml alginic acid solution
  2. Put suspension in a sterile syringe and use it to produce droplets in 100ml of the 100mM CaCl2/BaCl2 solution or use the microfluidic device
  3. Put the alginate beads over a filter into a falcon tube for storage

Fabrication of a Chrome-Mask

xxxxxx

Protocol:
  1. Design structure with AutoCAD and save it as a dxf file
  2. Load the dxf file to the Heidelberg Laserwriter
  3. Clean chrome-mask with acetone in ultrasonic bath
  4. Spin-coat mask with photoresist S1813 at 4000rpm and 2000rpm acceleration for 1min
  5. Pre-bake at 90°C for 2min
  6. Adjust chrome-mask on the Laserwriter stage
  7. Turn the vacuum on with the white valves
  8. Convert the dxf file to a job data for the Laserwriter
  9. Select a proper filter and laser head
  10. Start exposure
  11. Develop mask in MF319 for 1min
  12. Chrome-etching for 1-3min
  13. Control the etching process with an optical microscope

Fabrication of PDMS-Devices

Martin Trebbin

  1. Put pour PDMS oligomer solution (SYLGARD 184) and 1/10 (of its weight) curing agent on the SU-8 master template inside an aluminum foil coated petri dish
    1. Stir well, remove big bubbles with a pipette tip an then expose inside a desiccator for 1-2h
    2. Cure in oven at 75°C for 2h
  2. Cutting
    1. Rough cut
    2. Fine cut
  3. Drill holes with a biops-cutter with a 0.75mm needle diameter
  4. Clean PDMS pieces with isopropanol
    1. Rinse isopropanol on the pieces, gently rub the surface with a finger
    2. Dry with air jet
    3. Leave the cleaned pieces in a fume hood to dry in ambient atmosphere
  5. Activate PDMS surface with the low pressure plasma chamber (13.56MHz, 10Watt)
    1. Power on
    2. Ventilation of the chamber
    3. Open hatch carefully and insert sample in the middle of the chamber
    4. Turn ventilation off
    5. Turn pump on
    6. Open gas valve
    7. Wait until pressure sets stable at 0.38mbar
    8. Set power up to 10Watt and time to 100s
    9. Press generate
    10. Turn the pump off and the ventilation on
    11. Open hatch and remove sample with gloves
  6. Attach the bottom/up PDMS pieces onto another
    1. Drop filtered Milipore water on the bottom
    2. Put the upper part (with the holes) on the bottom part
    3. Adjust the two pieces with the help of an optical microscope
  7. Attach the PDMS to glass
  8. Dry out oven at 40°C for 2h or overnight