Difference between revisions of "Template:VirginiaNotebook1"

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<li>Miniprep GFP+VhB in pSB1C3</li>
 
<li>Miniprep GFP+VhB in pSB1C3</li>
 
<li>Transform SOD+HAO+Cyt, SOD+Cyt, and VhB+Cyt</li>
 
<li>Transform SOD+HAO+Cyt, SOD+Cyt, and VhB+Cyt</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa5">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 
</ul>
 
</ul>
 
</span>
 
</span>
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<ul>
 
<ul>
 
<li>Miniprep and Nanodrop: VhB+Cyt, SOD+Cyt, VhB+HAO/Cyt</li>
 
<li>Miniprep and Nanodrop: VhB+Cyt, SOD+Cyt, VhB+HAO/Cyt</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa7">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa8">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 
</ul>
 
</ul>
 
</span>
 
</span>
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</ul>
 
</ul>
 
</span>
 
</span>
 +
 +
<span class="targetspan" id="sa12">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
  
 
<span class="targetspan" id="sa13">
 
<span class="targetspan" id="sa13">
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<ul>
 
<ul>
 
<li><i>Pc. denitrificans</i> preculture made</li>
 
<li><i>Pc. denitrificans</i> preculture made</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa16">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 
</ul>
 
</ul>
 
</span>
 
</span>
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<li>Transformation of AMO + pSB1C3</li>
 
<li>Transformation of AMO + pSB1C3</li>
 
<li>Competent Cell Check + Transformation of <i>Pc. denitrificans</i></li>
 
<li>Competent Cell Check + Transformation of <i>Pc. denitrificans</i></li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa18">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa19">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa20">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
 +
 +
 +
<span class="targetspan" id="sa21">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 
</ul>
 
</ul>
 
</span>
 
</span>
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</ul>
 
</ul>
 
</span>
 
</span>
 +
 +
<span class="targetspan" id="sa25">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
 +
  
 
<span class="targetspan" id="sa26">
 
<span class="targetspan" id="sa26">
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<li>Culture of Sod in <i>E. coli</i> and BBa_K608010 in <i>E. coli</i></li>
 
<li>Culture of Sod in <i>E. coli</i> and BBa_K608010 in <i>E. coli</i></li>
 
<li><i>Pc. denitrificans</i> transformation with GFP</li>
 
<li><i>Pc. denitrificans</i> transformation with GFP</li>
 +
</ul>
 +
</span>
 +
 +
<span class="targetspan" id="sa29">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 +
</ul>
 +
</span>
 +
 +
 +
<span class="targetspan" id="sa30">
 +
Lab work:
 +
<ul>
 +
<li>No experiments performed this day</li>
 
</ul>
 
</ul>
 
</span>
 
</span>

Revision as of 05:34, 1 November 2017


Notebook


Lab work:
  • Media Creation:
  • Ampicillin stock solution created
  • Chloramphenicol stock solution created
  • 50% glycerol stock solution created
  • LB Broth created
  • Transformation Buffer made and filter sterilized
  • MgCl2, CaCl2, MnCl2, KCl, and PIPES stock solutions created
 Lab work:
  • Created LB plates with no antibiotics
 Lab work:
  • Created LB media with chloramphenicol
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • Created LB plates with ampicillin
  • Created Stop Buffer
 Lab work:
  • E. coli DH5 alpha pre-cultured overnight in 3 mL of LB with MgCl2
  • Another 500 mL LB+MgCl2 created as backup
Lab work:
  • Completed competent cell preparation for DH5 alpha
 Lab work:
  • E. Coli DH5 pre-cultured in 3 mL LB
  • Competent DH5 alpha 50% glycerol stocks made and stored in -80 degree C freezer
  • 1 mL of the rest of the pre-culture inoculated in 500 mL LB+MgCl2 and incubated in 18C shaker in PLSB lab.
  • Creation of another 500 mL LB+MgCl2 as a new backup media.
  • Another pre-culture of E. Coli DH5 was also incubated at 37C in the gilmer lab in case the main culture at PLSB was contaminated expectedly.
 Lab work:
  • OD values of the E. Coli DH5 culture incubated at 37C shaker after overnight in the morning
      • Blank Value = 0.018
      08:16 Value = 0.047
      09:00 Value = 0.051
      10:00 Value = 0.057
      11:00 Value = 0.030
      12:00 Value = 0.073
      12:30 Value = 0.116
  • The DH5 cell culture was then centrifuged and the pellets were resuspended twice in transformation buffer. DMSO was added to the cell media and the cell culture was aliquoted into 500 uL. The aliquots were freezed with liquid nitrogen and stored in -80C.
  • The cells were thawed on ice and the DNA from the competent cell kit was centrifuged. The plasmid backbone from the kit was transformed into the DH5 cells and incubated and spread around plates for overnight culture.
  • Competent Cell Check: First set of plates didn’t meet our expectation in terms of colony density, transformation of the same set of competent cells was executed again
 Lab work:
  • Second transformation attempt with the same competent cell stock was successful with ideal results
  • Competent cell check calculations
 Lab work:
  • First day of Interlab Lab Study
  • Positive and negative controls and 1-6 test plasmids transformed in E. Coli DH5 alpha
  • 16 chloramphenicol plates were used to culture different concentrations of E. Coli cells, 8 with 200 pg/uL and 8 with 20 pg/uL
 Lab work:
  • Plates had many colonies on them
  • We picked two colonies of each device and inoculated them, then put in 37 degrees C 220 rpm shaking incubator
  • More Agar plates with chloramphenicol supplement were created
 Lab work:
  • The calibration of the plate reader was done with the measurement of OD600 reference point and fluorescein standard curve
  • The different bacteria strains were pipetted into the well plate and the OD level of the cells were measured
  • Due to the time constraint, the cells were chilled at -20 degrees C overnight and not diluted to OD=0.02 yet
 Lab work:
  • As the chill was later determined to have possible effects on the accuracy of the measurements, the bacteria strains were re-cultured overnight and will be diluted and incubated for the second time on the next day
 Lab work:
  • 10 DNA plasmids from the DNA distribution kit were transformed into competent E. Coli DH5 alpha cells
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • LB made for transformed E. coli culture which was used to inoculate the cells in preparation for glycerol stocks tomorrow
  • N. europaea media stocks made for culturing
      • May be irregularities in the composition of the media, as it took very long for pH to increase, also N. europaea Broth step #2 had a unusually low initial pH
 Lab work:
  • Prepared for miniprep procedure
  • Created N. europaea growth media
 Lab work:
  • Miniprep completed
  • N. europaea culturing begins
 Lab work:
  • N. europaea and Pc. denitrificans in starting culture
 Lab work:
  • Transformations in preculture
 Lab work:
  • Made Pc. denitrificans plates and Pc. denitrificans glycerol stocks, replated transformed E. coli
 Lab work:
  • No experiments performed this day
 Lab work:
  • Plating N. europaea
      • Plated 10 plates, 5 from one 3.5 mL aliquot, 5 from the other
      Put in incubator at 37 degrees C then moved to incubator at 26 degrees C
  • Glycerol stocks of 50% N. europaea
  • Plating Pc. denitrificans
      • 6x plates of Pc. denitrificans incubating at 37 degrees C
      4x plates of Pc. denitrificans incubating at 26 degrees C
  • Competent Cell Prep for Pc. denitrificans
      • Failed: no cell pellet retrieved after after the transformation buffer was added
  • Made agar (1L) for Pc. denitrificans: plated onto 40 plates, 100 mg ampicillin added to the 1L culture
  • Made liquid media for Pc. denitrificans then autoclaved
 Lab work:
  • Starting Culture Pc. denitrificans
      • 3 mL culture in falcon tubes with Pc. denitrificans from glycerol stocks
  • Re-grow N. europaea from glycerol stocks
      • 3 mL culture in falcon tubes with N. europaea from glycerol stocks in shaking incubator
 Lab work:
  • Pc. Denitrificans had no growth on most plates
  • One plate with growth had two phenotypes of colonies
  • Made Several Pc. Denitrificans plates with no antibiotics to dry overnight
  • Made N. europaea and Pc. denitrificans plates from glycerol stocks
 Lab work:
  • Made Pc. denitrificans liquid precultures
      • 3 mL of Pc. denitrificans preculture was added to 500 mL of the Pc.denitrificans broth and put in the shaking incubator (37 degrees C, 220 rpm)
  • Made SGM17 Media
  • Made N. europaea, Pc. denitrificans, and Kanamycin LB plates
  • Begin assembly plans for promoter characterization with GFP generators
 Lab work:
  • Gram Stains performed on the two phenotype plate
      • Lighter colonies- gram positive (left); darker colonies- gram negative(right)
  • Transformation of VhB, Sod, Nar to competent E.coli DH5 alpha and plated on LB plates with ampicillin
  • Plated competent E.coli on 10 LB kanamycin
  • Nanodropped BBa_E0240, concentration: 100.9 ng/uL
 Lab work:
  • Pc. denitrificans Electroporation Transformation:
      • Pc. denitrificans liquid culture measured OD600 of 0.945, 0.355, and 0.365
      Transferred liquid culture into 1/100 dilutions in SGM17 media without Glycine
      Created a 250 mL aliquot with 2% glycine supplement
  • Pc. denitrificans heat shock transformation and plated competent cells
  • Created N. europaea and E. coli liquid cultures
 Lab work:
  • SGM17 + Glycine Pc. denitrificans liquid culture had not changed color while the SGM17 cultures did
      • OD 600 for SGM17 + glycine is 0, 0.012, 0- no growth
      OD 600 for SGM17 varied from -1.577 to 2.659; SGM17 media may be making OD readings unreliable
  • N. europaea liquid culture has turbidity
  • N. europaea plates are not growing colonies
  • Created 50% glycerol stocks of DH5 alpha E. coli with VhB, Nar, and Sod
  • Pc. denitrificans liquid culture started
 Lab work:
  • Miniprep for three promoters (VhB, Sod, Nar) completed and stored at -20 degrees C
  • 50% Glycerol stocks made from remaining liquid culture
  • Competent Pc. denitrificanscells made
 Lab work:
  • No experiments performed this day
 Lab work:
  • Transformed DH5 E. coli with Pc. denitrificans Tat w/ GFP, N. europaea Tat w/ GFP, RFP and plated transformed cells
  • Performed PCR on BBa_E0240 to amplify 4 uL of DNA
      • Anneal temp was set at 55 degrees C and Elongation/melt temp at 95 degrees C, 30 cycles
  • Pc. denitrificans liquid culture in SGM17 + glycine: Centrifuged, washed with sucrose washing solution, pellet resuspended
 Lab work:
  • Plated N. europaea on N. europaea and LB plates
  • Made liquid precultures of N. europaea tat w/gfp, Pc. denitrificans tat w/ gfp, RFP in DH5 E. coli
  • Electroporation Transformation of Pc. denitrificans
      • 6 transformations with Nar (3 with glycine, 3 without glycine)
      Plated transformations on SR plates with ampicillin
 Lab work:
  • Completed miniprep of DH5 alpha with Pc. denitrificans tat w /gfp, N. europaea tat w/ gfp, RFP
  • Made 6 glycerol stocks each for Pc. denitrificans tat w /gfp, N. europaea tat w/ gfp, RFP
  • Made LB plates with no antibiotics
Lab work:
  • Made SR plates and media
 Lab work:
  • No experiments performed this day
 Lab work:
  • Biobrick Formation of TAT reporter circuits: Restriction Digests
      • Digested 2.2 uL of N. europaea TAT DNA with E and S
      Digested 2.0 uL of Pc. denitrificans TAT DNA with E and S
      Digested two separate reactions of 1.2 uL RFP Part DNA with E and X
  • Biobrick Formation of TAT reporter circuits: Gel Separation
      • 4 uL loading dye for 20 uL restriction digest
      Used 0.5 uL ladder and 4 uL dye, with rest molecular grade water for 20 uL total
      Left to Right: Ladder, Empty, Pc. denitrificans TAT, Pc. denitrificans TAT undigested, N. europaea TAT, N. europaea TAT undigested, RFP, RFP, RFP undigested
  • Biobrick Formation of TAT reporter circuits: Ligation Reaction
      • Performed using 4.5 uL of insert, 3 uL of the vector, 10.5 uL water, 0.75 uL T4 ligase, 2 uL 10X buffer
  • Started Pc. denitrificans liquid cultures
 Lab work:
  • Repeat Biobrick transformation of TAT reporter circuits
      • 5uL of DNA, 50 uL of competent cells
      Performed using two different protocols (with and without SOC media)
 Lab work:
  • Calibrated dissolved oxygen probe
  • Replated N. europaea tat and Pc. denitrificans tat both with RFP
  • Replated BBa_E0240 from glycerol stock to miniprep again
  • Transform Nar into Pc. denitrificans cells with electroporation
 Lab work:
  • HAO and CYT plasmids transformed into E. Coli DH5a and placed in the 37 degree C incubator
  • BBa_E0240 plated
  • Made negative controls for all liquid media
  • Pc. denitrificans with Nar grown without glycine had growth, Pc. denitrificans with Nar grown with glycine had no growth
 Lab work:
  • Biobrick Formation of GFP and Promoters: Restriction Enzyme Digest
      • Insertion of 4 promoters (SOD, VhB, pBAD & T7) into GFP Generator backbone
      GFP part digested with E & X (4 replicates); Promoters each digested with E & S
  • Biobrick Formation of GFP and Promoters: Gel Electrophoresis
      • Promoters ran off gel
  • HAOB transformed into E. coli DH5
  • Started preculture of Pc. denitrificans
  • Started precultures for Pc. denitrificans TAT and N. europaea TAT transformations
 Lab work:
  • Created three gels with six wells each
  • Miniprepped HAO and CYT
      • Made 50% glycerol stocks with remaining preculture
      Nanodrop: HAO-628.9 ng/uL; CYT-450.4 ng/uL
  • Started preculture HAOB
 Lab work:
  • No experiments performed this day
 Lab work:
  • Prepared two 0.8% agarose gels and one 2.0% agarose gel
 Lab work:
  • Measured OD to create Pc. denitrificans growth curve, ran into issues with nanodrop
  • Created 4% Nusieve GTG agarose gel
 Lab work:
  • Miniprepped HaoB, Pc. denitrificans TAT, and N. europaea TAT
  • Seaplaque gel: ladder, uncut gfp, 4x cut GFP
  • Nusieve gel: ladder, uncut VhB, cut VhB, uncut Sod, cut Sod, uncut T7, cut T7
 Lab work:
  • Miniprep RFP from the liquid E. coli culture
  • Plated Sod and VhB from glycerol stock
  • Prepared preculture for GFP using LB + Chloramphenicol
  • Transformed Pc. denitrificans TAT GFP + RFP and N. europaea TAT GFP + RFP into E. coli
 Lab work:
  • Miniprepped GFP
  • Ligated GFP vector with arabinose, Sod, and VhB inserts
  • Created LB+AMP, LB+Chloramphenicol, Pc. denitrificans Media
  • Sequenced Pc. denitrificans TAT with RFP and N. europaea TAT with RFP
 Lab work:
  • Miniprepped and made glycerol stocks for Sod, VhB, Pc. denitrificans TAT with GFP and RFP,N. europaea TAT with GFP and RFP
  • Transformed and plated Sod with GFP, arabinose with GFP, VhB with GFP, and control in E. coli DH5
 Lab work:
  • Created Pc. denitrificans growth curve
 Lab work:
  • Biobrick formation of inserts into standard vectors: Restriction Enzyme Digest
      • Inserts: Pc. denitrificans TAT GFP + RFP (500 ng); N. europaea TAT GFP + RFP (500 ng)
      Vector: pSB1C3 (250 ng)
      Enzyme Digest: Inserts and vectors cut with E & P (0.5uL each)
  • Biobrick formation of inserts into standard vectors: Gel Electrophoresis
      • Ladder, spacer, Pc. denitrificans TAT (undigested), Pc. denitrificans TAT, spacer, N. europaea TAT (undigested), N. europaea TAT
  • Biobrick formation of inserts into standard vectors: Ligation
      • Digested with 1uL Vector, 1.8 uL insert
      Created control with vector and no insert
      Incubated at room temperature overnight
 Lab work:
  • Transformation and plating of TAT sequences from the previous day in E. coli
  • Created SeaPlaque gels
  • Miniprepped SOD+GFP, Arabinose+GFP, VhB+GFP
  • Biobrick formation of HAO and CYT with promoters: Restriction Enzyme Digest
      • Inserts: VhB, SOD & arabinose promoters (500ng each)
      Vectors: Cytochromes, HAO full operon (250ng each)
      Enzyme Digest: Inserts cut with E & X, vectors cut with E & X
  • Biobrick formation of HAO and CYT with promoters: Gel Electrophoresis
      • SeaPlaque 0.8% for 1 hour at 60V
      Nusieve 4% for 2 hours at 40V
 Lab work:
  • Transformation of VhB, SOD, Arabinose with HAO and VhB, SOD, Arabinose with CYT into E. coli
  • Created precultures from plated TAT sequences
 Lab work:
  • Observed plates from the previous day
      • Top row: HAO + Sod, Cyt + Vhb, Cyt + arab, HAO ligation control
      Bottom row: Cyt + Sod, HAO + Vhb, HAO + arab, Cyt ligation control
  • Miniprep of N. europaea TAT GFP + RFP and Pc. denitrificans TAT GFP + RFP
 Lab work:
  • Miniprep of Promoters + CYT/HAO
  • Preculture of SOD/HAO biobrick
  • Biobrick Formation via 3A Assembly into pSB1k3: arab/HAO + arab/CYT; VhB/HAO + VhB/CYT
  • Restriction Enzyme Digest
      • 300 ng of insert 1 (digested with 0.5uL of E and S), 300 ng of insert 2 (digested with 0.5 uL of X and P), 100 ng of vector (digested with 0.5 uL of E, P & DpnI)
      2 uL Cutsmart, 20uL total rxn volume for master mix
      Incubated at 37 degrees C for 30 min, heat kill at 80 degrees C for 20 min, ligation overnight
 Lab work:
  • Transformation of HAO+CYT+VhB and HAO+CYT+Arab in E. coli DH5
  • Miniprep of HAO+SOD
  • Transformed Pc. denitrificans with VhB+GFP, Sod+GFP, Arab+GFP, N. europaea tat/Pc. denitrificans tat+RFP
  • New N. europaea culture started
 Lab work:
  • Re-transformed HAO/CYT with promoters(Vhb/Arab) into E. coli DH5
 Lab work:
  • 3A Assembly of HAO/Cyt + arab/sod promoters in PSB1K3: Restriction Enzyme Digest
      • 300ng of HAO+promoter, 300ng of Cyt+promoter, 100ng of pSB1K3
      30 min at 37 degrees C; heat kill at 80 degrees C for 20 min
  • 3A Assembly of HAO/Cyt + arab/sod promoters in PSB1K3: Two Ligation Reactions
      • 2uL of digest 1, 2uL of digest 2, 2uL of vector, 1uL of 10X Ligase buffer, 0.5uL of Ligase
      One set at 16 degrees C for 30 min, heat shock at 80 degrees for 20 min; other set overnight at room temperature
 Lab work:
  • Adjustments to ligation and transformation protocol:
      • Ligation: 30 min insert 1 and vector in ligation reaction, 30 min add insert 2 to reaction
      Transformation: 5uL of ligation product + 100uL of competent cells, add 1 mL of SOC media, spin down (8000 rpm for 1 min), aspirate 800-900mL
 Lab work:
  • Due to nonideal growth of transformed Pc. denitrificans on SR+Chlor plates, re-transformed Pc. denitrificans with ampicillin backbone TAT sequences and plated the transformation products on LB+Amp plates
 Lab work:
  • Gibson Assembly of AMO: Restriction Enzyme Digest + Ligation with pSB1C3 vector
 Lab work:
  • No experiments performed this day
 Lab work:
  • Transformation of XL1 blue cells: 20 uL AMO Bricks, 2 uL control gibson, 2 uL BBa_E0240 control, 2 uL water
Lab work:
  • Gibson Assembly failed, no growth on plates
 Lab work:
  • Biobrick Assembly of CYT+SOD, CYT+VhB, CYT/HAO+VhB in pSB1C3 vector: Restriction Enzyme Digest
      • Method 1: Digest 250 ng of pSB1C3 with E & P, Digest 500ng of insert (promoter+cytochrome) with E & P
      Method 2: Digest Cytochrome+VhB with X & P, Digest HAO+Vhb with E & S, Digest pSB1C3 with E & P
  • Biobrick Assembly of CYT+SOD, CYT+VhB, CYT/HAO+VhB in pSB1C3 vector: Gel Electrophoresis
      • Ladder, spacer, HAO + VHB cut, uncut, spacer, cytochrome + Vhb cut, uncut, spacer GFP cut, uncut
      Ladder, spacer, cytochrome + SOD cut, uncut, spacer, Vhb cut, uncut, spacer, pSB1C3
      Ligation overnight at room temperature
Lab work:
  • Biobrick Formation of VhB+GFP
  • Gibson Assembly of amoABC with pSB1C3 backbone
 Lab work:
  • One step biobrick formation of VhB+HAO/Cyt failed, nothing grew on the plates
  • Characterization of chassis Pc. denitrificans: kanamycin resistance
 Lab work:
  • Grow up glycerol stocks for:
      • Measurement GFP: Arab+GFP, Pc. denitrificans TAT, N. europaea TAT
      Biobrick formation: Arab+HAO, Arab+Cyt, VhB+HAO, VhB+Cyt
  • Pre-culture of VhB+GFP in pSB1C3
  • Transform AMO and pSB1C3
  • Biobrick formation of Sod+HAO and Sod+Cyt in pSB1C3
 Lab work:
  • AMO+pSB1C3 assembly failed
  • Miniprep GFP+VhB in pSB1C3
  • Transform SOD+HAO+Cyt, SOD+Cyt, and VhB+Cyt
 Lab work:
  • No experiments performed this day
 Lab work:
  • Miniprep and Nanodrop: VhB+Cyt, SOD+Cyt, VhB+HAO/Cyt
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • Assembled Arab+HAO+Cyt
 Lab work:
  • Gibson Assembly attempt
      • 2uL of each fragment (50ng/uL) and 2uL of pSB1C3(25ng/uL), 12uL mastermix
      thermocycle 50 degree C for 1 hour
 Lab work:
  • Gibson Assembly failed
 Lab work:
  • No experiments performed this day
 Lab work:
  • Culture N. europaea in dark in N. europaea media
      • Check morphologies under microscope, looks like N. europaea
  • Culture Pc. denitrificans in Pc. denitrificans media
 Lab work:
  • OD of Pc. denitrificans taken: Morning-0.03; Afternoon-1.05
  • OD of N. europaea taken: Morning-0.124; Afternoon-0.254
  • N. europaea and Pc. denitrificans media made
 Lab work:
  • Pc. denitrificans preculture made
 Lab work:
  • No experiments performed this day
 Lab work:
  • Transformation of AMO + pSB1C3
  • Competent Cell Check + Transformation of Pc. denitrificans
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
  • Looking into PCR and purification and getting ready to perform a test run with previously ordered primers for amplifying pSB1C3
 Lab work:
  • PCR for pSB1C3 vector using primer SB-prep-3P-1&2Ea
 Lab work:
  • Nanodrop of PCR product
  • Run gel with PCR product
 Lab work:
  • No experiments performed this day
 Lab work:
  • Pc. denitrificans Competent Cell Preparation
 Lab work:
  • Monitor OD of Pc. denitrificans culture and perform Competent Cell protocol
 Lab work:
  • Culture of Sod in E. coli and BBa_K608010 in E. coli
  • Pc. denitrificans transformation with GFP
 Lab work:
  • No experiments performed this day
 Lab work:
  • No experiments performed this day
 Lab work:
  • Performed PCR
  • Performed transformation
Lab work:
  • Competent Cell Check of Pc. denitrificans with GFP via electroporation
 Lab work:
  • Failed transformation, possible issue with electroporator
  • Planning for PCR of AMO fragments
  • Interlab Study data uploaded
  • Safety forms submitted
 Lab work:
  • Gibson Assembly of AMO
 Lab work:
  • PCR Purification of Gibson Assembly of AMO
 Lab work:
  • Growth of SOD and VhB promoters
  • Created glycerol stocks of the SOD and VhB promoters
  • Ran a miniprep of the SOD and VhB promoters
 Lab work:
  • Tested SR based plates
 Lab work:
  • Failed transformation with both tests
  • Positive control of empty chloramphenicol backbone was successful
  • Tested ampicillin and chloramphenicol with LB
 Lab work:
  • Nanodrop of AMO fragments PCR products
  • Gibson Assembly
  • Run gel of Gibson Assembly products
 Lab work:
  • Plated Pc. denitrificans
 Lab work:
  • Precultured Pc. denitrificans
 Lab work:
  • Cultured Pc. denitrificans in 1L Minimal Media
 Lab work:
  • Successful growth of transformed Pc. denitrificans with GFP+Arab
      • Precultured to be made into glycerol stocks
  • Monitor OD of Pc. denitrificans
  • Plated DH5a untransformed cells and DH5a cells containing medium promoter biobrick from distribution kit, our MN SOD GFP reporter construct, and the Vhb GFP reporter construct
  • All transformed cells were plated in LB+chloramphenicol media and transformed cells in plain LB media
  • Plates left overnight for single colony growth
  • PCR amplification of Gibson Assembly product- AMO (3ng/ul to 10 ng/ul)
 Lab work:
  • Pc. denitrificans Control Experiment
      • 1L culture reached OD 0.3
      Culture split into three 294ml aliquots
      Three volumetric flasks labeled Flask 1, 2, and 3 each receive one 294ml aliquot
      Initial ODs of each 294ml flask taken
      Initial [NH4+ ], [NO3+], and [DO] recorded for each flask at two minute intervals
      At t=0 minutes, 6ml [NH4] spike solution added to each flask
      Theoretical [NH4] of each flask brought to 50mM, an established value for average [NH4] in wastewater treatment plants
      [NH4], [NO3], and [DO] measured in each flask at intervals of two minutes
      At each two minute, three measurements are taken, one for each flask
      After the measurement, each sensor is washed with deionized water and rotated to the next flask
      This experimental design staggers measurements so that accurate changes in nitrogen containing compounds are measured
      Rotation and measurement proceeds until t=90 minutes
  • Plate N. europaea
  • Single colonies were picked from plates at approximately 10:00 pm and suspended in 50mL liquid cultures of the same volume
  • Cultures were allowed to grow overnight
 Lab work:
  • Precultured N. europaea
  • Overnight cultures were transferred into new media which was allowed to grow for 6 hours before being divided into 3.5 mL portions for experimentation
  • Every 1 hour, a set of tubes was covered in parafilm to simulate deprivation of oxygen
  • At the end of 6 hours, all tubes were tested with the DO probe and the readings recorded
  • All tubes then had a 1mL sample extracted for DO reading in cuvettes
  • Then a 200uL sample of each tube was put into a PCR optical well plate for testing
 Lab work:
  • Cultured N. europaea in 1L Minimal Media
 Lab work:
  • N. europaea Control Experiment
      • 1L culture reached OD 0.3
      Culture split into three 294ml aliquots
      Three volumetric flasks labeled Flask 1, 2, and 3 each receive one 294ml aliquot
      Initial ODs of each 294ml flask taken
      Initial [NH4+ ], [NO3+], and [DO] recorded for each flask at two minute intervals
      At t=0 minutes, 6ml [NH4] spike solution added to each flask
      Theoretical [NH4] of each flask brought to 50mM, an established value for average [NH4] in wastewater treatment plants
      [NH4], [NO3], and [DO] measured in each flask at intervals of two minutes
      At each two minute, three measurements are taken, one for each flask
      After the measurement, each sensor is washed with deionized water and rotated to the next flask
      This experimental design staggers measurements so that accurate changes in nitrogen containing compounds are measured
      Rotation and measurement proceeds until t=90 minutes
Lab work:
  • Plated E. coli
 Lab work:
  • Confirmatory digest of promoter-enzyme-terminator plasmid miniprepped on 10/9
  • Sequencing of promoter-enzyme-terminator plasmid miniprepped on 10/9
  • Made minimal medium plates with nothing, 3mM leucine, or 3mM CBZ-leu
  • Precultured E. coli
 Lab work:
  • Cultured E. coli in 1L Minimal Media
 Lab work:
  • digest, gel, ligation of parts into CAM backbones
  • E. coli Control Experiment
      • 1L culture reached OD 0.3
      Culture split into three 294ml aliquots
      Three volumetric flasks labeled Flask 1, 2, and 3 each receive one 294ml aliquot
      Initial ODs of each 294ml flask taken
      Initial [NH4+ ], [NO3+], and [DO] recorded for each flask at two minute intervals
      At t=0 minutes, 6ml [NH4] spike solution added to each flask
      Theoretical [NH4] of each flask brought to 50mM, an established value for average [NH4] in wastewater treatment plants
      [NH4], [NO3], and [DO] measured in each flask at intervals of two minutes
      At each two minute, three measurements are taken, one for each flask
      After the measurement, each sensor is washed with deionized water and rotated to the next flask
      This experimental design staggers measurements so that accurate changes in nitrogen containing compounds are measured
      Rotation and measurement proceeds until t=90 minutes
 Lab work:
  • Took overnight cultured plates and suspended in LB or LB+Chloramphenicol media at 12:30
  • Removed from media at 4:30 and was able to get all cultures into test tubes by 5:00, volume for each tube was ~3.5 mL
  • First round of caps put on at 5:00
  • Took OD 600 of each culture
  • Capped another set of tubes every hour
  • Day 2 VhB and DH5 alpha swapped
  • Shaker settings: 37 degrees C, 220 rpm
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