Team:Aachen/Contribution

Contribution

What M.A.R.S. passes on to the next generations

Making contributions for future iGEM teams rightly plays a crucial role in the iGEM competition as it enables upcoming scientists to spend more time on a fresh idea. They do not need to redo the work or the mistakes of previously perticipating iGEM teams.

We want to make our contribution by providing some unseful lab instructions.

Additionally, we put the sequence of the following parts we have used in our project on the iGEM parts registry:

• I1-60GFPHis - ATPase Inhibitor with GFP and His-Tag
• I1-60 - ATPase Inhibitor
• MBP-1 - Polystyrene-binding peptide
• Domain Z - Stiff spacer domain
• MBP-1 - LCI fusion protein - Connector between polystyrene coated magnetic particles and liposomes

Lab instructions

Practical advice for the work in the wet lab

Due to the corona pandemic, our actual laboratory work could only start in the beginning of June. Even though we had prepared ourselves as best as possible by doing research and looking for protocols, we had to face various challenges and problems. With the following definitions of terms, tips and laboratory instructions, we would like to ease the lab preparation of future teams, so that more time can be invested in what is really important: the project.

Click on the arrowheads to get practical lab tips for different activities.
The glossary will help you if a term is unknown to you.

Glossary ➤

Autoclaving

Sterilization of culture media and laboratory materials through thermal treatment in overpressure range, using gas-tight, lockable pressure vessels. The aim is not to guarantee the killing of germs, but to reduce the probability of contamination of the sterile goods by reducing the number of cells to 10^-6. Moist heat even enables bacterial spores to be killed by swelling, which reduces their resistance. The required time interval t is different depending on the germ.

Centrifugation

Separation process that uses the principle of inertia. The sedimentation speed depends on the relative centrifugal force, in most cases a multiple of the gravitational constant g.

Clean Bench

Laminar flow work cabinet or similar workspaces which provide filtered air across the work surface to protect against contamination. The clean bench protects the work on the work surface (e.g. when creating microorganism cultures), but not the user or the environment from aerosols created on the work surface.

Competence

Ability of cells to take up DNA that is freely present in the surrounding medium; it is therefore a prerequisite for the transformation of bacteria. Only certain types of bacteria show natural competence. Various treatment methods (e.g. using heat, chemicals or electricity) also enable artificial competence to be achieved.

Cryostock

Storage of cells or tissues by freezing, e.g. in liquid nitrogen. Due to the resulting cold rigor and the resulting greatly slowed down metabolic processes, it is possible to maintain the vitality of the cells/tissues almost indefinitely.

Culture

Scheduled artificial cultivation and reproduction of microorganisms and viruses as well as plant, animal and human cells or tissues under controlled conditions. Common culture processes and methods: pure culture, mixed culture, nutrient medium culture, liquid culture, stitch culture, enrichment culture, selective culture, fermentation.

Dialysis

Physical process for separation of low molecular weight substances from solutions of macromolecular/colloidal compounds through the exclusive diffusion of the low molecular weight substances through a semipermeable membrane by using a concentration gradient.

French Press

A cell suspension is compressed in a pressure vessel using a piston pump to a pressure of 69 to 138 MPa. By opening a needle valve with a small diameter, the suspension expands, creating shear forces that tear biomembranes. The strength of the shear forces can be adjusted via the pressure.

Generation time

Doubling time; time span in which the number of cells doubled in microorganisms under growth conditions. Approximate guide values under optimal conditions:

• Bacteria: 15-60 min
• unicellular Eukaryotes (e.g. yeasts): 90-120 min

Innoculation

Targeted transfer of viable cells/microorganisms (inoculum, see Preculture) in a larger nutrient medium or an organism for the purpose of reproduction.

Insert

DNA fragment that was ligated into cloning vector.

Lysis

Cell disruption (or homogenization) through (active) damage or dissolution of the outer cell membrane using mechanical (e.g. French Press, Ultrasonic, Vortex, mortar) or chemical (e.g. hypotonic buffers, Lysozyme and Triton X-100) methods or means. The result is called the lysate.

Medium

Nutrient or culture media are used for the controlled cultivation of microorganisms, cells, tissues or small plants with a certain supply of nutrients. A distinction is made between liquid and gelled (“solid”) media. With the addition of antibiotics or special substrates, additional selection can be made.

Molecular Cloning

Methods for obtaining and identical amplification of DNA by integrating the desired DNA fragment into a vector (e.g. a plasmid). The use of various organisms (e.g. Escherichia coli, algae, fungi) as hosts enables a cost-effective and precise alternative to in vitro methods (e.g. PCR), which also enables the production of large quantities.

Functional cloning identifies similar DNA sequences, positional cloning identifies neighbouring DNA sequences.

pH meter

Measuring device for determining and displaying the pH value of a solution. The determination takes place via electrochemical means, in the lab often with the help of a glass electrode.

Plasmid

Small, usually circular, autonomously replicating, double-stranded DNA molecules that do not belong to actual chromosome and are found in bacteria and archaea (rare in eukaryotes). The genes contained on plasmids can provide a selection advantage (e.g. antibiotic resistance). Each plasmid contains at least one sequence that serves as the origin of replication (ORI) and must be compatible with the strain of microorganism to enable replication of the plasmid. Different types of plasmids can coexist in one and the same cell (number depends on the organism in question). Due to their properties, they can be used as vectors to duplicate or express genes. The specific adaptation of the plasmid as a vector is known as vector design.

Plating out

Dilution technique, which is used to grow single, separate colonies on solid media, e.g. agar plates. Single colonies can for example be used to determine macroscopic properties, the number of cells in a suspension or to produce clones.

Preculture

Process for production of inoculation material (inoculum) as preparation for cell reproduction on a large scale by means of stepwise enlargement in standing or shaking cultures.

Transformation

In addition to transduction and conjugation, transformation is one of three options for gene transfer in prokaryotes. Free DNA is taken up by naturally or artificially competent cells. It is used as a substep in molecular cloning after the vector insert construct has been designed. In this way, the desired DNA section (the insert) can be duplicated very frequently and inexpensively.

General tips for (first) time in the lab ➤

• Get a lab book and immediately document everything you do.
• Schedule a weekly meeting for the lab team to review the work done and the work that is to be done – talk to each other!
• Find a common sharing platform (e.g. Microsoft Teams).
• As soon as they are established, write down your experiments directly in the form of a protocol and update them regularly if necessary.
• Check early before the start of an experiment whether all the necessary materials are available so that you can obtain/order them on time.
• Seriously: talk to each other! About everything – what you do, what you want to do, what is there, what is missing...
• Ask for help if you are not sure about something.
• Always wear a lab coat, and additionally gloves and safety goggles, if needed.
• Wash your hands (with soap) before and after work.
• Label everything you have (bottles with media, tubes, agar plates, etc.) with at least: name of (chemical) content, cell culture, date, (group) name.
• Inform yourself about the dangers and risks associated with the chemicals you use. This also includes their disposal.
• Always have a “waste beaker” ready at the workplace for used media, liquid cultures that are no longer required, residual chemicals, etc.
• Prepare a negative control to exclude contamination of media or materials (e.g. when preparing precultures).
• Culture vessels should be kept closed as far as possible.
• Spilled cultures must be removed immediately, and the area needs to be disinfected (70% ethanol, bacillol for smaller areas).
• Liquids and materials that have come into contact with genetically modified organisms (GMO’s) must be collected separately and autoclaved.
• Always leave your workplace tidy and clean.
• Adhere to your local biosafety regulations

Formulas you will need ➤

Calculation of the amount of substance (n), mass (m) or molar mass (M): $$ n =\frac{m}{M} \; \widehat{=} \; m = n \cdot M \; \widehat{=} \; M = \frac{m}{n}$$ Calculation of the concentration (c), the amount of substance (n) or the volume (V): $$ c =\frac{n}{V} \; \widehat{=} \; n = c \cdot V \; \widehat{=} \; V = \frac{n}{c} $$ Calculation of density (ρ), mass (m) or volume (V): $$ \rho =\frac{m}{V} \; \widehat{=} \; m = \rho \cdot V \; \widehat{=} \; V = \frac{m}{\rho} $$ Calculating the ratios of two components for a mixture: $$ c_1 \cdot V_1 = c_2 \cdot V_2 $$ Substance proportion (x_i): $$ x_i = \frac{n_i}{n} $$ Mass fraction (w_i): $$ w_i = \frac{m_i}{m} $$

Clean Bench ➤

Tips

• Organize your setup to be able to reach everything without touching too much, e.g. with the sleeve of your lab coat.
• Make sure that pipetted solution doesn’t touch the tip of the micropipette while pipetting.
• If the tip touches anything except your substance (media, culture, etc.) and the sterile inside of your vessel, discard it.

Prepare sterile workingplace

• Disinfection working surface with surface disinfectant.
• Prepare a waste bag.
• Have all the materials you need for your work ready, e.g. micropipettes, sterile tips, media, tubes, beakers, petri dishes, etc.
• Sterilize them by surface disinfection when placing in Clean Bench.
• Have paper towels within reach.
• To reduce contamination, disinfect the tip of your micropipette.
• Wait about 15 min before actually starting work at Clean Bench, so everything inside gets decontaminated and sterilized by laminar flow.
• Label everything while waiting.
• Disinfect your working surface again when starting with work.
• Disinfect gloves.
• When you are done, clean up the working space and disinfect the working surface.

Pipetting techniques ➤

• for usage of modern pipettes, like micropipettes
• tips are normally for single usage

Tips and tricks

• best results by using tips offered/recommended by manufacturer of pipette
• when aspirating the medium, hold pipette in as vertical position as possible
  → volume deviations can occure as angle of inclination increases
• pre-wetting the tip improves pipetting accuracy in all techniques by rising the humidity in tip
  → causes less evaporation
• stripping of droplets adhering to tip avoids transfer of additional liquid
• stack two tips on top of each other to make it longer and reach even into deep vessels

Forward Pipetting

1	2	3
Press button up to 1st stop. Immerse tip a few millimeters into liquid.	Release button slowly. Tip fills up.	Dispense liquid by pressing up to 1st stop. Then blow out rest by pressing up to 2nd stop.

Starting position	1	2	3	4
1st stop	⬇	⬆	⬇
2nd stop			⬇	⬆

	Forward Pipetting	Reverse Pipetting
Notes	most used, less concentration needed
For what?	standard solutions, e.g. water, buffer, diluted saline solutions/acids/alkalis, etc.	viscous liquids, e.g. glycerin, solutions with high vapor pressure (e.g. hot water)
How?	suction of liquid without using overstroke, subsequent delivery with overstroke	suction of liquid using overstroke, subsequent delivery without overstroke → rest of liquid remains in tip and is discarded!

Reverse Pipetting

1	2	3
Press button up to 2nd stop. Immerse tip a few millimeters into liquid.	Release button slowly. Tip fills up.	Dispense liquid by pressing up to 1st stop. Rest of liquid remains in tip.

Starting position	1	2	3	4
1st stop	⬇		⬇	⬆
2nd stop		⬆

Autoclaving ➤

Tips

• Only fill containers about 2/3 full with liquid as they can boil/foam.
• Leave lids half-closed on bottles to avoid excessive pressure build-up.
• Fresh autoclaved items are always hot! Only touch with heat resistant gloves.
• Wait with releasing the pressure in order to prevent delayed boiling

Medium ➤

Tips

• Avoid condensation on the surface of culture media.
• Best leave a stirring rod in bottles with media. While autoclaving, it will mix the solution. After autoclaving, you don’t need to open the bottle again to stir it properly.
• Media with agar should be poured promptly into petri dishes or else.
• Stir or shake it again before you pour it.
• If you don’t have the time to pour it immediately, you can store media with agar for about a day at 70 – 80°C.
• If you add agar later to an already mixed medium, you need to autoclave it again.

Culture ➤

Tips

• Use the clean bench as you workspace
• Make sure your medium covers the needs of your culture

Overnight Pre-cultures ➤

Escherichia coli

work space
clean bench

Material

• LB-medium
• corresponding antibiotics (concentration: )
• agar plate with strain
• sterile (glas) test tube
• (Micro-)Pipette
• (toothpick)

Conservation
37°C for 16-18h, best shaking

Procedure

1. pipette 5 mL LB-medium into test tube
2. add 5 μL of antibiotics
3. pick one colony of agar plate with toothpick/tip of pipette and throw it in test tube
4. leave it shaking at 37° C over night

Saccharomyces cerevisiae

work space
clean bench

Material

• YEP-medium
• corresponding antibiotics (concentration: )
• agar plate with strain
• sterile (glas) test tube
• (Micro-)Pipette
• (toothpick)

Conservation
28° C for 1-2 days, best shaking

Procedure

1. pipette 5 mL YEB-medium into test tube
2. add 5 μL of antibiotics
3. pick one colony of agar plate with toothpick/tip of pipette and throw it in test tube
4. leave it shaking at 28° C over 1-2 nights

Plating out ➤

Tips

• Glow or flame inoculation loops and needles to sterilize them before and after work.
• Let them cool to avoid destroying organisms by burning.
• For quick cooling, press it onto the agar in a free space on the edge.
• Use a maximum of 0.2 mL per agar plate for plating out a suspension with a Drigalski Spatula.

Fractionated Smear ➤

work space:

• clean bench

Materials

• Cell culture
• Agar plates
• Bunsen burner
• Inoculation loop

Procedure

1. Prepare your workspace on the Clean Bench.
2. Mark four quadrants on the bottom of the petri dish(es) (from the outside).
3. Glow out the inoculation loop and let it cool.
4. Briefly dip the inoculation loop in the culture solution to obtain cells.
5. Rub it in a snake pattern in the first quadrant. Just slide gently over the agar surface so as not to destroy it.
6. Glow out the inoculation loop and let it cool.
7. Pull it through the first quadrant and rub the collected cell material in a snake pattern gently in the second quadrant.
8. Repeat 6 and 7 for the third and fourth quadrant by pulling the inoculation loop through the second/third quadrant.
9. Glow out the inoculation loop and let it cool.
10. Incubate them.

Cell harvest ➤

Material

• Cell culture
• 50 mL falcon tube
• Buffer or medium
• Waste beaker

Procedure

1. Aliquot cell culture to 50 mL falcon tubes.
2. Set centrifuge to 4°C.
3. Centrifuge at 4°C, 4000 rpm for 10 min.
4. Decant supernatant carefully.
5. Resuspend pellet in 2 mL buffer or medium.
6. Combine all resuspended pellets in one falcon.
7. Centrifuge again at 4°C, 4000 rpm for 10 min.
8. Carefully decant supernatant.
9. Store pellet at -20°C or further use immediately.

Centrifugation ➤

Tips

• Due to the different types of centrifuges and centrifugation methods, a briefing and extensive examination of the necessary properties is essential for the success of an experiment.
• Even with small benchtop centrifuges, make sure that the rotor loaded with the samples is balanced in that opposing tubes have identical weights. The bigger and faster the centrifuge, the more important is the balancing.
• If the number of sample tubes is odd, use a tube with water with the same weight as its counterpart.
• Some centrifuges tend to get hot after a while, so it’s helpful to turn on cooling while centrifuging, even if your sample doesn’t need to be kept cool.

Lysis ➤

Tips

• Larger pieces of tissue should first be crushed in the mixer.
• Physical processes often lead to an increase in temperature – control and possible cooling is necessary.
• Chemical processes are usually gentler than mechanical ones.

French Press ➤

Tips

• First, ultrasonicate the cells so that there are no lumps in the cell solution.
• Always store your lysate in ice.

Material

• Ethanol
• Cell suspension
• Double distillate water

Procedure

1. Push start and let the ethanol out of the cabin.
2. Wash French Press three times with ethanol and three times with water. While washing with water, close the cabin and put the pressure tube on top of the cabin. Be careful because of the pressure.
3. Make a system pressure test. Therefore, increase the air pressure to a maximum of 40 bar with upregulating the air regulator. Decrease the air regulator again to zero. Do this three times. There should be liquid in the cabin and tube.
4. Let the water out and check if there is no bubble in the tube.
5. Load the cabin with your cells before all the water got out. Your cell suspension should not contain any solid particles! Avoid any bubble formation.
6. Increase the pressure until 1000 and 1100 bar and refill the cabin with your sample to “recycle” them. Repeat only a few times because of the heat formation.
7. Collect your sample and save it on ice until you start the next cycle.
8. After finishing the cycles, decrease the pressure to zero and wash the system.
9. For washing, fill in water and wash after that again with water but put on the high pressure to clean the system properly. Avoid bubble formation.
10. Repeat step 3 and 4.
11. Wash the cabin three times with ethanol and pressure on like in step 3.
12. As a last step fill the cabin again with ethanol and leave it like this for the next person.

Ultrasonic Lysis ➤

Tips

• If you have to enter the room during ultrasonication, always wear hearing protection.
• It is best to always remember to cool the sample with ice – especially with thermosensitive proteins.
• If the supernatant is yellowish, the sonication was probably too strong. Lysis presumably took place, but the proteins were also destroyed.

Material

• Sonotrode (for needed volume)
• Tube with cell culture
• Beaker with ice

Procedure

1. Settings according manufacturer and protocol.
2. Fix sonotrode with clamp so that horizontal movements are no longer possible.
3. Prepare the tube with cell culture in the middle of the beaker with ice.
4. Place the sonotrode straight into the cell culture so that it’s still a thumb’s width away from the bottom of the vessel. The sonotrode must not touch the vessel wall, otherwise it could melt.
5. Start the ultrasonic and leave the room.
6. When the ultrasonication is finished, clean the sonotrode – preferably with a surface disinfectant that does not grease.

pH meter ➤

In general

• Electrode is always stored in KCl solution (to leave diaphragm potential neutral and conductive) and should never get dry!
• Always control if pH meter measures correct pH of KCl solution before usage. If not: exchange solution.
• pH electrode: lever to open and close à open to measure
• Always rinse electrode with distilled water before measuring another solution/storing it again in KCl.
• If you think you rinsed enough – rinse a little bit more 😉. It provides contamination of solutions.
• Best always calibrate pH meter before measuring actual solution.
• Always stir liquid while measuring/adjusting it.
• Never hit electrode with stirring rod!
• Always wait a few seconds until the pH reading is constant.

Measuring

1. Take electrode out of KCl solution and rinse it with distilled water (into waste beaker).
2. Carefully remove the last drop on the tip with paper towel.
3. Attach electrode to setup by fixing in needed position with clamp.
4. Tip of electrode with diaphragm has to be submerged in liquid.
5. Calibration with solutions in acidic, neutral and basic range (e.g. pH 4, 7 and 9):
   • Combination of calibration solutions depends on pH you want to measure/you want to adjust to.
   • Calibrate with solutions that enclose the desired pH (e.g.: solution to be measured: 7,5 – calibration with 7 and 9 (because 7,5 is in between)).
   • Always stir solutions before measuring.
   • Always start with ph 7, rinse electrode and calibrate the other value.
   • If needed, adjust display at pH meter as specified by manufacturer.
6. Rinse electrode off.
7. Measure pH value of solution.
8. If needed, adjust pH with acid or base.
9. Rinse the electrode off when you’re done.
10. Store in KCl solution.

Dialysis ➤

Tips

• The larger the surface area of the membrane in relation to the liquid to be dialyzed, the faster the dialysis will proceed.
• A continuous or at least frequent exchange of the solvent increases the dialysis speed by maintaining the concentration gradient.

Cryostock ➤

Storage of cells or tissues by freezing, e.g. in liquid nitrogen. Due to the resulting cold rigor and the resulting greatly slowed down metabolic processes, it is possible to maintain the vitality of the cells/tissues almost indefinitely.

Materials

• Cell Culture
• 70% glycerol
• Tube with screw cap
• Micropipette + tips
• (liquid nitrogen)

Procedure

1. Mix 500 µL of culture with 500 µL of 70% glycerol in a tube with screw cap by gently pipetting up and down.
2. Freeze in liquid nitrogen or store directly at -80°C.

Inoculation ➤

Tips

• Workplace: Clean Bench
• Always use fresh, sufficiently large inoculum with good cell density (to reach phase of exponential growth).
• The nutrient medium should always be fresh and contain all the necessary growth substances.
• Pay attention to optimal environmental conditions (e.g. temperature, ventilation, light, etc.).

Parts

Everything is made from its parts

During our project, we designed several parts that we wanted to express and use in the wet lab. These parts could not be found in the parts registry. Thus, we decided to make them available for future iGEM Teams in the iGEM parts registry to retrieve the sequence if they need the proteins coded by these parts.

Name	Type	Short Description	Designer	Length
BBa_K3456000	Composite	I1-60GFPHis - ATPase Inhibitor with GFP and His-Tag	Lina Schmidt	992 bp
BBa_K3456001	Coding	I1-60GFPHis	Lina Schmidt	183 bp
BBa_K3456002	Coding	MBP-1	Lina Schmidt	105 bp
BBa_K3456003	Coding	Domain Z	Lina Schmidt	174 bp
BBa_K3456004	Composite	MBP-1 - LCI fusion protein	Lina Schmidt	423 bp

I1-60GFPHis

ATPase Inhibitor with GFP and His-Tag

The first of these parts is BBa_K3456000. It is a composite part coding for the first 60 residues of the bovine ATPase inhibitor IF1 followed by an eGFP- and a His-Tag. By binding to the ATPase, it inhibits the hydrolytic function of the ATPase but not the ATP synthesis. For this purpose, only the first 60 amino acid residues of this protein are needed. The whole part can be used to purify an ATPase with an affinity purification via the His-Tag. Its expression is regulated by a Lac-Operon consisting of LacI, LacO and a T7 promoter and terminator.

I1-60

ATPase Inhibitor

The second part, whose sequence has been contributed to the part registry, is the basic coding part BBa_K3456001. The sequence of the eGFP- and the His-Tag have already been entered in the parts registry. We only needed to add the sequence coding for the first 60 residues of IF1 in order to be able to create the composite part BBa_K3456000. These first 60 residues of IF1 have been uploaded as the basic coding part BBa_K3456001.

MBP-1

Polystyrene-binding peptide

The third sequence we contributed to the parts registry (BBa_K3456002) codes for MBP-1, which is an antimicrobial peptide originating from Zea mays (Maize). It can bind to polystyrene-coated magnetic particles. Thus, we planned to use it as an anchor peptide that connects the polystyrene beads to our linker peptide. We used this part in the composite part BBa_K3456004.

Domain Z

Stiff spacer domain

The fourth part whose sequence we entered in the parts registry codes for the stiff spacer Domain Z. Its part name is BBa_K3456003. We used this domain to connect the anchor peptides MBP1 and LCI. Thus, it is also a basic part for the composite part BBa_K3456004 coding for the MBP-1 - LCI fusion protein.

MBP-1 - LCI fusion protein

Connector between polystyrene coated magnetic particles and liposomes

The composite part BBa_K3456004 is the fifth and last part whose sequence we contributed to the parts registry. It codes for the MBP-1 - LCI fusion protein, which can connect polystyrene-coated magnetic particles and liposomes consisting of 16:0-18:1 PC (POPC)(1-palmitoyl-2-oleoyl-glycero-3-phosphocholine). It consists of the anchor peptide MBP-1, the stiff spacer Domain Z and the anchor peptide LCI. We already found the sequence of the LCI peptide in the parts registry. The sequences of MBP1 and the spacer Domain Z have been contributed by us.