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Protocols

Genetic Engineering

• In this section of our protocol pages you can read more about our methods for cloning and BioBrick assembly.
  • Genetic engineering is a basic tool of synthetic biology. With the help of standardized DNA building blocks (BioBricks) it is fairly easy to create new and modify existing natural systems. The methods we have used in our project to create BioBricks and to modify, mutate, transform and analyse DNA are presented in this section.

Whole Genome Isolation

• For the isolation following kit has been used: Fermentas GeneJET™ Plasmid Miniprep Kit
• Standard Protocol:
  • Centrifuge 10 mL of over-night liquid culture
  • Resuspend pellet in 800 µL of resuspension solution
  • Ribolyse three times for 60 s with 6500 rpm (30 s break between every run, 400 mg beads)
  • Centrifuge 3 min at 10,000 rpm
  • Transfer 500 µL of supernatant into new 2 mL tube
  • Add 500 µL of lysis buffer, invert 4 - 6 times
  • Add 700 µL of neutralisation buffer, invert 4 - 6 times
  • Centrifuge 10 min at 12,000 rpm
  • Transfer 2 x 600 µL of supernatant into spin column (centrifuge down each time for 30 s at 12,000 rpm)
  • Two wash steps with 750 µL of washing buffer
  • Dry column
  • Elute in 75 µL of elution buffer

Generating electrocompetent cells

• Material:
  • 550 mL LB-Medium
  • 1 L cooled bidest. H2O
  • 150 mL cooled 10 % glycerine
  • 10 pre-cooled 50 mL Falcons

• Protocol:
  • Inoculate 2x3 mL LB with bacterial stock; incubate over night at 37 °C and 200 rpm
  • Inoculate 2x250 mL LB with the over night cultures in 1-litre-flask at 37 °C and 140 rpm
  • Incubate until OD600 0,4-0,6
  • Cool the culture 15-30 minutes on ice
  • Onwards all steps at 4°C
  • Divide the cultures into cooled 50 mL Falcons and centrifugate at 4000 rpm, 4 °C for 15 minutes, make sure to slowly accelerate and deccelerate
  • Discard supernatant
  • Resuspend pellet in 5 mL cooled bidest H2O (and don't get frustrated while doing it, keep shaking gently)
  • Pool two suspensions each, add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
  • Discard supernatant
  • Resuspend pellet in 5 mL cooled bidest H2O
  • Add bidest H2O up to 50 mL and centrifugate again (see centrifugation above)
  • Discard supernatant
  • Resuspend pellet in 5 mL cooled 10 % glycerine
  • Transfer suspensions in two 50 mL Falcons and centrifugate again (see centrifugation above)
  • Discard supernatant
  • Add volume of 10 % glycerine that is approximately equal to the volume of the pellet and resuspend
  • Divide cells in 50 μL aliquots and freeze in liquid N2 immediately
  • Store at -80 °C

Transformation via electroporation

• Thaw 50 µL competent E.coli cells on ice, dilute with icecold 50 µL glycerine (10 %) if necessary
• Add 0.5-5 µL plasmid to 50 µl electrocompetent cells
• Store cells on ice for 1 minute
• Electroporate at U = 2.5 kV, C = 25 µF, R = 400 Ώ
• Transfer transformation reaction to 450 µL SOC-Medium and shake 1 h at 37 °C
• Centrifuge 2 min at 2000 rpm and plate on selective LB-Medium
• Incubate over night at 37 °C

Transformation of Single Step (KRX) Competent Cells by Promega

• Using [http://www.promega.com/~/media/Files/Resources/Protocols/Technical%20Bulletins/101/Single%20Step%20Competent%20Cells%20Protocol.ashx protocol E. coli KRX single step competent cells by Promega]

• Remove Single Step (KRX) Competent Cells from –70 °C, and place on ice for 5 minutes or until just thawed.
• Add 1–50 ng of DNA (in a volume not greater than 5 μL) to the Single Step (KRX) Competent Cells. Move the pipette tip through the cells while dispensing. Quickly flick the tube several times. Do not vortex!
• Immediately return the tubes to ice for 5–30 minutes
• Heat-shock cells for 15–20 seconds in a water bath at exactly 42 °C. Do not shake.
• Immediately place the tubes on ice for 2 minutes.
• Add 450 μL of room-temperature SOC-Medium to each transformation reaction, and incubate for 60 minutes at 37 °C with shaking (approximately 225 rpm). For best transformation efficiency, lay the tubes on their sides and tape them to the platform.
• For each transformation reaction, we recommend plating 100 μL of undiluted cells and 1:10 and 1:100 cell dilutions on antibiotic plates. Incubate the plates at 37 °C over night.

Standard BioBrick Assembly

• modified from [http://openwetware.org/wiki/Silver:_BB_Strategy Silver lab]:

• This assembly method can be used for BioBricks which are bigger than 150 bp. The BioBrick should be at least 500 bp bigger or smaller than the backbone. The BioBrick, which complies with these conditions, is used as the insert and is assembled into the prefix or suffix of the other used BioBrick, called vector. So you have to differentiate between a prefix and a suffix insertion.

Suffix Insertion

• Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x Tango buffer, 0.5 µL XbaI, 1 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
• Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL SpeI, 0.5 µL PstI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
• Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.

Prefix Insertion

• Digestion of insert: at least 700 ng DNA / 10 µL volume, 1 µL 10x BamHI buffer, 0.5 µL EcoRI, 0.5 µL SpeI. Digest for 2 h at 37 °C, afterwards inactivation for 20 min at 80 °C. Clean up the insert via gel electrophoresis. When cutting the insert out of the gel try to avoid staining or exposure to ultraviolet light of the insert.
• Digestion of vector about 700 ng DNA / 10 µL volume, 1 µL 10 x Tango buffer, 0.5 µL EcoRI, 0.5 µL XbaI. Digest for 2h at 37 °C, afterwards inactivation for 20 min at 80 °C. Add 1 µL SAP (shrimp alcaline phosphatase) and 1.2 µL 10 x SAP buffer, incubate for 1 h at 37 °C. Clean up the vector with a PCR clean-up kit.
• Ligation: after digestion and clean-up: 50 - 200 ng of vector, 3 - 10 fold molar access of insert, 20 µL ligation volume, 2 µL T4-Ligase-Buffer, 1 µL T4-Ligase. Incubate for 20 - 30 min at room temperature, afterwards inactivation for 5 min at 70 °C. Then: store at -20 °C or transform.

Variations

• A digestion over night is possible. If you digest over night use only 0.1 µL restriction enzyme.
• It is also possible to use PCR product as insert. Digest after PCR with corresponding restriction enzymes and clean up with PCR clean-up kit. This could lead to higher yields of insert DNA because a lot of DNA gets lost during the gel electrophoresis clean up.
• Sometimes some BioBricks are hard to assemble. Then you have to clean up the vector by gel electrophoresis as well.

Gibson assembly

• Modified from [http://www.nature.com/nmeth/journal/v6/n5/full/nmeth.1318.html Gibson et al. (2009)]

• This assembly method is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules. By coordinating the activity of a 5‘ exonuclease, a DNA polymerase and a DNA ligase two adjacent DNA fragments with complementary terminal sequence overlaps can be joined into a covalently sealed molecule, without the use of any restriction endonuclease.

Preparation of DNA molecules for in vitro recombination

• Generate the complementary sequence overlaps by PCR using the Phusion DNA-polymerase. If necessary add 5 M Betain in the reaction mix by reducing the amount of H₂O to decrease the number of false PCR products.
• Identify the PCR products of interest by gel electrophoresis with known DNA standards.
• Extract the PCR products from the gel by cutting out the DNA fragments and clean them up by using a commercial clean up kit.

In vitro recombination

• assembly mixture
  • • 320 µL 5x isothermal reaction buffer
    • 0.64 µL of 10 U mL^-1 T5 exonuclease (for DNA molecules overlapping by greater than 150 bp add 3.2 µL of 10 U ml^–1 T5 exonuclease)
    • 20 µL of 2 U mL^-1 Phusion DNA polymerase
    • 160 µL of 40 U mL^-1 taq DNA ligase
    • add ddH₂O water up to a final volume of 1.2 mL
    • aliquote 15 µL of the reagent-enzyme mix and store it at –20 ˚C
• Thaw 15 µL assembly mixture aliquot and keep it on ice until use.
• Add 5 µL of the purified DNA molecules in equimolar amounts (between 10 and 100 ng of each DNA fragment).
• Incubate the resulting mixture at 50 ˚C for 15 to 60 min, with 60 min being optimal.
• Transformation (heatshock or electroporation) without cleaning up the assembly product.

Restriction analysis

• Digest BioBrick of interest: about 400 ng DNA / 10 µL volume, 1 µL 10x orange buffer, 0.5 µL NotI or PstI. Digest for 2 h at 37 °C. NotI is used to determine the length of the BioBrick and the plasmid backbone, PstI ist used to determine the length of the BioBrick in the plasmid backbone.
• Gel electrophoresis: add 2 µL loading buffer to every digestion mix, apply about 100 - 200 ng DNA / pocket in gel. Don't forget to apply the uncut BioBrick as well. A good agarose concentration for BioBricks between 0.2 and 3 kb is 1.5 %. The smaller your BioBrick of interest is the higher the agarose concentration should be and vice versa. The gel electrophoresis is made with TAE-buffer. Be sure that you melt your agarose gel in the same buffer you use for the electrophoresis later.

Colony PCR

• Pick one colony with a sterile tip and elute it in 100 µL ddH₂0 or medium
• Store the colony in 4 °C while colony PCR is running
• One reaction mix contains:
  • 10 µL 5x buffer
  • 2 µL MgCl₂ (25 mM stock)
  • 1 µL dNTPs
  • 0.5 µL primer mix (prefix/suffix primers or sequencing primers)
  • 35.25 µL ddH₂O
  • 0.25 µL GoTaq polymerase (Promega)
  • 1 µL template
• PCR program:
  • Start: 3 min, 98 °C
  • 30 cycles of:
    • 30 s, 98 °C
    • 30 s, 55 °C
    • 30 s / 1 kb template, 72 °C
  • Finish: 5 min, 72 °C

• Gel electrophoresis: check the fragment size
• Plate the correct colony

Analytics

• Protocols for the analytical methods we used.
  • DNA and proteins are very small and cannot be seen by the naked eye. To control the success and the results of your upstream and downstream processes, analytical methods are necessary that give reliable results to make DNA or proteins in any way visible for you. The analytical methods we used in our project can be found in this section.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

This analytical method can be used for separation and identification of proteins according to their electrophoretic mobility. The mobility is a function of length of the molecular weight. Proteins that have identical charge per unit mass due to binding of SDS results in an equal electrophoretic mobility.

Pouring the polyacrylamide gel

• Make a master mix for the stacking and separating gel without adding ammonium persulfate and TEMED.
• Aliquote 6,5 mL for each separating and 2,5 mL for each stacking gel.
• Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form. Leave about 2 centimeters below the bottom of the comb for the stacking gel.
• Layer isopropanol on top of the gel.
• Leave the separating gel at room temperature for >60 minutes to polymerize.
• Remove isopropanol and wait until the surface is dry.
• Add ammonium persulfate and TEMED to each separating gel aliquote and pour the solution quickly into your gel casting form.
• Insert comb without getting bubbles stuck underneath
• Leave the gel at room temperature for >60 minutes to polymerize.

• For storage
  • Remove sealing and store the gel wrapped in moistened paper towel at 4°C.

Preparing the sample

• Mix your protein mixture 4:1 with Laemmli-buffer (30 mL protein solution + 10 mL Laemmli-buffer)
• Heat for 5 minutes at 95 °C.

Running the gel

• Remove sealing, put the polymerized gel into gel box and pour SDS running buffer into the negative and positive electrode chamber.
• Remove comp without destroying the gel pocket.
• Pipet the sample into the gel pockets, adjusting the volume according to the amount of protein in your sample. Make sure to include a lane with molecular weight standards (PageRuler Prestained Protein Ladder™ (Fa. Fermentas)) to determinate the molecular weight of your sample.
• Connect the power lead and run the stacking gel with 10 mA until the blue dye front enters the separating gel.
• Raise amperage up to 20 mA for running the separating gel.
• When the distance of the lowest molecular weight standard lane to the gel end is down to 0.5 cm stop the electrophoresis by turning off the power supply.

Polyacrylamide gel staining

Colloidal Coomassie Blue staining

Modified staining protocol from [http://http://newjournal.kcsnet.or.kr/main/j_search/j_abstract_view.htm?code=B021105&qpage=j_search&spage=b_bkcs&dpage=ar Kang et al., 2002].

• agitate the staining solution at 37 °C over night to form the colloids
• After finishing the SDS-PAGE remove gel from gel casting form and tranfer it in to a box.
• Add 100 mL of the stainig solution to your polyacrylamid gel.
• Incubate the gel in the solution at room temperature until the protein bands got an intensive blue color. Shake the gel continuously during incubation.
• Remove the staining solution
• wash the gel with dH₂O
• Incubate the gel in ddH₂O (2-6 h) for bleaching the background. Shake the gel continuously during incubation. If necessary replace the colored water with new one.

Tryptic digest of gel lanes for analysis with MALDI-TOF

Note:

• Make sure to work under a fume hood.
• Do not work with protective gloves to prevent contamination of your sample with platicizers.

Reaction tubes have to be cleaned with 60 % (v/v) CH₃CN, 0,1 % (v/v) TFA. Afterwards the solution has to be removed completely followed by evaporation of the tubes under a fume hood. Alternatively microtiter plates from Greiner® (REF 650161) can be used without washing.

• Cut out the protein lanes of a Coomassie-stained SDS-PAGE using a clean scalpel. Gel parts are transferred to the washed reaction tubes/microtiter plate. If necessary cut the parts to smaller slices.
• Gel slices should be washed two times. Therefore add 200 µL 30 % (v/v) acetonitrile in 0,1 M ammonium hydrogen carbonate each time and shake lightly for 10 minutes. Remove supernatant and discard to special waste.
• Dry gel slices at least 30 minutes in a Speedvac.
• Rehydrate gel slices in 15 µL Trypsin-solution followed by short centrifugation.
• Gel slices have to be incubated 30 minutes at room temperature, followed by incubation at 37 °C over night.
• Dry gel slices at least 30 minutes in a Speedvac.
• According to the size of the gel slice, add 5 - 20 µL 50 % (v/v) ACN / 0,1 % (v/v) TFA.
• Samples can be used for MALDI measurement or stored at -20 °C.

Trypsin-solution: 1 µL Trypsin + 14 µL 10 mM NH₄HCO₃

• Therefore solubilize lyophilized Trypsin in 200 µL of provided buffer and incubate for 15 minutes at 30 °C for activation. For further use it can be stored at -20 °C.

Preparation and Spotting for analysis of peptides on Bruker AnchorChips

• Spot 0,5 - 1 µL sample aliquot
• Add 1 µL HCCA matrix solution to the spotted sample aliquots. Pipet up and down approximately five times to obtain a sufficient mixing. Be careful not to contact the AnchorChip.

Note: Most of the sample solvent needs to be gone in order to achieve a sufficiently low water content. When the matrix solution is added to the previously spotted sample aliquot at a too high water content in the mixture, it will result in undesired crystallization of the matrix outside the anchor spot area.

• Dry the prepared spots at room temperature
• Spot external calibrants on the adjacent calibrant spot positions. Use the calibrant stock solution (Bruker’s “Peptide Calibration Standard II”, Part number #222570), add 125 µL of 0.1% TFA (v/v) in 30% ACN to the vial. Vortex and sonicate the vial.
• Mix the calibrant stock solution in a 1:200 ratio with HCCA matrix and deposit 1 µL of the mixture onto the calibrant spots.

NADH-Assay

• This method has been used for measurement of intracellular NADH concentration.

• Protocol:
  • Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
  • Centrifugate (5 min at 5000 g) of 6-10 mL overnight culture (OD 4-6), adjust volume between different samples for the approximation of the number of cells
  • Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer
  • Resuspend pellet in 1 mL PBS buffer
  • Cell disruption by Ribolysation (3 x 30 sec at 6500 rpm)
  • Centrifugation for 10 min at maximum speed
  • Store supernatant at - 20 ° C or direct measurement with Tecan Infinite® M200 platereader

• Tecan Infinite® M200 platereader parameters:
  • Sample volume = 100 μL clear supernatant
  • Excitation = 340 nm
  • Emission = 460 nm
  • Concentration calculation by NADH calibration curve

Hexadecan Assay

• This assay has been used to measure cell membrane hydrophobicity.

• Protocol
  • Inoculate an overnight culture (30 mL with 1 mL of pre-culture)
  • Centrifugate (5 min at 4000 g) of 2 mL overnight culture (OD 4-6)
  • Discard supernatant and wash pellet 3 times with 1 mL of PBS buffer
  • Resuspend pellet in 1 mL 0,9% NaCl and measurement of OD600
  • Add x μL of washed cells to 0,9% NaCl for a final volume of 3 mL . Final OD600 should be approximately 0,3 (denoted as A0, calculate exact value)
  • Add 3 mL Hexadecan and vortex for 60 sec
  • Incubation for 15 min
  • Discard the upper organic phase and measure OD600 of the aqueous phase (denoted as A)
  • Hydrophobicity can be calculated using the equation: affinity [%] = 100 x [1 – (A/A0)]

Cold osmotic shock

• Release of periplasmic protein fraction from E. coli by cold osmotic shock

• Modified protocol from Neu & Heppel (1965)
  • Centrifuge E. coli cell suspension for 5 min at 14,000 g (4 °C) to collect the cells
  • Discard the entire supernatant
  • Resuspend the cells in ice-cold cell fractionating buffer 1. The resulting volume should be 1/4 of the former suspension volume
  • Incubate for 20 min on ice. Invert the suspension at regular intervals to counteract sedimentation
  • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  • Discard the entire supernatant
  • Resuspend the cells in ice-cold cell fractionating buffer 2. The resulting volume should be 1/4 of the former suspension volume
  • Incubate for 10 up to 20 min on ice under regular invertion
  • Centrifuge the cell suspension for 15 min at 14,000 g (4 °C)
  • Save the supernatant, which contains the periplasmatic proteins and membrane proteins when using Cell fractionating buffer 2.2 and 2.3
  • If the periplasmatic protein fraction is turbid, re-centrifuge and filter it through a 0.2 µm filter

Bisphenol A analysis

LC-ESI-qTOF-MS(-MS)

HPLC method

• Column: C18 reverse phase column (Knauer [http://beta.knauer.net/products/column-detail-view/productdetail/vertex_plus_column_50_x_2_mm_blueorchid_175_18_c18-1.html Blue Orchid])
  • dimension: 50 x 2 mm
  • Pore size: 175 Å
  • Particle size: 1.8 µm
• Flow: 0.4 mL min^-1
• Column temperature: 30 °C
• Gradient:
  • 0 - 1.05 min: 45 % acetonitrile
  • 2.55 min: 95 % acetonitrile
  • 6.00 min: 95 % acetonitrile
  • 6.15 min: 45 % acetonitrile
  • 12.00 min: 45 % acetonitrile
• VWR Hitachi LaChrom ULTRA HPLC equipment
• Software: HyStar 3.2, HyStarPP, mircrOTOF Control

Ionization method

• Using Bruker Daltonics micrOTOF_Q
• ESI in negative mode
• Mass range: 50 - 1500 m/z
• End plate offset: - 500 V, 107 nA
• Capillary: 2500 V, 4 nA
• Nebulizer: 3 bar
• Dry gas: 8 L min^-1
• Quadrupole
  • Ion energy: 5 eV
  • Low mass: 100 m/z
• Collision energy: 10 eV
• Collision RF: 150 Vpp
• Transfer time: 70 µs
• Pre puls storage: 7 µs

MS-MS

• Isolated mass: 243.1 +/- 0.1
• Collision energy: 30 eV

Cultivation

Cultivation in liquid LB-Medium

Materials

• Chemicals, enzymes and kits we used in our lab work.
  • Chemical and biological reactions need defined conditions to work as expected. The chemicals, enzymes, kits, buffers and media we used in our project are listed in this section.

Used enzymes

Enzyme	Producer
AgeI	[http://www.fermentas.de/product_info.php?info=p247 Fermentas]
DpnI	[http://www.fermentas.de/product_info.php?info=p296 Fermentas]
EcoRI	[http://www.fermentas.de/product_info.php?info=p336 Fermentas]
GoTaq DNA-polymerase	[http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega]
KOD Hotstart DNA-polymerase	[http://www.merck-chemicals.com/germany/life-science-research/kod-hot-start-dna-polymerase/EMD_BIO-71086/p_iFCb.s1O874AAAEj2Bl9.zLX Novagen]
NgoMIV	[http://www.neb.com/nebecomm/products/productR0564.asp NEB]
OneTaq DNA-polymerase	[http://www.neb.com/nebecomm/products/productM0480.asp NEB]
Pfu DNA-polymerase	[http://www.promega.com/products/pcr/routine-pcr/pfu-dna-polymerase/ Promega]
PstI	[http://www.fermentas.de/product_info.php?info=p458 Fermentas]
Phusion HF DNA-polymerase	[http://www.finnzymes.com/pcr/phusion_high_fidelity_dna_polymerase.html Finnzymes]
Shrimp alcaline phosphatase	[http://www.fermentas.de/product_info.php?info=p592 Fermentas]
SpeI	[http://www.fermentas.de/product_info.php?info=p202 Fermentas]
T4-DNA-Ligase	[http://www.fermentas.de/product_info.php?info=p580 Fermentas]
T5 exonuclease	[http://www.neb.com/nebecomm/products/productM0363.asp NEB]
taq DNA Ligase	[http://www.neb.com/nebecomm/products/productM0208.asp NEB]
taq DNA-polymerase	[http://www.bioline.com/h_prod_detail.asp?itemid=219 Bioline]
XbaI	[http://www.fermentas.de/product_info.php?info=p247 Fermentas]

Used Kits

Function	Name
Molecular Cloning	[http://www.fermentas.de/product_info.php?info=p1620 Fermentas CloneJET™ PCR Cloning Kit]
Plasmid purification	[http://www.fermentas.de/product_info.php?info=p874 Fermentas GeneJET™ Plasmid Miniprep Kit]
Plasmid purification	[http://www.promega.com/b/de/minis/default.aspx?utm_source=Promega&utm_medium=Online&utm_campaign=Online_DEPureYield100Preps_Promega_HP_Banner Promega PureYield™ Plasmid Preps]
PCR Cleanup	[http://www.mn-net.com/tabid/10745/default.aspx Macherey Nagel NucleoSpin® Extract II]
PCR Cleanup	[http://www.promega.com/products/dna-and-rna-purification/dna-fragment-purification/wizard-sv-gel-and-pcr-clean_up-system/ Promega Wizard® SV Gel and PCR Clean-Up]
PCR core system	[http://www.promega.com/products/pcr/routine-pcr/gotaq-pcr-core-systems/ Promega GoTaq® PCR Core System I]

Media, buffer, solutions etc.

Chloramphenicol stock solution

• Solubilize 20 mg mL-1 Chloramphenicol in 100 % Ethanol
• Store at -20 °C

DNA loading buffer

• 50 % (v/v) glycerol
• 1 mM EDTA
• 0.1 % (w/v) bromphenol blue
• Solve in ddH2O

LB medium

• 10 g Trypton
• 5 g yeast extract
• 10 g NaCl
• 12 g Agar-Agar (for plates)
• Adjust pH to 7.4

M9 minimal medium

• For 1 L M9 mineral medium 867 mL sterile water is needed(for plates add 16 g Agar-Agar as well)
• Then add in the following order:
  • 00 µL 1 M CaCl2
  • 1 M CaCl2
  • 1 M CaCl2-H20
  • 14.70 g/100 mL

• 100ml 5x M9 salt solution
  • 10x M9 salt solution
    • Na2HPO4-2H2O 75.2 g/L
    • KH2PO4 30 g/L
    • NaCl 5 g/L
    • NH4Cl 5 g/L

• 20 % glucose
  • 20 % (w/v)glucose 200 g/L
  • For 500 mL stock solution add 100 g glucose to 440 mL water. Autoclave for 15 at 121°

• 1 M MgSO4 </p>
  • 1 M MgSO4-7H20 24.65 g/100 mL

• 1 mL Biotin (1 mg/mL)
  • Biotin (1 mg/mL) 50 mg/50 mL
  • For 50 mL stock solution dissolve 50 mg biotin in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.

• 1 mL Thiamin (1 mg/mL)
  • Thiamin-HCl (1 mg/mL) 50 mg/50 mL
  • For 50 mL stock solution dissolve 50 mg thiamin-HCl in 45 mL water. Add water to a final volume of 50 mL. Sterilize the solution over a 0.22-µm filter. Prepare 1 mL aliquots and store at -20°.

• 10 mL 100x trace elements solution
  • 100x trace elements solution
    • EDTA 5 g/L 13.4 mM
    • FeCl3-6H2O 0.83 g/L 3.1 mM
    • ZnCl2 84 mg/L 0.62 mM
    • CuCl2-2H2013 mg/L 76 µM
    • CoCl2-H2O 10 mg/L 42 µM
    • H3BO3 10 mg/L 162 µM
    • MnCl2-4H20 1.6 mg/L 8.1 µM
    • Dissolve 5g EDTA in 800 mL water and adjust the pH to 7.5 with NaOH. Then add the other components in the quantities mentioned below and add water to a final volume of 1L. Sterilize the solution over a 0.22µm filter</p>

• Unknown
  • 498 mg FeCl3(anhydrous)
  • 84 mg ZnCl2
  • 765 µL 0.1 M CuCl2-2H20 1.70 g/100 mL
  • 210 µL 0.2 M CoCl2-H2O 4.76 g/100 mL
  • 1.6 mL 0.1 M H3BO3 0.62 g/100 mL
  • 8.1 µL 1M MnCl2-4H20 19.8 g/100 mL

SOC medium

• Add the following components for 900 ml of distilled H2O:
  • 20 g Trypton
  • 5 g Bacto Yeast Extract
  • 2 mL of 5 M NaCl
  • 2.5 ml of 1 M KCl
  • 10 ml of 1 M MgCl2
  • 10 ml of 1 M MgSO4
  • 20 ml of 1 M glucose

Adjust to 1L with distilled H2O. Sterilize by autoclaving.

Biotin 500x Stock

• Dissolve 20 mg biotin in 100 mL of 0.05 M NaOH solution and filter sterilize
• Store at 4 °C
• Durable for one year

TAE-Buffer

• 1 L of 50x TAE buffer
  • 242.48 g Tris
  • 41.02 g Sodiumacetate
  • 18.612 g EDTA
  • Adjust pH to 7.8 with acetic acid
  • Solve in dH2O

• • Dilute 20 mL 50x stock in 1L dH2O for 1x Buffer for PAGE

Cell Fractioning Buffers

CFB 1 (pH 8)

• 0.2 M Tris
• 200 g L -1 sucrose
• 0.1 M EDTA

CFB 2.1 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4

CFB 2.2 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4
• 1% Trition
• 2% SDS

CFB 2.3 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4
• 1% Trition
• 0.2% SDS

CFB 2.4 (pH 8)

• 0.01 M Tris
• 0.005 M MgSO4
• 1% Trition
• 1% SDS

Contents 1 Genetic Engineering 1.1 Whole Genome Isolation 1.2 Generating electrocompetent cells 1.3 Transformation via electroporation 1.4 Transformation of Single Step (KRX) Competent Cells by Promega 1.5 Standard BioBrick Assembly 1.5.1 Suffix Insertion 1.5.2 Prefix Insertion 1.5.3 Variations 1.6 Gibson assembly 1.6.1 Preparation of DNA molecules for in vitro recombination 1.6.2 In vitro recombination 1.7 Restriction analysis 1.8 Colony PCR 2 Analytics 2.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 2.1.1 Pouring the polyacrylamide gel 2.1.2 Preparing the sample 2.1.3 Running the gel 2.2 Polyacrylamide gel staining 2.2.1 Colloidal Coomassie Blue staining 2.3 Tryptic digest of gel lanes for analysis with MALDI-TOF 2.4 Preparation and Spotting for analysis of peptides on Bruker AnchorChips 2.5 NADH-Assay 2.6 Hexadecan Assay 2.7 Cold osmotic shock 2.8 Bisphenol A analysis 2.8.1 LC-ESI-qTOF-MS(-MS) 3 Cultivation 3.1 Cultivation in liquid LB-Medium 4 Materials 4.1 Used enzymes 4.2 Used Kits 4.3 Media, buffer, solutions etc. 4.3.1 Chloramphenicol stock solution 4.3.2 DNA loading buffer 4.3.3 LB medium 4.3.4 M9 minimal medium 4.3.5 SOC medium 4.3.6 Biotin 500x Stock 4.3.7 TAE-Buffer 4.3.8 Cell Fractioning Buffers 4.3.8.1 CFB 1 (pH 8) 4.3.8.2 CFB 2.1 (pH 8) 4.3.8.3 CFB 2.2 (pH 8) 4.3.8.4 CFB 2.3 (pH 8) 4.3.8.5 CFB 2.4 (pH 8)