Team:UoA NewZealand/WetLab

Wet Lab

Overview and Results

11/8 First Result ever: Found that the DE3 E. coli cells we were using already had a plasmid with chloramphenicol resistance. Couldn’t tell using the iGEM competency test (which added a plasmid with chloramphenicol resistance) how competent our cells were, as those transformed with the iGEM plasmid would have been selected against. Switched to DH5 alpha cells for further transformations.

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Do we have any results of competency of our DH5 alpha cells?
  • Were they chemically competent?
    • They were, calculations to the competency on page 12 of lab book 06/09
    • Also other competent cells - 09/09
      • gels/photos would be gr9
  • Optional: mention the fricking RE sites in the middle of the construct?
  • 16/08 (labbook) - Optional: talk about issues with ligation/understanding provided igem protocol
  • Gel - 31/08

  • 03/09 - 04/09 - made new chemically competent cells
  • Gained PCR skills 07/09, 08/09

Cells passed competency test, but we weren't seeing any colonies following transformation, switched to electroporation as recommended by our PI, as it would remove more steps that could go wrong in chemical transformation.

23/09 Kan backbone + PETase End colonies grew on plates 1) Kan plate and competent cells - no growth 2) + 3) - PETase end + kanamycin backbone - many small colonies 4) negative control - kan plate and no cells - no growth 5) plain agar plate with cells ‘transformed’ with PETase end (to see growth of cells after transformation) - lawn of cells

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Miniprep of kan backbone + PETase end -> 27/09, gel page 20, used another miniprep protocol 29/09 - still no DNA

  • Could be due to overgrowing colonies
    • Unlikely, cells displayed kanamycin resistance when innoculated in higher concentration (80ng/ul vs 50ng/ul) and when grown on fresh plates
    • Grew cells on agar poured with kanamycin stocks from Kyle for elimination of bad antibiotic stock as a variable- still grew
  • Could be due to fault in construct - EcoRI site that would have cut up the PETase end
  • Self-ligation of the backbone?
  • Backbone insertion into the DNA?
  • WHY

Decided to use fresh DH5 alpha cells and to ditch the faulty construct

03/10 Third Result: electroporation results Decided to use another backbone (pSB1C3) as gel (page 34 - 04/10) showed that PCR of kan backbone not entirely pure and was worried with the previous results of the plasmid being able to insert into the genome/being low copy number/self-ligating. Successfully transformed cells with plasmid given by one of our advisors & confirmed with miniprep page 44 - therefore transformation is not the problem

  • 1 = ampicilin plate with cells transformed with peroxyredoxin plasmid (A positive control, Plasmid from Kyles stocks)- many big colonies
  • 2 = kan backbone (no insert) transformants - no growth
  • 3 = col backbone with PETase start -no growth
  • Did some transformations with cells provided by our PI - as they were known to be electrocompetent. Also used a plasmid with a multiple cloning site we could use for our construct, pET-DUET to see whether that would take up our construct any easier.
  • 4 = chloramphenicol plate with col backbone digest at 9/10 dilution (no insert) - lawn of plates - issue with antibiotic plating/spreading
  • 5 = ampicilin plate + petduet + PETase - no growth
  • 6 = canvas
  • 7 =

3 = chlorampheicol plate with col backbone + PETase start insert -

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Forth Results: start monitoring on the digestion and ligation concentration - put on gel to see results.

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=> 2 positive controls are positive, one colony growth in negative control and two colonies in insert + backbone (PETDUET)

Lab Notes

Date: 25/07/16

People in lab: Jess, Grace, David, Irene, Thai, Peter Goal: Training via purifying protein for Kyle (you’re welcome) * Made filtered water for columns * Collected transformed bacteria from –80C freezer * David & Irene (afternoon) * Prepared Lysis buffer and Sec Buffer * Jess & Grace (afternoon) * Lysed cells using French press. * X * Lysate prepared for centrifugation - increase concentration - Imac column prepared. * {IMAC elution dropped, therefore didn’t run sec on AKTA}

Date: 1/8/16–3/8/16

Date: 1/8/16-3/8/16 PEOPLE: Jess GOAL: Make chemically competent cells Made chemically competent cells using protocol on Slack (link) Incubated overnight Prepared * Cacl2 (0.1M) / H20 / Glycerol 15% * CaCl2 = 0.441g * H20 = 25.5mL * Glycerol = 4.5 mL

Date: 9/08/16

Morning: People: Jess & Grace Goal: Ligation and Digestion {{was using old IGEM protocol - results will have failed }} Protocol: 3A Assembly with linearised Plasmid Backbone - IGEM (link) * 10am - Jess gone through brief * Made up three master mixes as specified. Afternoon: 3–4 pm People: May & ??? Goal: Prepare Plates * Made LB agar plates for competency testing: * 9 x Chloramphenicol plates * 3x 0.5 pg (picograms) * 3x 10 pg * 3 x 50 pg
* 20mL agar to 20uL chloramphenicol per plate (original conc?) * 4 x kanamycin plates for transformation * 20mL agar to 20uL kanamycin (original conc???) * 1x agar plate w/o antibiotic (control)

Date: 11/09/16

People: Jess, David, Grace, Irene Commenced - 10am Goals: Create correct ligation products * Note: Previous protocol used for ligation incorrect, used new protocol Protocol: Protocol for 11/08 (link) * Made Enzyme Master Mix * Created 3 small bottles of Soc & autoclaved * Digested Backbone & insert (which ones????) - assuming the IGEM kan one + PETase start * Ligation carried out with digestion product, ligase buffer and t4 DNA ligase {and we all know how well that went- no ATP }

Date: 12/09/16

People: ???? (Jess?) Goals: Check transformation * Checked colonies: * Competency worked - colourless colonies {little did we know……. They were already chloramphenicol resistant} * Transformed Bacteria (PETases) - no colonies * Incubated overnight to see if would get new growth

Date: 13/09/16

People: Jess & May Goals: Check transformation - create new ligation? Jess? * Checked PETase plates * 2x100uL no colonies * 2x200uL - some colonies - yellow and white * {what does this mean someone plz} * Stored old PETase competent cells in 37C incubation overnight - check for more growth

Date: 16/08/16

People: Grace & Jess Goal: Troubleshooting™ * Ran DNA gel to see whether ligation/insertion was working * 0.9 % agarose gel with Ethididum Bromide Used * PETase insert ~ 1kbp * Plasmid ~2kbp * Gel image in book * Ran 1kb ladder * 11/8 PETase ligation (start?) * Faint band around 2kbp - plasmid only * 15/8 PETase ligation (start?) * No bands seen on gel * PETase insert (start?) * Stronger band around 1kb * Concluded that ~something~ was wrong with ligation

Date: 17/08/16

People: David * Nanodrop spec on plasmid backbone (which one - IGEM kan?) * 32.05ng ul–1 concentration

Date: 29/08/16

People: David? * Followed 19th August protocol for Plasmid construction. Have completed digestion and ligation steps * Made 500mL Agar * Plates to be spread are stored in second drawer down at lab bench * Problem : Accidentally had 6 minutes of incubation at 37C after heat kill cycle. This happened during digestion. * 0.8ul of T4 DNA ligase buffer added + 3.2 ul of water. (1:5 dilution) * ATP ? 1/10 concentration - was ACTUALLY ? 1/11 concentration * ATP stock at 10mM needed at 0.1 mM

Date: 30/08/16

People: David, Irene, Jess May & Peter joined in later * Carried out PETase (Start & End) transformations. Also conducted competency test. * Used protocol Protocol for 30/08) * Alterations; * Original iGEM competency plasmid concentrations used as well as serial dilution, plasmids at concentrations of (ng/uL): * 50, 20, 10, 5, 09, 5x10^–2, 5x10^–3, 5x10^–4, 5x10^–5 * Cells incubated for 2 hours rather than 30 minutes prior to plating
* Shaken at 180rpm * Plated out transformed cells (and competency)

Date: 31/08/16

People: David, Thai & Irene (10am) * Plates collected from incubator * All competency plates have high growth * DE3 strain already has Col resistance * No growth on PETase plates * Ran gel with plasmid constructs * Gel on page 10 of lab book * (no notes on gel, will summarise here) * Appeared to be no ligation products for Start, ligation for End showed faint band at ~100 kbp

Date: 01/09/16

People: Irene & David Protocol: 01/09 protocol * Made up stock of LB & glycerol * Plated DH5αα alpha cells on kanamycin and chloramphenicol to check if resistance already present. Plain agar control. * Prepped for making electrocompetent cells: seeded culture of DH5αα alpha overnight.

Date: 02/09/16

People: Thai & Jess Protocol : 02/09 protocol The day things fucked up :’< * Didn’t make up chemically competent cells - ran out of time, everything had to be chucked out. Good chat.

Date: 03/09/16 - 04/09/16

People: Maggie * Made chemically competent cells * Used protocol from Shaun’s Lab guide (found in lab folder)

Date: 05/09/16

People: Irene, David & May Protocol: 05/09 protocol * DH5αα alpha cells from Maggie were transformed with plasmids for PETase from previous week’s ligation. * Competency test also carried out * Incubated cells

Date: 06/09/16

People: Irene & then May * Made up LB and glycerol * Results of Competency and Transformation * No growth of PETase start or end (on antibiotic?) * Plain agar plates with PETase cells didn’t grow either * Competency test successful * Lots of calculations to calculate competency of maggie’s cells (page 12)

Date: 07/09/16

People: David & Jess * Ran PCR of PETase start, end and IGEM kanamycin backbone * Original concentrations of PETase start and end (nanodrop) * PETase start - 19.96 pg/uL (average) * PETase end - 31.73 pg/uL (average)

Date: 08/09/16

People: Irene * Ran gel of PCR products (page 14) * Ladder leaked into neighboring well * Showed there was PCR product of the inserts - backbone not as successful * Contamination of buffer - faint smear at longer bp lengths * Nanodroped concentrations - unsuccessful * (blanked with control - would have DNA primers and dNTPs so wouldn’t be the best blank)

Date: 09/09/16

People: David * DH5ααalpha competency - chemically competent cells? * Measured OD600 of 0.3 - returned to incubation room to bring to OD600 of 1 * PCR products: * Made new buffer to blank nanodrop with (what was it)?? * Machine gave warning that absorbance too high in blank * Measured concentrations in book - page 15 * Prepared to run gel - 8 lanes but ran out of time

Date: 20/09/16

People: Irene * Nanodropped PCR products - confirm DNA concentration * PETase start: 19.8ng/uL * PETase end: 18.3 ng/uL * PETase end: 18.450 ng/uL * Ran gel of PCR products - page 17 * Shows start and end have been PCR’d successfully (yay) * PCR protocol in drawer, changes: * PCR was made to volume of 25uL - with 1uL of construct and 1.5uL of each primer * Extension time = 70 seconds * Annealing temp = 65C * Transformed cells with PETase start and End * David’s cells and Maggie’s cells

21/09/16

People: David, Irene & Thai ???/ PETase - no growth on Kan plates, put back in incubator in case any transformed cells were slow growers. Kan backbone transformation had lawn of growth.?

Date: 22/09/16

People: Jess + Grace (morning) * Made up stocks of LB agar * One colony from previous transformation potentially successful * Was red - contaminant from competency test? * Plated out colony onto plates with and without kanamycin * Afternoon? -> Undertook digestion and ligation of PETase start + end

Date: 23/09/16

People: Irene, Grace, Thai & Maggie Goal: Electrotransformation funtimes Protocol: Electrotransformation as put on slack * Tried transforming PETase start and end ligation products into our electrocompetent cells * Used 2uL of ligation product - PETase start arced, but PETase end didn’t * Incubated transformed PETase end bacteria in tube at 37 C for 1 hour * Made up plates for plating (extra were made, those used are below) * 20 mL of agar to 20 uL of kanamycin (antibiotic plate) * At end of incubation, centrifuged transformed bacteria to resuspend in agar * Very small pellet observed -few bacteria * Plated as follows * 1 plate without kan and without transformants - negative control * 1 plate without kan + plated with PETase end * 1 plate with kan + plated with PETase end * 1 plate without kan + plated with DH5ααalpha (?) * 1 plate with kan + plated with DH5αα alpha (?)

Date: 24/09/16

People: Maggie: * Checked transformed plates * No growth on negative controls * Small colonies on kan + PETase end plate - incubated a little longer to try and grow colonies a little longer

26/09/16

People: Maggie, Jess, David + Grace * PETase end grew (yay) * Maggie and Jess started miniprep following kit instructions * Miniprep yielded no results - appears to be no Plasmid present in PETase end cells

28/09/16

People: Jess, Grace + Irene - morning * Streaked out 4 colonies from PETase end transformation onto kan plates * Afternoon: Maggie + Jess * Made liquid broth cultures of 4 colonies for miniprep * Previous miniprep DNA too low to show up on gel * Gel on page 20

Date: 29/09

People: Maggie + Grace (morning) Protocol: Maggie’s miniprep * Tried different miniprep protocol of PETase end colonies grown previous day * Left the streaked colonies from previous day in incubation room to grow more. * Carried out miniprep * DNA concentration of plasmid via nanodrop non-existent * (~–7.5 ng/uL to –0.5 ng/uL) * Decided to up concentration of antibiotic to put more selective pressure on bacteria with plasmid * Made up 6 liquid cultures with 80ug/mL kanamycin (up from previous plates/liquid cultures - 50ug/mL) * Left in incubation room overnight for miniprep next day

Date: 30/09/16

People: David (???) * Miniprepped the PETase end liquid cultures that were grown with higher kanamycin concentration * Again, no results, no plasmid DNA * The start of Troubleshooting™ v 2.0 * Want to transform with: * Empty IGEM plasmid * New plasmid + Construct * Transformation with Kyle’s plasmid - peroxiredoxin * Plate transformed cells onto plates with Kyle’s kanamycin * Plated up cells onto Kyle’s kanamycin plates: * 4 PETase end on kyle’s kan plates * 1 DH5αα alpha (no transformation) plated on kyle’s kan * 1 control of plain agar, spread with DH5αα alpha (no transformation)

Date: 1/10/16

People: May, David & Grace * Created new digestion mixtures * Realised all PETase constructs except start have second EcoRI site (and Not 1 sites) * Only transform PETase start with EcoRI - will have to use XbaI for other constructs * Decided to do transformations with kanamycin backbone and chloramphenicol backbone (pSB1C3) (kan was running low on stock and why tf not at this point in time lmao) * pSB1c3 - has chloramphenicol resistance, same RE sites, and pUC19 derived ori * Digest: * Mastermix - may have extra BSA * Excess NEB in tube after adding to master mix - not sure why * Created three digests: * 1 - 6 uL water / 2 uL kan backbone / 4 uL MM * 2 - 6 uL PETase start (PCR) / 2uL chlor backbone / 4 uL MM * B - 4 uL MM / 4 uL water * Ligation: * Created 3 ligation products: - note, forgot to add ATP again like idiots :P * 1 - 4uL digest 1 / 1 uL T4 DNA ligase buffer / 0.5 uL T4 ligase / 4.5 uL water * 2 - 4uL digest 2 / 1 uL T4 DNA ligase buffer / 0.5 uL T4 ligase / 4.5 uL water * B - 4 uL digest B / 1 uL T4 DNA ligase buffer / 0.5 uL T4 ligase / 6.5 uL water * Incubated - 16C/30 mins + 80C/20 mins * Prepped plates for spreading on transformants * 3 x kanamycin plates * Kanamycin backbone only transformants (1) * 3 x chloramphenicol plates * Chloramphenicol backbone + PETase start transformants (2) * 3 x ampicillin plates * Peroxiredoxin plasmid transformation (Kyle’s) * Agar had started to set when we were plating - ampicillin will be at a slightly lower concentration * 4 x plain agar plates * 1 x each for each transformant + 1 for control * Electrotransformation * (Grace had to leave, David remained behind after brief walk through transformation protocol) * Used Shaun’s electrotransformation protocol * Transformations: * Kanamycin backbone only: * 40 uL our cells + 1 uL ligation 1 * kV = 1.82 / T = 6.10 ms * Peroxiredoxin: * 40 uL our cells + 0.5 uL plasmid (to conserve) * kV = 1.80 / t = 6.10 ms * PETase (diluted- how dilute?) ( to try and prevent arcing): * 40 uL our cells + 1 uL ligation product 2 * kV + t not taken * Incubated for 1 hour in 37 C * Plated for overnight culture: * Ampicillin agar x 3: * 2 x original concentration of peroxiredoxin plasmids * 1 x spun and resuspended cells * Expect: colonies, if not = cells/transformation not viable * Kanamycin agar x 3: * 2 x original concentration of kanamycin backbone transformants * 1 x spun and resuspended cells * Expect: no colonies, if not = backbone can be expressed without an insert * Plain Agar + Kan backbone: * 2 x original concentration of kanamycin backbone transformants * 1 x spun and resuspended cells * Expect: colonies, if no colonies are present, then the cells/transformation is not viable * Col Agar + PETase Start x3 : Col Agar + Diluted PETase Start * If colonies are present : Successful transformation of Col resistance at least. Complete mini-prep to extract the plasmid. Colonies should be white. * If no colonies are present : Cells, transformation or ligation are erroneous * Any red colonies have the Col backbone but no insert. Any white colonies have an insert disrupting the RFP. * Plain Agar + PETase Start + Dilution x1 * Expect: colonies, if no colonies are present, then recheck everything!

Date : 2/10/2016

People : David and Grace

To do today : * Gel * Nanodrop * PCR

Gel set with EtBr * 3 g Agarose * 30 mL Buffer * 2 uL EtBr

Nanodrop

Result
Blank with Ligation Buffer
PETase Ligation Product		 195.05 (Weird! Plasmid concentration 25 uL)
194.70
Blanked with DiH2O Primers: Anti 89.15 ng/uL 88.80 ng/uL
Primers : Pre 91.3 ng/uL 90,95 ng/uL

Gel to Run

2 Start
Start
End
B
Buffer (LB)
Skipped
L2 PETase Ligation product
Went into 2 wells ^
PETase Insert
PETase Digestion product 5uL
Col Backbone 2.9 uL backbone, 1uL dye
Skipped Lane
Ladder 6uL

WE! FORGOT! TO! ADD! ATP! (to ligation)

Gel shows that ligation didn’t work (no ATP lmao)

Date: 3/10/2016

People : ??

  • To run Experiment: Into cells of known competency (given by Shaun)

  • Into our cells
    • PETase start
    • Col Backbone + Insert
    • PET duet + Insert
  • In Shaun’s cells
    • Digested Col backbone
    • Peroxiredoxin plasmid
  • In both cells
    • Peroxiredoxin plasmid

  • Bluescript could not be found, so PET duet was used instead, given by Gk.

  • PET duet requires EcoRI / XBaI and PSTI

  • PCR of PETase Start + PETase End

  • PCR Mix
    • KAPA Hot Start Ready Mix 25 uL
    • Primer (Forward) 1.5 uL
    • Reverse 1.5 uL
    • Template DUT (?) 0.25 uL (6.5 ng of template)
    • Water 21.75 uL
    • Total 50 uL

  • x3 for each of PETase Start and PETase End - Tubes are labelled PCR E and PCR S

    • Initial Denaturation 95 C, 3 minutes
    • Denaturation 98 C, 20 seconds }
    • Annealing 65 C, 15 seconds } 25 cycles
    • Extension 72 C, 20 seconds }
    • Final Extension 72 C, 1 minute

Nanodrop of PCR Products

NB / Peroxy Plates : restreak and see if they grow

—— table image here —–

* Going to store in iGEM fridge as is (10x concentration) 
* Labelled PCRS 1-3 = Start PETase 
* PCRE  1-3 = End PETase
* Approx 48 uL left in tubes (before transformation)


* Run gel of PCR products

1 uL PCR product 1 uL Loading Dye 4 uL of MQ Water

PCR PETase Start seems to have 3 bands 1 @ 1 Kbp 1 @ 700 bp 1 @ 500 bp

  • Digestion

  • NEB Buffer diluted x 10

  • Master Mix :
    • 200 uL NEB
    • 2 uL BSA (possibly ola?)
    • 2 uL ECO RI
    • 2 uL PstI
    • 74 uL Water
  • Digest:
    • Col backbone + PETase Start (d KP)
    • pET Duet + PETase Start (d CP)
    • Col backbone (d C)
    • Just MM + Water ( d MM)
    • (Out of Kan backbone)

  • d CP : 4 uL MM, 6 uL PETase Start, 2 uL Col backbone

  • d PP : 4 uL MM, 6 uL PETase Start, 2 uL Pet Duet

  • d C: 4 uL MM, 6 uL DI H2O, 2 uL Col backbone

  • d MM : 4 uL MM, 8 uL DI H2O | 12 uL

  • Digestion was Run :
    • 37 C / 30 minutes digest
    • 80 C / 20 minutes heat kill
    • Sat @ 15 C for 10 minutes or so

Yesterday’s Peroxidoxin cells have been streaked onto a new plate spread with Amp.

Date : 04/10/2016 People : Jess and Grace

  • Going to ligate Digest products of yesterday
    • Diluted T2 ligation buffer stock
    • 3 uL 10x conc. T4 ligation buffer

  • 30 uL 1 x concentration T4 ligation buffer - must add separately

  • LCP
    4 uL dCP 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water

    LPP 4 uL dPP 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water

    LC 4 uL dC 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water

    LM 4 uL dMM 1 uL T4 ligation buffer 0.5 uL T4 DNA ligase 1 uL ATP 3.5 uL MQ Water

BBPCR - For backbone PCR (Kanamycin backbone) 25 uL Hotstart KAPA ReadyMix 10 mM 1.5 uL Forward Primer } Stocks are 10x concentration 10 mM 1.5 uL Backward Primer }Kan Backbone —> MQ Water 21.5 uL

Ligation Product Conc.

LCP 2564.5 ng/uL LPP 1377.60 ng/uL LC 971.55 ng/uL LM 1174.3 ng/uL

~ 11:30 a.m., ran PCR on Kan backbone

Electroporation Irene, David & May came in

Gel of Ligation Products : —– table image here —–

Prepared antibiotic dilutions for transformation plates

Antibiotic stock solutions 1000x concentration (50 mg/mL) Working Concentration (50 ug/mL) Dilution -> 1uL antibiotic stock 1 mL MQ water / ethanol

  • Kanamycin
  • Chloramphenicol
  • Ampicillin

Washing Electroporation Cuvettes -> Water, ethanol, hydrogen peroxide

Gel of Digested Products

—– table image here —–

    {DNA Gel picture)

Put back in to keep running (2x DNA Gel Pictures)

Transformation: 1. Col backbone + PETase (ECP) 2. PET DUET + PETase (EPP) 3. Col backbone (EC) 4. Peroxidoxin (Prx) (in tube of conc. 1ng/uL labelled PRX)

Transformed cells :

Shaun’s DH5α, Stats unknown, Incubation began at 5:45 CP PP C Prx

iGEM DH5ααα

CP (2.49 kV, 6.10 ms) PP (2.49 V, 5.80 ms) C (2.49 V, 6 ms) Prx (Unknown)

Have all been plated up: Shaun’s on 1/10 & 9/10 plates, our iGEM cells just 100% on a plate. Restreak of peroxyredoxin cells (Kyle)

Date: 05/10/2016 People : Grace (morning)

Checked transformants Small colonies on peroxyredoxin plates (yay) Lawn of cells on chloramphenicol backbone only plates (chloramphenicol was diluted with water, this may have affected its growth) Other plates = no colonies Use Shaun’s cells from now on, our DH5ααα are probably too dilute to use

Results : Col backbone (negative control) = lawns of cells Col backbone + PETase = N/A PETDUET + PETase = N/A Peroxiredoxin (positive control) = ~100 colonies

Col has been plated on agar with water to dilute instead of ethanol, BUT no biggies .. cuz Col backbone + PETase has nothing growing on it….

Need to miniprep peroxidoxin to figure out whether the steps after ligation have been right

Why are stuffs growing on negative control? Hell knows Shaun can’t figure it out either

Improvement for next digestion : Separate backbone and insert in different tubes for digestion

New protocol : Day plans not used anymore but updates Standard protocol folder set up

To do :

Run minipreps of colonies on plates - Peroxiredoxin cells - Col backbone LB inoculated 4x Col, 2x Prx Do digest of stocks Col backbone Col + Insert Kan Kan + Insert pET DUET pET DUET + Insert Buffer

Master Mix has been made

Date : 06/10/2016 People : Jess, Grace, Irene

Following Digestion / Ligation protocol 06/10 Digesting insert + backbones separately 3x PETase Start - “PET” 1 x each backbone K = Kanamycin C = chloramphenicol D = pET Duet

Diluted PETase start PCT tube 1 as for digestion concentration needs to be ~20 ng/uL Original = 290.42 ng/uL 1/10 dilution = 29.04 ng/uL 8mL left

Kan backbone (iGEM) PCR Nanodrop
332.95 ng/uL 333.15 ng/uL 332.70 ng/uL Diluted 1/10 to give ~ 33.2 ng /uL → 16 uL left

Miniprep liquid cultures haven’t grown - left in incubation room. Make up 3x new preparations of our peroxi transformants with 1% amp in LB Make up 3x Kyle’s peroxi transformants with 1% amp in LB 15 uL amp -> 15mL LB Kyle’s amp is already diluted - don’t use our diluted stock. Started incubation at 11:00 a.m

Creating 6 Ligation Products

LPD 1 uL of digest pET DUET 3 uL of PETase Start digest I uL ligation buffer, 0.5 uL ligase 3.5 uL of MQ Water , 1uL ATP

Assume buffer, ligase, ATP and MQ is same for next ligations if not changed

LPC 1 uL digest chloramphenicol backbone 3uL of PETase Start digest

LPK 1 uL digest kanamycin backbone 3uL of PETase insert

LD 4 uL pET DUET

LC 4 uL chloramphenicol backbone

LK 4 uL kanamycin backbone - negative control

Making up big mix of ligation mix 24.5 uL MQ Water 7 uL ligation buffer 3.5 uL T4 DNA ligase 7 uL ATP 42 uL total - 6uL into each tube

Washed electroporation cuvettes 100 % ethanol x2 washes Make sure they are dry for transformation

David made 1L of glycerol in prep. For making electrocompetent cells tomorrow

7 Transformations PK (Kan backbone + PETase) DP (Duet + PETase) CP (Chlor. Backbone + PETase) K (Kan backbone) → Neg. Control Prx (Peroxiredoxin Plasmid) → Pos. Control D (Duet backbone) → Neg. Control C (Chlor backbone) → Neg. Control Pn (pET DUET - not digested → Pos. Control

Each of the 8 transformation tubes have been filled with: 40 uL Shaun’s DH5αα cells (except Pn which had a little less) 2 uL of DNA product (except for Prx, which had 1 uL)

Transformation: —– table image here —– Began incubation at 37C @ 300rpm at 5:02 p.m.

Gel of Digest & Ligation Products

—– table image here —–

Meanwhile: 2x 10mL LB bottles inoculated with DH5α 1 plate streaked with DH5α (LB Agar)

Gel removed at ~ 8:15 & imaged (Picture)

Date: 07/10/2016 People: Jess, Grace & David

Results from yesterday’s transformations: Growth on : pET DUET without ligation Peroxiredoxin transformants (small)

Growth on outer edges of some plates Antibiotic spreading errors Kan backbone (both) Kan backbone + insert (both) Col bckbone + insert (1/10th) pET Duet without ligation (9/10th)

Potential growth on : PET DUET with PETase insert - 9/10th Faint lawn of cells Going to restreak out for cells forming faint lawn on PET DUET on plates with infused ampicillin 1x w/ amp no plating 2 x w/ amp + streaked colonies 1 x w/o amp + no plating 1 x w/o amp + streaked colonies 15 mL agar to plate → 15 uL amp (not dilute) Incubated in 37C incubator in our lab!!!!

Grace’s miniprep liquid cultures of KYle’s and our peroxiredoxin transformants has grown - ready for miniprep

Miniprep time - Grace & David Started miniprep with liquid stock of Kyle’s successful peroxiredoxin + our transformed peroxiredoxin (Shaun’s cells) Mainly to check MAggie’s protocol works + to see that we can miniprep Miniprep of iGEM 1 + 2 are mixed. Should be fine, all from same colony. Miniprep protocol - no visible DNA pellet - continued on anyway

Nanodrop Results

—– table image here —–

David restarted miniprep with “super easy plasmid extraction” (?) Visible pellets formed - looks positive

Nanodrop results (blanked with TE)

—– table image here —–

Date: 09/10/2016

People: David, Grace, Irene & Jess Transformation & Digestion: Transformed cells with: Kan backbone Kan + PETase start Col backbone Col + PETase start pET DUET pET DUET + PETase start Peroxiredoxin Digest buffer Ligation buffer Digested - seperately with 2 enzymes each to improve efficency Kan backbone Col backbone petDUET PETase Innoculated 6x seperate pET DUET PETase insert (from 7/10) colonies for miniprep

Date: 10/10

People: Irene & Grace (morning) 1 miniprep innoculum grew Digest pET DUET with single RE - see efficency ECORI, PSTI & XBAI

Date: 11/10

PEOPLE: Grace Checked liquid cultures or pETDUET & PETase from 10/10 - left in incubator for more growth Ran gel of single RE pET DUET digests prepared on 10/10 Lane 1 = 1kb+ ladder Lane 2 + 3 = pET-DUET (not cut) - 5uL and 10uL - bands at 8kbp + 3 kbp = forms of plasmid (nicked and supercoiled) Lane 4 + 5 = pET-DUET (cut with XbaI) - 5uL and 10uL - bands at 8kbp + 3 kbp = forms of plasmid (nicked and supercoiled), faint band at 5kbp = linearised plasmid Lane 6 + 7 = pET-DUET (cut with PstI) - 5uL and 10uL - bands at 8kbp + 3 kbp = forms of plasmid (nicked and supercoiled), band at 5kbp = linearised plasmid Lane 9 + 10 = pET-DUET (cut with EcoRI) - 5uL and 10 uL - band at 5kbp = linearised plasmid, faint bands at 8kbp and 3 kbp Decided to not use XbaI in constructs and to increase digestion time to 1.5 hours to give PstI more chance to linearise plasmid.

Date: 12/10

PEOPLE: David, Irene & Grace Nanodrop of purified pET DUET digest (double RE digest) - average concentration = 3.5 ng/uL Prepared Ligation mixtures for transformation - Col backbone only, Col backbone + PETase, Kan backbone, Kan backbone + PETase, pET DUET + PETase, pET DUET Ligated for 30 minutes and then 20 minute heat kill Prepared digest with 3 RE DALL (EcoRI, SacI + PstI) - SacI should cleave in between EcoRI and PstI - prevent religation - 5uL of pET DUET stock -> 1210ng total, 15 uL water, 18uL master mix, 0.4uL of each enzyme DE (EcoRI) - 250ng pET DUET, 9.8uL mastermix, 0.2uL EcorI DS (SacI) - 250ng pET DUET, 9.8uL mastermix, 0.2uL SacI DP (PstI) - 250ng pET DUET, 9.8uL mastermix, 0.2uL PstiL PETase start digest = PAll (EcoRI + Psti+I) - 1000ng of DNA total, 0.4uL enzyme each. Digested all products for 1.5 hours.

Date: 14/10

PEOPLE: David, Grace & Irene Grace & Irene: Purified pET DUET that had been digested with 3 restriction enzymes (EcoRI, SacI and PstI) with size exclusion purification (link) Nanodropped to find concentration after resuspension in water - 8.21 ng/uL David: Ligated with purified pET DUET (triple digest), 240ng of vector to 144ng of PETase construct (for a 1:3 molar ratio). Made up three ligation mixtures: L1 - 1uL ligase buffer, 0.5uL T4 ligase, 1uL ATP, 15uL pET-DUET (purified triple digest) and 3uL PETase L2 - 1.25uL ligase buffer, 0.625 uL T4 ligase, 1.25 uL ATP, 15 uL pET-DUET (purified triple digest) and 3 uL PETase L3 - 2uL ligase buffer, 0.1uL T4 ligase, 2uL ATP, 10uL pET-DUET (purified triple digest) and 5uL MQ water Ligated for 30 minutes with 20 minutes heat kill ---- Ran gel of digest products and this day’s ligation. 1 = KB+ ladder 2 = PETase 3 = DUET Digest (EcoRI, SacI & PstI) 4 = DUET Digest (EcoRI) 5 = DUET Digest (PstI) 6 = DUET Digest (SacI) 7 = PETase digest (EcoRI & PstI) 8 = L1 9 = L2 10 = L3 Carried out Electroporation from ligation mixture from 12/10 of PETase start + chloramphenicol backbone, PETase start kanamycin backbone and PETase start pET-DUET (double digest), also transformed chloramphenicol backbone, kanamycin backbone, pET-DUET and advisor’s plasmid of peroxiredoxin by themselves as controls and plated them out onto appropriate plates

Date: 15/10

PEOPLE: Jess Checking transformation result from previous day Growth on positive control, pET DUET (1 colony) and pET-DUET (double digest) + PETase insert (2 colonies) Innoculated colonies from pET-DUET (double digest) / insert for miniprep Chemically transformed cells provided by Maggie L1, L2 and L3 from previous day (pET-DUET = purified triple digest) following John Taylor’s standard protocol (link) Heat shocked at 47C instead of 37C - technical error Plated cells at 1/10th dilution (100uL of broth) and at 9/10th dilution (centrifuged cells, removed supernatant and resuspended into 100uL for plating)

Date: 16/10

PEOPLE: David, Grace & Maggie Minipreped pET-DUET (double digest) + PETase transformants from 14/10 - using Super Easy Plasmid Purification protocol (link) Nanodrop showed that there was successful DNA extraction (2 samples were run, one had concentration of 241.15 ng/uL, other had concentration of 201.62 ng/uL) Ran gel of plasmids, digested with XbaI, EcoRI + Hind3 (seperately) - but showed a smear with no definitive results.

Date: 17/10

PEOPLE: Irene, Jess, David & Grace Ran miniprep following Super Easy Plasmid Purification for pET-DUET (triple digest) + PETase transformants from 15/10 All colonies had some absorbance spectra in range of DNA Transformed BL21(DE3) cells using miniprep plasmids from 16/10 pET-DUET (double digest) + PETase

Date: 18/10

PEOPLE: David, Irene, Jess & Grace BL21(DE3) cells transformed on 17/10 were unsuccessful - pET-DUET (double digest) + PETase were unsuccessful - further transformants will refer solely to the pET-DUET (triple digest) and PETase from 15/10 Run digest of miniprep products from previous day using EcoRI and Hind3 (separately) to run on gel Wasn’t very easy to see which plasmids on gels had the insert in plasmid as pET-DUET wouldn’t show and some plasmids didn’t show up on gel at all. Transformed all plasmids from 17/10 miniprep into BL21(DE3) strain for next day using Frankie’s protocol (link)

Date: 19/10

PEOPLE: Jess, Grace & David Transformed plates from previous day all showed growth on ampicilin plates = successful transformation into expression strain As gels of plasmids were widely inconclusive, decided to try and PCR out the insert in the plasmids to see which colonies had the insert. 1 = 1 kb+ ladder 2 = PETase 3 = pET DUET 4 - 14 = Miniprep of colonies 3-13 15 = PCR mixture (negative control) - might be contaminated Colonies 4, 6, 8, 9 + 10 had bands at same level as PETase - most likely to have correct insert

Date: 20/10

PEOPLE: Grace Prepped liquid cultures with ampicilin for protein expression Innoculated single colonies from BL21(DE3) transformants 4, 6, 8, 9 & 10 as they all showed bands in previous night where PETase would be expected, also innoculated colony 11 as it shared a band with pET-DUET and could act as a control Once cells reach OD600 of 0.5 - will induce protein expression in half Colony #.1 = cells grown without IPTG || Colony #.2 = cells innoculated with 1mM IPTG Grew cells for just over 3 hours before adding IPTG to the #.2 colonies. Final OD600 Readings 4.1: 0.486 | 4.2: 0.622 | 6.1: 0.390 | 6.2: 0.706 | 8.1: 0.432 | 8.2: 0.433 | 9.1: 0.395 | 9.2: 0.533 | 10.1: 0.560 | 10.2: 0.637 | 11.1: 0.456 | 11.2: 0.555 |

Protocol

Digestion (2 Separate for Plasmid vs Construct)

  • Enzyme Master Mix (Makes 25 ul)
    • 5 ul NEB Buffer
    • 0.5 ul BSA
    • 0.5 ul EcoRI-HF
    • 0.5 ul SpeI (Pst1 for PETDUET)
    • 18 ul dH20
  • Digest Plasmid Backbone (tube 1) (1 for Kan, 1 for Chlor, 1 for Duet)
    • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
    • Add 4 ul of Enzyme Master Mix
  • Digest Construct (tube 2) x 3
    • Add 4ul of construct (25ng uL)
    • Add 4 ul of Enzyme Master Mix
  • Put tubes 1 and 2 into PCR machines incubate: Digest 37C/30 min, heat kill 80C/20 min

Ligation (1 Reaction tube)

  • Add 1ul of digested plasmid backbone (25 ng)
  • Add 3ul of digested construct
  • Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase. (Get ligase buffer from Shaun)
  • Add 0.5 ul T4 DNA ligase
  • 1mM ATP
    • 1uL Ribo ATP not deoxyribo ATP
  • Add 3.5 uL MQ water
  • Incubate: Ligate 16C/30 min, heat kill 80C/20 min

Transformation (Chemical)

  • Take competent E.coli cells from –80 degrees

Transformation (Electroporation)

Plating

  • 20–15 ml of LB agar + 20–15 ul of complement antibiotics (50ng/ul)
  • 100 ul of cells from the transformed cells

iGEM RECIPES

Chloramphenicol / Kanamycin Plates

Before pouring plates, make sure that the agar is cooled enough so it is still liquid, but not hot enough to burn your hand. Work in sterile conditions (fume hood)

For two plates : 1. In a 50mL Falcon tube, add 40mL of liquid agar and pipette in 40uL of antibiotic solution. 2. Mix gently. 3. Pour 20mL into both plates.

DNA Gel

Note: Ethidium bromide is dangerous, gloves must be worn in preparation of DNA gel.

  1. Mix 0.3g of DNA gel powder and 30mL of liquid buffer in a conical flask
  2. Microwave at medium power for 2x 30 seconds. Swirl in between.
  3. Microwave for another 15 seconds at medium power. The solution should have no cloudiness / be completely clear.
  4. Pour into set. Pipette in 2uL of ethidium bromide (stored in a light-sensitive Eppendorf tube) and swirl with your (gloved) finger,
  5. Throw glove out.
  6. Put in the cast to set the wells and leave to harden.

T4 DNA Ligase Buffer

We dont need this right?

Luria Broth - 500ml MQ water

0.5% Yeast Extract - 2.5g 1% Tryptone - 5g 1% NaCl - 5g

Agar (500ml Luria broth)

Add 1.2% agar powder to LB - 6g

Calcium Chloride Solution - 30ml MQ water

CaCl2 0.1M - 0.441g

Digest Master Mix - 25µl MQ water

5µl NEB buffer 0.5µl BSA 0.5µl Restriction Enzyme 1 0.5µl Restriction Enzyme 2 18.5µl Filtered MQ water

SOC Buffer - 20ml

2% Tryptone - 0.4g 0.5% Yeast Extract - 0.1g 10mM NaCl - 0.01168g 2.5mM KCl - 0.00373g 10mM MgCl2 - 0.04066g 10mM MgSO4 - 0.04929G 20mM Glucose - 0.07206g

Making Electrocompetent Cells

Make up 500ml LB. Make 1L of 10% glycerol Pick a colony from cell streak and add to 5ml of LB - Put in shaker at 37oC overnight

Add the 5ml culture to the 500ml LB Shake at 37oC - grow cells until OD600 = 0.5 - 0.7 (takes a few hours) Put cells, the 10% glycerol solution and all centrifuge bottles and tubes on ice

Transfer cell culture to a cold centrifuge bottle and spin at 4000 × g for 15 min at 4oC. Carefully pour off and discard the supernatant Gently resuspend the pellet in 500ml ice-cold 10% glycerol

Centrifuge at 4000 × g for 15 min at 4oC. Carefully pour off and discard the supernatant. Gently resuspend the pellet in 250ml of ice-cold 10% glycerol

Centrifuge at 4000 × g for 15 min at 4oC. Carefully pour off and discard the supernatant Resuspend the pellet in approx. 20ml of ice-cold 10% glycerol

Centrifuge at 4000 × g for 15 min at 4oC Carefully pour off and discard the supernatant Resuspend the cells in 1 – 2 ml of ice-cold 10% glycerol ( (Cell concentration should be about 1 – 3 × 1010 cells/ml, so if you harvested the cells at OD600=0.5 resuspend in 1ml, if at OD600=0.7 resuspend in 2ml).

Aliquot out this cell suspension (50μl per aliquot) into 1.5ml microcentrifuge tubes and snap freeze in liquid nitrogen. Store in –80oC freezer.

Transformation (Electrocompetent cells)

For each sample to be transformed: place a 1.5 ml microcentrifuge tube and an electroporation cuvette (0.1 cm) on ice. Thaw an aliquot of competent cells on ice In the cold 1.5 ml tube, mix 40 μl of competent cells with at least 100ng of DNA (2ul of 50ng/ul) Mix well and incubate on ice for approx 1 min

Turn the MicroPulser on and set for your cuvette size (Ec2) Transfer this mixture of cells and DNA to the cold electroporation cuvette and tap the mixture to the bottom Place the cuvette in the chamber, pulse once

Remove the cuvette from the chamber and immediately add 1 ml of LB to the cuvette. Quickly but gently resuspend the cells. Transfer this cell suspension to a 13 ml sterile tube and incubate at 37oC for 1 hour, shaking at 225 rpm.

Check and record the pulse parameters on the MicroPulser. The time constant should be close to 5 milliseconds. The field strength can be calculated as actual volts (kV) / cuvette gap (cm).

After 1 hour incubation, spread 100 μl of the transformation onto an LB + antibiotic plate (1/10th). Spin the remainder of the transformation down and discard the supernatant. Resuspend the pellet in 100 μl LB and spread on another LB + antibiotic plate (9/10th). Put both plates in the 37oC room overnight.

Making Chemically-competent Cells

Pick a single bacterial colony of your strain and transfer into 10 ml LB (5 ml aliquots) in a sterile falcon tube Incubate overnight at 37 °C, 180 rpm

Inoculate 50 ml LB in 250 ml sterile flask with 500 μl seed overnight culture Incubate culture at 37 °C for 1.5 – 3 h, 180 rpm until OD600 = 0.4 – 0.6 Remove cells when growth has reached appropriate OD and place flask on ice to cool cells Cells MUST be kept on ice at all times to achieve maximum efficiency of transformation

Transfer of bacterial cells to icecold falcon tubes and centrifuge at ~3000 rpm, 5 min, 4 °C Carefully decant supernatant, then discard. Stand inverted tubes on paper towel for 1 min to drain last traces of media Take care that the pellet does not fall out of the falcon tube

Resuspend pellet in 25 ml ice cold 0.1 M CaCl 2 by pipetting up and down with 10 ml pipette Incubate on ice for 1 h

Centrifuge at 4000 xg for 8 min at 4 °C Carefully remove supernatant Resuspend cell pellet (pipette up and down) in 3 ml of ice-cold 0.1 M CaCl 2 , 15% (v/v) glycerol Aliquot 100 μl into autoclaved eppendorf tubes and snap freeze using liquid Nitrogen and store at 80 °C

Transformation (Chemically competent cells)

Thaw an aliquot of chemically competent cells on ice. Add DNA (approx. 0.5 ng DNA) to the cells and gently mix with the pipette tip. Incubate on ice for 30 min. Heat-shock for 2 min at 42oC. Add 1 ml LB (no antibiotic). Incubate for 1 hour at 37oC.

After this incubation, spread 100 μl of the transformation onto an LB + antibiotic plate (1/10th). Spin the remainder of the transformation down and discard the supernatant. Resuspend the pellet in 100 μl LB and spread on another LB + antibiotic plate (9/10th). Put both plates in the 37oC room overnight.