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| | </ul></html> | | </ul></html> |
| - |
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| - | {{Team:Aachen/BlockSeparator}}
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| - |
| |
| - | = 2D detection of IPTG and HSL =
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| - |
| |
| - | == Chip production ==
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| - |
| |
| - | === Cell preparation ===
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| - |
| |
| - | # over night culture of sensor cells (50 mL in a 250 mL flask with) max. 16 h
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| - | # centrifuge all 50 mL by 3000 g for 10 min at RT (21 °C).
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| - | # discard the supernatant
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| - | # re-suspend the pellet in 1 mL tempered (~21 °C) LB-medium .
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| - |
| |
| - |
| |
| - | === Agar preparation ===
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| - |
| |
| - | # autoclave 50 mL medium with 1.5 % (w/v) agarose (has to be multiplied with the number of chips prepared).
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| - | # cool it down to 45 °C in a water bath.
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| - |
| |
| - |
| |
| - | === Chip preparation ===
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| - |
| |
| - | # mix the cooled medium with the cells by inverting gently.
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| - | # pour it in the chip form, avoiding bubble formation (!).
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| - | # wait for approximately 20 min until the agar has solidified.
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| - | # cut out the chips with a scalpel.
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| - | # put two chips into a labeled petri dish and store additional 4 chips in labeled petri dishs in the refrigerator.
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| - | # incubate two chips for 1 h at 37 °C prior to induction.
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| - |
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| - |
| |
| - | <center>
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| - | {{Team:Aachen/Figure|Aachen 14-10-09 flowsheet chip manufacturingV8 ipo.png|title=Sensor-chip manufacturing|subtitle=XXX|width=900px}}
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| - | </center>
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| - |
| |
| - | == Measurement of fluorescence ==
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| - |
| |
| - | {{Team:Aachen/BlockSeparator}}
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| - |
| |
| - | = Culture medium and culture conditions =
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| - | == Media ==
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| - | === LB medium ===
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| - | # weight components
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| - | #: '''5 g/L NaCl'''
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| - | #: '''10 g/L tryptone'''
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| - | #: '''5 g/L yeast extract'''
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| - | #: (15 g/L agar for plates)
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| - | # fill up to 1 L with deionized water
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| - | # '''mix well''' by shaking
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| - | # autoclave
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| - | ## autoclaving tape, caps slightly unscrewed
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| - | ## base of the pot has to be covered with deionized water
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| - | ## close lid
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| - | ## heat '''level 3 until the pressure valve opens'''
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| - | ## reduce '''heat level to 1.5'''
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| - | ## set timer to '''20 minutes'''
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| - | ## turn heater off
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| - | ## '''wait until the pressure valve retracts''' (30-45 minutes)
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| - | ## open, close caps & shake
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| - | # for plates, wait until you can touch the bottle ('''<60 °C''', clean bench!)
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| - | # add antibiotics (1 µL/mL) and '''shake''' (gloves!)
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| - |
| |
| - |
| |
| - | === TB medium ===
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| - | # components 1:
| |
| - | #: '''4 mL/L glycerol'''
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| - | #: '''12 g/L tryptone'''
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| - | #: '''24 g/L yeast extract'''
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| - | # fill up to 900 mL with deionized water
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| - | # '''mix well''' by shaking
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| - | # autoclave
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| - | # components 2:
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| - | #: '''0.17 M KH<sub>2</sub>PO<sub>4</sub>'''
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| - | #: '''0.72 M K<sub>2</sub>HPO<sub>4</sub>'''
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| - | # dissolve in 100 mL deionized water and sterilize it by passing it through a filter
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| - | # after autoclaving and cooling down, add sterile phosphate solutions
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| - |
| |
| - |
| |
| - | === Hartmans minimal medium (HM) ===
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| - |
| |
| - | === M9 minimal medium (M9) ===
| |
| - | <center>
| |
| - | {| class="wikitable centered"
| |
| - | ! '''Components for 1 L''' !! '''Volume'''
| |
| - | |-
| |
| - | | bidest. water || style="text-align:right"| 778.667 mL
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| - | |-
| |
| - | | 10x Salt solution || style="text-align:right"| 100 mL
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| - | |-
| |
| - | | Magnesiumsulfatehaptahydrate (10 mM) || style="text-align:right"| 100 mL
| |
| - | |-
| |
| - | | Glucose 20% (w/v) || style="text-align:right"| 20 mL
| |
| - | |-
| |
| - | | 1000x Trace elements || style="text-align:right"| 1 mL
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| - | |-
| |
| - | | Thiamin (1 mM) || style="text-align:right"| 0.333 mL
| |
| - | |}
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| - |
| |
| - |
| |
| - | {| class="wikitable centered"
| |
| - | | colspan="3"| '''10x Salt solution'''
| |
| - | |-
| |
| - | ! '''Component''' !!''Final concentration''' !! '''Concentration in stock solution'''
| |
| - | |-
| |
| - | | BisTris || style="text-align:right"| 95 mM || style="text-align:right"| 200,000 mg/L
| |
| - | |-
| |
| - | | Ammmonium chloride || style="text-align:right"| 60 mM || style="text-align:right"| 32,100 mg/L
| |
| - | |-
| |
| - | | Sodium citrate || style="text-align:right"| 12.5 mM || style="text-align:right"| 27,000 mg/L
| |
| - | |-
| |
| - | | Monopotassium phosphate || style="text-align:right"| 3 mM || style="text-align:right"| 4,170 mg/L
| |
| - | |-
| |
| - | | Dipotassium phosphate || style="text-align:right"| 0.7 mM || style="text-align:right"| 1,590 mg/L
| |
| - | |-
| |
| - | |}
| |
| - |
| |
| - | {| class="wikitable centered"
| |
| - | | colspan="3"| '''1000x Trace elements'''
| |
| - | |-
| |
| - | ! '''Component'''!!'''Final concentration''' !!'''Concentration in stock solution'''
| |
| - | |-
| |
| - | | Iron(III) chloride || style="text-align:right"| 50 mM || style="text-align:right"| 13,515 mg/L
| |
| - | |-
| |
| - | | Calcium chloride || style="text-align:right"| 20 mM || style="text-align:right"| 2,220 mg/L
| |
| - | |-
| |
| - | | Manganese(II) chloride || style="text-align:right"| 10 mM || style="text-align:right"| 1,258 mg/L
| |
| - | |-
| |
| - | | Zinc sulfate || style="text-align:right"| 10 mM || style="text-align:right"| 1,615 mg/L
| |
| - | |-
| |
| - | | Cobalt(II) chloride || style="text-align:right"| 2 mM || style="text-align:right"| 260 mg/L
| |
| - | |-
| |
| - | | Copper(II) chloride || style="text-align:right"| 2 mM || style="text-align:right"| 269 mg/L
| |
| - | |-
| |
| - | | Nickel(II) chloride || style="text-align:right"| 2 mM || style="text-align:right"| 259 mg/L
| |
| - | |-
| |
| - | | Sodium molybdate || style="text-align:right"| 2 mM || style="text-align:right"| 412 mg/L
| |
| - | |-
| |
| - | | Sodium selenite || style="text-align:right"| 2 mM || style="text-align:right"| 346 mg/L
| |
| - | |-
| |
| - | | Boric acid || style="text-align:right"| 2 mM || style="text-align:right"| 124 mg/L
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| - | |-
| |
| - | | Hydrochloric acid || style="text-align:right"| 1 mM || style="text-align:right"| 20 mL
| |
| - | |-
| |
| - | |}
| |
| - | </center>
| |
| - |
| |
| - |
| |
| - | === SOC ===
| |
| - | # components
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| - | #: '''0,5 % yeast extract'''
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| - | #: '''2 % tryptone'''
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| - | #: '''10 mM NaCl'''
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| - | #: '''2.5 mM KCl'''
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| - | #: '''20 mM MgSO<sub>4</sub> '''
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| - | # fill up with deionized water
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| - | # adjust to pH 7.5 with NaOH
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| - | # after autoclaving, add 20 mM sterile glucose solution (filter sterilization)
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| - |
| |
| - | {{Team:Aachen/BlockSeparator}}
| |
| - |
| |
| - | = Molecular biological methods =
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| - | == Cloning ==
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| - | === Restriction Digest ===
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| - |
| |
| - | === Ligation ===
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| - |
| |
| - | === Gibson Assembly ===
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| - |
| |
| - | The Gibson Assembly was conducted according to the protocol published by [https://www.neb.com/protocols/2012/12/11/gibson-assembly-protocol-e5510 New England Biolabs].
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| - |
| |
| - | # Set up the reaction according to the table below on ice (2-3 fragment assembly).
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| - | # Incubate samples in a thermocycler at 50°C for 15 minutes when 2 or 3 fragments are being assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.
| |
| - | # Transform NEB 5-alpha Competent E. coli cells with 2 μl of the assembly reaction, following the transformation protocol.
| |
| - |
| |
| - | <center>
| |
| - | {| class="wikitable" style="text-align: right;"
| |
| - | |-
| |
| - | | '''Total Amount of Fragments''' || 0.02-0.5 pmols
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| - | |-
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| - | | '''Gibson Assembly Master Mix (2X)''' || 10 µl
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| - | |-
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| - | | '''Deionized H<sub>2</sub>O''' || 10-X µl
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| - | |-
| |
| - | | '''Total Volume''' || '''20 µl'''
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| - | |-
| |
| - | |}
| |
| - | </center>
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| - |
| |
| - | == Transformation ==
| |
| - | === Heat Shock ===
| |
| - | # thaw cells on ice
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| - | # add 1 µL of plasmid DNA
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| - | # incubate on ice for 30 min
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| - | # heat shock at 42 °C for 60 s
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| - | # incubate on ice for 5 min
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| - | # add 200 µL of SOC media
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| - | # incubate at 37 °C for 2 h
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| - | # plate 20 and 200 µL on plates supplemented with the appropiate antibiotic
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| - |
| |
| - |
| |
| - | === Electroporation ===
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| - | # add 1 μL plasmid to electrocompetent cells
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| - | # put DNA/ cell suspension in electroporation cuvette
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| - | # wipe dry the electroporator
| |
| - | # use a small plastic pipette to place the cells
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| - | # pulse: 2.5 kV, 200-400 Ω, 25 μF (for ''E.coli'')
| |
| - | # immediatly add 1 mL LB and incubate for 2 h at 37 °C
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| - | # plate 50 μL on selective medium plate
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| - | # centrifuge the rest (3000 g, 20 min), discard supernatant, re-suspend the pellet in 50 μL LB and plate it on selective medium plate
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| - |
| |
| - | == PCR ==
| |
| - |
| |
| - | We have used several different types of PCR throughout our project:
| |
| - |
| |
| - | * colony PCR
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| - | * check PCR
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| - | * gradient PCR
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| - | * SOE PCR
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| - | * touchdown PCR
| |
| - | * QuikChange(Ligation-During-Amplification)
| |
| - |
| |
| - | The scope of appplication as well as the conduct are described below.
| |
| - |
| |
| - |
| |
| - | === Colony PCR /Check PCR ===
| |
| - | '''With GoTaq Mast Mix'''
| |
| - |
| |
| - | * 12.5 µl GoTaq Master Mix
| |
| - | * 1 µl primer_F
| |
| - | * 1 µl primer_R
| |
| - | * pick colony with tip and suspend in PCR tube
| |
| - | * 9.5 µl ddH<sub>2</sub>O
| |
| - |
| |
| - | <center>
| |
| - | {| class="wikitable"
| |
| - | ! parameter !! duration !! temp [°C] !!
| |
| - | |-
| |
| - | | denature||5:00||95 ||
| |
| - | |-
| |
| - | | '''anneal'''||00:30||56 || rowspan="3" | 30 cycles
| |
| - | |-
| |
| - | | '''elongate'''||01:00 per kb||72
| |
| - | |-
| |
| - | | '''denature'''||00:30||95
| |
| - | |-
| |
| - | | elongate||05:00||72 || rowspan="2" |
| |
| - | |-
| |
| - | | store||forever||8
| |
| - | |}
| |
| - | </center>
| |
| - |
| |
| - |
| |
| - | === gradient PCR ===
| |
| - |
| |
| - | === SOE PCR ===
| |
| - |
| |
| - | === touchdown PCR ===
| |
| - |
| |
| - | === QuikChange ===
| |
| - |
| |
| - | {{Team:Aachen/BlockSeparator}}
| |
| - |
| |
| - | = Analytical methods =
| |
| - |
| |
| - | == Agarose gel electrophoresis==
| |
| - |
| |
| - | Separation of DNA or RNA
| |
| - |
| |
| - | # take 5µl of the PCR product
| |
| - | # mix with 1µl loading dye
| |
| - | # apply onto agarose gel together with a marker
| |
| - | # run at 120°C for 40 minutes for a full gel
| |
| - |
| |
| - | == SDS-PAGE ==
| |
| - | === Cell preparation ===
| |
| - | * lysis cell pellet in lysis buffer
| |
| - | * centrifuge for 15 min at 13.000 rpm
| |
| - | * mix the supernatant with 2x lammli buffer with β-mercaptoethanol
| |
| - | * denatured for 5 min at 95 °C
| |
| - | * sample to the gel
| |
| - |
| |
| - | For some SDS-PAGEs, we used BioRad ready made gels.
| |
| - |
| |
| - | The recipe of the self-made SDS is as follows:
| |
| - |
| |
| - | === 1.5x Buffer ===
| |
| - | * 1.5 M Tris-Cl pH = 8.8
| |
| - | * in 1 L is 40 ml 10 % SDS
| |
| - |
| |
| - | === Gels ===
| |
| - | <center>
| |
| - | {| class="wikitable" style="text-align: right;"
| |
| - | !
| |
| - | !! style="border-left: 2px solid #404040;" colspan="3"|0.75 mm 12 % RUNNING Gel
| |
| - | !! style="border-left: 2px solid #404040; background-color:#8ebae5;" colspan="3"|1 mm 4 % STACKING Gel
| |
| - | |-
| |
| - | |
| |
| - | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
| |
| - | | style="border-left: 2px solid #404040;"| '''1x''' || '''2x''' || '''4x'''
| |
| - | |-
| |
| - | | '''H{{sub|2}}O'''
| |
| - | | style="border-left: 2px solid #404040;"| 1.65 mL || 3.3 mL || 6.6 mL
| |
| - | | style="border-left: 2px solid #404040;"| 1.5 mL || 3 mL || 6 mL
| |
| - | |-
| |
| - | | '''1.5x Gel Buffer'''
| |
| - | | style="border-left: 2px solid #404040;"| 1.3 mL || 2.6 mL || 5.2 mL
| |
| - | | style="border-left: 2px solid #404040;"| 0.65 mL || 1.3 mL || 2.6 mL
| |
| - | |-
| |
| - | | '''30 % Acrylamide (37.5:1)'''
| |
| - | | style="border-left: 2px solid #404040;"| 2 mL || 4 mL || 8 mL
| |
| - | | style="border-left: 2px solid #404040;"| 0.325 mL || 0.65 mL || 1.3 mL
| |
| - | |-
| |
| - | | '''10 % APS'''
| |
| - | | style="border-left: 2px solid #404040;"| 50 µL || 100 µL || 200 µL
| |
| - | | style="border-left: 2px solid #404040;"| 25 µL || 50 µL || 100 µL
| |
| - | |-
| |
| - | | '''TEMED'''
| |
| - | | style="border-left: 2px solid #404040;"| 10 µL || 20 µL || 40 µL
| |
| - | | style="border-left: 2px solid #404040;"| 5 µL || 10 µL || 20 µL
| |
| - | |-
| |
| - | |}
| |
| - | </center>
| |
| - |
| |
| - | ==Run gel==
| |
| - | * apply the prepared samples together with a protein marker on the gel
| |
| - | * run the gel for 10 min at 60 V and after that for ca. 60 min at 120 V
| |
| - |
| |
| - | == Bradford assay ==
| |
| - |
| |
| - | Determination of protein concentration
| |
| - |
| |
| - | == Measurement of fluorescence ==
| |
| - |
| |
| - | The measurement of fluorescence was performed using the Synergy Mx (BioTek) microplate reader and the Gen5 software.
| |
| - |
| |
| - | * volume of sample in each well: 100µl
| |
| - | * measure GFP fluorescence at an excitation wavelength of 496 nm and an emission wavelength at 516 nm
| |
| - |
| |
| - | == Measurement of optical density ==
| |
| - |
| |
| - | Depending on the number of samples, two different devices were used for measurement of optical density, the Unico Spectrophotometer 1201 (Fisher Bioblock Scientific) and the Synergy Mx (BioTek) microplate reader.
| |
| | | | |
| | {{Team:Aachen/Footer}} | | {{Team:Aachen/Footer}} |
"
