iGEM 2017 — Team Virginia

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# Team:Virginia/Notebook

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# Notebook

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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
* ![](https://static.igem.org/mediawiki/2017/3/36/Try_1_CCC--Virginia.png)

Lab work:

* Second transformation attempt with the same competent cell stock was successful with ideal results
* ![](https://static.igem.org/mediawiki/2017/9/91/Try_2_CCC_Plates--Virginia.jpeg)
* Competent cell check calculations
* ![](https://static.igem.org/mediawiki/2017/1/11/CCC_Calculations--Virginia.png)

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

![](https://static.igem.org/mediawiki/2017/1/12/Two_Phenotypes--Virginia.jpeg)
* 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)

![](https://static.igem.org/mediawiki/2017/1/1c/Gram_Collage.png)
* 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. denitrificans*cells 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

![](https://static.igem.org/mediawiki/2017/9/9d/Seaplaque_Gel_1--Virginia.jpeg)
* Nusieve gel: ladder, uncut VhB, cut VhB, uncut Sod, cut Sod, uncut T7, cut T7

![](https://static.igem.org/mediawiki/2017/a/a4/Nusieve_Gel_1.jpeg)

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
* ![](https://static.igem.org/mediawiki/2017/2/2f/PD_growth_curve_1--Virginia.png)

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

![](https://static.igem.org/mediawiki/2017/3/3b/TATs_gel--Virginia.jpeg)
* 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

![](https://static.igem.org/mediawiki/2017/b/b9/HAO_CYT_promoters--Virginia.jpeg)
* 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

![](https://static.igem.org/mediawiki/2017/f/fa/Gel_1--Virginia.jpeg)

Ladder, spacer, cytochrome + SOD cut, uncut, spacer, Vhb cut, uncut, spacer, pSB1C3

![](https://static.igem.org/mediawiki/2017/2/2f/Gel_2--Virginia.jpeg)

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:

* Flux Balance Analysis discussed with Professor Papin and finalized.

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
* Modeling: Flux Variability Analysis suggests that several essential reactions are inevitably improved by inserting the nitrification device. The goal is to find which reaction is the sort of "limiting reaction" that gets enhanced, resulting in all other flux enhancements.
* Modeling: Secondary goal: check the presence and correctness of denitrification reactions already present in the model.

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:

* No experiments performed this day

Lab work:

* Modeling: metabolic script was made more user-friendly (documentation added).* Modeling: introduced individual and simultaneous knockouts, mostly for validation purposes. Should we add NO-reductase to the model?* Modeling: More research into COBRA gapfilling and *escher*

Lab work:

* Competent Cell Check of *Pc. denitrificans* with GFP via electroporation

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

Lab work:

* Failed transformation, possible issue with electroporator

* Planning for PCR of AMO fragments
* Interlab Study data uploaded
* Safety forms submitted
* Modeling: dipeptide uptake changes significantly in the synthetic strain. A mystery to be solved?

Lab work:

* Gibson Assembly of AMO

Lab work:

* PCR Purification of Gibson Assembly of AMO

Lab work:

* No experiments performed this day

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:

* No experiments performed this day

Lab work:

* Failed transformation with both tests
* Positive control of empty chloramphenicol backbone was successful
* Tested ampicillin and chloramphenicol with LB
* Modeling: troubleshot some issues with metabolic model. When it came to denitrification, turns out it had more holes than swiss cheese! Thinking of how to reconcile environmental conditions with the steady-state model that we have at hand.

Lab work:

* Nanodrop of AMO fragments PCR products
* Gibson Assembly
* Run gel of Gibson Assembly products

Lab work:

* No experiments performed this day

Lab work:

* Plated *Pc. denitrificans*

Lab work:

* Precultured *Pc. denitrificans*

Lab work:

* No experiments performed this day

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)
* Modeling: bioreactor modeling grand prix begins. Some of the references containing important measurements are unavailable because they're hidden behind a ?-wall of some Australian university. Need to figure out how to find the best fit estimates for the parameters in the equations I'm planning to add.

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:

* No experiments performed this day

Lab work:

* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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:

* No experiments performed this day

Lab work:

* No experiments performed this day
* First poster templates were developed* Modeling: thinking through stoichiometric matrix for the amended biorecator model. Figuring out how to do parameter fitting.

Lab work:

* No experiments performed this day

Lab work:

* No experiments performed this day

Lab work:

* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

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* No experiments performed this day

Lab work:

* Some bricks transformed into some *E. coli** Top-left part of the poster is finished, at least tentatively

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
* More poster editing and modeling

Lab work:

* Modeling: parameter fitting for DE's fails -- solver stalls. Can we brute-force it?

Lab work:

* No experiments performed this day

Lab work:

* No experiments performed this day

Lab work:

* Modeling: after some thinking, brute-force on anything that's larger than 3-4 parameters is just silly. Absolutely don't want to simulate 10^18 nonlinear solutions, a lot of which are going to stall anyway. Eh.

Lab work:

* SCOOPED: Discovery of a paper that says HAO does **not** work the way we originally thought

Lab work:

* Modeling: finished the modified model after the talk. Searching for some interesting things to simulate. Parameters were fit using a submodel.

Lab work:

* No experiments performed this day

Lab work:

* No experiments performed this day

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* No experiments performed this day

| May | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **S** | **M** | **T** | **W** | **T** | **F** | **S** |
|  | 1 | 2 | 3 | 4 | 5 | 6 |
| 7 | 8 | 9 | 10 | 11 | 12 | 13 |
| 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 21 | 22 | 23 | 24 | 25 | 26 | 27 |
| 28 | 29 | 30 | 31 |  |  |  |

| June | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **S** | **M** | **T** | **W** | **T** | **F** | **S** |
|  |  |  |  | 1 | 2 | 3 |
| 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 |
| 18 | 19 | 20 | 21 | 22 | 23 | 24 |
| 25 | 26 | 27 | 28 | 29 | 30 |  |

| July | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **S** | **M** | **T** | **W** | **T** | **F** | **S** |
|  |  |  |  |  |  | 1 |
| 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| 9 | 10 | 11 | 12 | 13 | 14 | 15 |
| 16 | 17 | 18 | 19 | 20 | 21 | 22 |
| 23 | 24 | 25 | 26 | 27 | 28 | 29 |
| 30 | 31 |  |  |  |  |  |

| August | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **S** | **M** | **T** | **W** | **T** | **F** | **S** |
|  |  | 1 | 2 | 3 | 4 | 5 |
| 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 13 | 14 | 15 | 16 | 17 | 18 | 19 |
| 20 | 21 | 22 | 23 | 24 | 25 | 26 |
| 27 | 28 | 29 | 30 | 31 |  |  |

| September | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **S** | **M** | **T** | **W** | **T** | **F** | **S** |
|  |  |  |  |  | 1 | 2 |
| 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| 10 | 11 | 12 | 13 | 14 | 15 | 16 |
| 17 | 18 | 19 | 20 | 21 | 22 | 23 |
| 24 | 25 | 26 | 27 | 28 | 29 | 30 |

| October | | | | | | |
| --- | --- | --- | --- | --- | --- | --- |
| **S** | **M** | **T** | **W** | **T** | **F** | **S** |
| 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| 8 | 9 | 10 | 11 | 12 | 13 | 14 |
| 15 | 16 | 17 | 18 | 19 | 20 | 21 |
| 22 | 23 | 24 | 25 | 26 | 27 | 28 |
| 29 | 30 | 31 |  |  |  |  |

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# Team:Virginia/Experiments

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---

# Protocols

---

## *P. denitrificans* Protocols

Competent Cell Preparation

1. Culture P. denitrificans in SGM17 medium until OD600=0.6-0.8.
2. \*\*\*The following procedure should be done in a cold room at 0-4˚C.\*\*\*
3. Harvest by centrifuge 250mL samples each in four centrifuge bottles at 5000xg at 4 C. Decant supernatant
4. Completely resuspend the pellets in 50mL of 0.5 M sucrose solution with 10% glycerol, for each bottle
5. Combine the resuspended cells into one centrifuge bottle,ncounterbalance with water in another centrifuge bottle
6. Centrifuge at 5000xg at 4 C
7. Decant supernatant and resuspend the pellets with 100mL of 0.5 M sucrose solution with 10% glycerol. Counter-balance bottle should have 100mL of water
8. Centrifuge the two bottles at 5000xg at 4 C
9. Decant out supernatant and resuspend the resulting pellets in 10mL of 0.5 M sucrose solution with 10% glycerol
10. Aliquot the cells into microcentrifuge tubes
11. Flash freeze the aliquoted cells with liquid nitrogen and store at -80 C

Transformation via Electroporation

1. Thaw and mix 50 uL portions cells with 5 uL DNA- Transfer to ice cold electroporation cuvette- Electroporate with single pulse set at 25 uF and 2.0 kV for 4.5 to 5 ms- The electroporator as a Gene-Pulser by BioRad connected to a 200-(-) resistor also by biorad- IMMEDIATELY following electroporation: GENTLY resuspend the cells with 0.95 mL ice cold SGM17 containing 20 mM MgCl2, 2 mM CaCl2 and incubate on ice for 5 minutes- Incubate cells at 30 C for 2 hours in a shacking incubator.- Take 100 uL portions and plate.

P. denitrificans Culture

**P. denitrificans Nutrient Agar**1. Combine 23g 3 Nutrient Agar/Broth Agar Medium Nutrient Agar with 1000 mL deionized water, then autoclave
   3g Nutrient Agar Composition Beef Extract, 5g peptone, 15g agar

**P. denitrificans Nutrient Broth**1. Combine 8g Medium Nutrient Broth with 1000 mL deionized water, pH to 6.8 +/- 0.2, then autoclave
   3g Nutrient Agar Composition Beef Extract, 5g peptone

## Measurement Protocols

Mn-Sod, VhB and T7 Promoter Characterization

**Isolate Single colonies**1. Select glycerol stock cultures from -80 C storage to begin- Thaw on ice for 15 minutes and observe when the cell stock begins to melt- Take a plastic p-loop or other spreading instrument and spread on a selective media agar plate containing the appropriate antibiotics
       If the culture is P. denitrificans, culture on PD media or standard LB
       If the culture is E. coli use LB media
       If using untransformed bacteria, equivalent non-selective media should be used
     - Incubate both P. denitrificans and E. coli overnight at 37 C- Isolate single colonies after overnight incubation and resuspend in 60 mL volumes of liquid selective LB media with appropriate antibiotics

**Preculture for Device Testing**1. Allow the resuspended liquid culture to incubate for 4 to 6 hours in a shaking incubator at 37 C and 220 rpm to reach late- to mid-exponential phase of growth, look for an OD 600 value of ~0.6- Obtain a 1 mL sample and measure the OD600. If at a satisfactory level, then proceed with device testing- Divide the 60 mL pre-cultures into 3.5 mL aliquots in 14 mL falcon RB tubes or comparable sterile culture tube
       Prepare one tube for each hour that the planned test calls for, mark each tube with the number of hours it will remain unsealed and permeable to oxygen
       At the start of testing, cover the tubes marked “0 hours” with parafilm and leave sealed for the duration of testing
     - Start incubation in a shaking incubator at 37 C and 220 rpm
       Every hour stop the shaking incubator and cover the next tube with parafilm. Repeat this step until all tubes except the last ones are covered
       Allow the final set of tubes to incubate unsealed for an hour and stop the incubation

**Device Measurements**1. Starting with the unsealed tube, use a vernier Dissolved Oxygen (DO) membrane probe or comparable measurement system to measure the do levels
   Do this for all samples that were left open for a given time interval before moving to the next
   In between DO measurements, clean the probe with ethanol and then rinse with deionized water to minimize cross-contamination between samples
2. Immediately take three 150 uL samples of the culture and dispense into appropriate wells in an optical PCR well plate- Take a 500 uL sample and dilute with 500 uL deionized water for a total volume of 1 mL in a spectrophotometer cuvette to make a final 1:2 dilution. Store on ice if possible.- Once all tubes have been unsealed and measured for DO content, seal the PCR optical well plate with an optical grade adhesive cover- After all samples for optical fluorescence measurement have been taken, remove cuvettes from storage to measure and record OD600 absorbance values

**PCR Fluorescence Measurement**1. Immediately after all samples have been collected and the well plate covered, run a pre-created protocol for fluorescence measurement- The protocol should have three measurement cycles set at 30 seconds and 37 C- After measurements are made by the optical reader, export the data for analysis
       f the Bio Rad MyiQ Optical Reader is used, copy and paste the relative fluorescence unit table presented by the output and export to either a .txt or .excel format

**Disposal**1. Disinfect all Cuvettes with bleach or ethanol before washing with tap/sink water. Then rinse with deionized water and wipe dry- All remaining tubes should be sterilized with bleach or ethanol and drained down an appropriate lab sink. All tubes are to be disposed of in a lab waste receptacle- All PCR well plates should be sterilized disposes of similarly

Measuring Efficiency of Ammonium Conversion and Nitrate Production Over Time

**Minimal Media Preparation**1. Combine:
   1L deionized water
   0.2g MgSO4
   0.8g NH4Cl
   1.0g NH4SO4
   0.5g NaCl
   5.2g K2HPO4
   2.0g KH2PO4
2. Autoclave solution- Add 5.0g glucose

**Experimental Procedure**1. Prepare 1L minimal media- Dissolve 6.68g NH4Cl in 50 mL deionized water (spike solution)- Preculture experimental bacteria overnight in minimal medium at 30oC in an erlenmeyer flask- Culture experimental bacteria overnight in minimal media
         When conducting experiment with pBAD promoter, add 0.15g arabinose 3-4 hours before experimentation begins
       - Remove three aliquots of 294 mL when culture reaches OD=0.3 suspension. Use three separate flasks labeled Flask 1, 2, and 3- Put the experimental flasks on hot plates with magnetic stirrer and stabilize the temperature at 30oC; stirrers function as aerators- Measure initial [ammonium], [nitrate], [DO] concentrations in three beakers using ammonium, nitrate, and dissolved oxygen probe at T=0 (THIS WILL REQUIRE A FULL ROTATION OR 6 MINUTES)- Use 1mL of each sample to measure the OD- Get initial thermometer readings; adjust plates as necessary- After the min 6 sample, quickly spike with 6 mL of spike solution to create 50mM ammonium cultures (culture volume is now 300ml)- At T=6min measure [ammonium], [nitrate], [DO] after spike solution added- Continue to rotate probes between flasks 1, 2, and 3 at two minute intervals for 90 minutes- Measure OD at 30 minute intervals at times t=0, t=30, t=60, t=90

## *N. europaea* Protocols

N. europaea Media Prep

**Step 1**1. Put 900 ml of deionized water in a 2-liter Erlenmeyer flask- Add 3.3 g (NH4)2SO4 (50mM)- Add 0.41 g KH2PO4- Add 0.75 ml 1 M MgSO4 stock solution- Add 0.2 ml 1 M CaCl2 stock solution- Add 0.33 ml 30 mM FeSO4 /50 mM EDTA stock solution- Add 0.01 ml 50mM CuSO4 stock solution- Autoclave solution

**Step 2**1. Put 400 ml of deionized water in a beaker. Add: 27.22 g KH2PO4, 2.4 g NaH2PO4- Adjust pH to 8.0 with 10 N NaOH, and bring it to a final volume of 500 ml with deionized water- Autoclave in 100 ml fractions in 250-500 ml Erlenmeyer flasks

**Step 3**1. Prepare 500 ml of 5% (w/v) Na2CO3 (anhydrous)- Sterilize it in an autoclave

**Growing N. europaea**1. Add 1 x 100 ml aliquot of solution prepared in Step 2 to the flask prepared in Step 1- Now add 8 ml of the solution prepared in Step 3 to the flask prepared in Step 1- Finally add 10 ml of 3-day old culture to the flask prepared in Step 1, and incubate it on a rotary shaker (100-150 RPM) at 30°C in darkness- The bacterium should reach approximately OD600nm~0.1 in three to four days

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# Team:Virginia/Results

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---

# Results

---

#### Summary

---

Our summer project was designed to improve the process of nitrogen removal in the tertiary sewage treatment stage of raw sewage processing. Currently, a fairly common design is to spatially separate the processes of nitrification and denitrification so that distinct bacterial populations within activated sludge can perform their respective biochemical processes independently. Our project proposes that both processes be temporally separated and housed under a single organism. To achieve this, we attempted to transform the genes encoding for proteins responsible for nitrification into *Paracoccus denitrificans*, a known denitrifier in active sludge.

![](https://static.igem.org/mediawiki/2017/8/81/FinalCircuit.png)
By the end of summer experimentation, we were able to transform most, but not all of the nitrification genes into *Pc. denitrificans*. Our central composite circuit consists of genes for HaoA and HaoB, proteins that catalyze the oxidation of hydroxylamine into nitrite ions. It also includes CycA and CycX, genes encoding for two c-type cytochromes involved in shuttling electrons from the ammonia oxidation reactions to terminal electron acceptors. We were, however, unable to create the full AMO circuit from the G-Blocks for AmoA, B, and C into *Pc. denitrificans*. We experienced a lot of difficulty in creating circuits through Gibson Assembly. These genes would have encoded for a heterotrimeric protein complex that would have catalyzed the oxidation of ammonia into hydroxylamine.

In addition to the ammonia oxidation circuit we developed, we also designed a series of reporter circuits to test the function of several promoters in P. denitrificans compared to E. coli. We were able to transform *Pc. denitrificans* with a composite part consisting of Mn-SOD, an oxygen-inducible promoter, and GFP. With this reporter circuit, we attempted to characterize promoter activity in response to different dissolved oxygen levels by measuring relative fluorescence at the absorbance spectra of GFP compared to control untransformed cells.

In addition to Mn-SOD, a new promoter added to iGEM, we also tested the promoter activities of VhB and T7 in transformed E. coli cells. These promoters were connected to the coding sequences for GFP in a composite part. Again, all promoters were tested at various dissolved oxygen levels. These were tested against untransformed E. coli cells. Finally, we accounted for the fact that different oxygen levels may lead to different growth rates, so we divided relative fluorescence units (RFU) values by the OD600 of a given sample that was measured.

#### Characterization of Chassis

---

There were three attempts to generate a well-characterized growth curve for P. denitrificans under our laboratory conditions. The first resulted in a culture reaching a growth plateau at an OD600 of around 0.4. The growth plateau was reached between five and six hours after inoculation with a preculture of Paracoccus cells into a 1 L liquid culture in PD media (see protocol/link). Growth was completely plateaued by ten hours. The error bars shown are 95% confidence intervals. The OD600 values of this round of measurement was done with a NanoDrop machine that was potentially not capable of accurately determining the cell density.

![](https://static.igem.org/mediawiki/2017/0/0e/Virginia_PDGC1_David.png)

The second attempt yielded extremely different results. Again, the culture was grown in a shaking incubator at 33.0 C at 220 rpm. The same PD media was used to grow the culture. The plateau was at an OD600 between 2 and 2.5, Exponential growth ended at 10 hours just as it did in the first trial. Mid-exponential growth was reached at four to five hours just as in the first trial as well. This round of measurement was done with Spectronic Genesys 2, which detected the OD600 of the cell culture more precisely than NanoDrop did.

![](https://static.igem.org/mediawiki/2017/3/35/Virginia_PDGC2_David.png)

The last attempt was unsuccessful. The culture reached a plateau at an OD600 of ~0.15 before gradually falling to 0.13. This failure was indicative of the challenge in maintaining a stable P. denitrificans culture.

![](https://static.igem.org/mediawiki/2017/9/9b/Virginia_PDGC3_David.png)

As a conclusion, the P.D. incubated in 1L of P.D. medium would have exponential growth in the during the first 10 hours of incubation and would then reach the carrying capacity after 20 hours. The OD600 value went up to around 1.8~2.0 at the end of the experiment after the growth curve plateued. For the purpose of P.D. competent cell preparation, 1L of P.D. medium culture will reach the ideal OD600=0.6~0.8 after approximately 5 hours of incubation.

##### Mn-Sod

While Mn-SOD has been described in literature as an oxygen-inducible promoter element in *E. coli*, the results of experimentation described under protocols (citation, link) have been inconclusive. Following the previously described protocol, bacterial cell cultures were grown from single colonies for four hours to reach early- to mid-exponential growth phase (shown to be ~4 hours in the literature and our own results). After four hours, colonies were transferred into 14 mL tubes. Because equipment to modulate the dissolved oxygen environment was not available, tubes were covered after a specified amount of time and kept closed until the end of experimentation to simulate differential oxygen levels. Bacteria in tubes that are covered for longer periods of time will consume the available oxygen over time, resulting in lower dissolved oxygen levels. After the experimental window closes, all relevant parameters were measured (DO, OD600, and RFU output from the optical plate reader) simultaneously.

The first trial was conducted on 9/25/2017 and cultures were grown for a total of four hours before being transferred into 14 mL falcon tubes. At four hours, tubes were opened and tested for DO with a Vernier Instruments DO probe and for OD600 with a Genesys 2 spectrophotometer in the order from most hours spent open to least. The reasoning behind this is that the measurement process was time-consuming and exposing cultures that had spent more time open to atmospheric oxygen would minimize the bias caused by re-exposure, as they would be less sensitive than cultures that had more time in an anaerobic environment to changes caused by unsealing tubes. Immediately after all tubes were opened and samples were taken from each strain and time point, the well plate was sealed with an an optical adhesive cover and measured by a BioRad MyiQ optical plate reader.

When plotted against hours spent open and exposed to open atmosphere, a steady increase in the relative fluorescence can be observed. When compared against the DO content (in mg/L), however, the opposite trend is observed. For the relationship between DO and RFU, the coefficient of determination is 0.445, corresponding to a correlation coefficient of -0.667. This indicates that the observed RFU of bacteria containing the Mn-SOD reporter biobrick is negatively correlated with DO with a linear relationship of moderate strength. The relationship between the RFU and time spent exposed to atmospheric oxygen, while positive, was much weaker. The coefficient of determination (R2) was only 0.023, corresponding to a correlation coefficient (R) of 0.152. This is indicative of a weak linear relationship.

![](https://static.igem.org/mediawiki/2017/6/63/Virginia--MnSod1-1.png)

![](https://static.igem.org/mediawiki/2017/5/54/Virginia--MnSod1-2.png)

**Figure 1:** OD600 normalized RFU values between all measurement replicates over time and DO saturation/content (mg/L).

The same pattern is also observed in results taken from the second trial on 10/22/17. This time, bacterial cell cultures were grown at 37 C in a shaking incubator (220 rpm) for four hours before being transferred into smaller 3.5 mL culture volumes. All aliquots were grown and a new tube was covered every hour as described above, except cultures were grown for a total of six hours. As in the DH5a strain tested on 9/25/17, the strain transformed with the Mn-SOD reporter plasmid showed increasing fluorescence over time but a decreasing RFU value as the observed DO content increased. The coefficient of determination and correlation coefficient for the relationship between RFU value and time spend open were 0.503 and 0.709 each. This indicates that the relationship is positive and moderately strong. The relationship between DO and RFU values was negative and the coefficient of determination and correlation coefficient were 0.374 and -0.612. This is indicative of a moderately strong negative linear relationship.

![](https://static.igem.org/mediawiki/2017/d/d1/Virginia--MnSod2-1.png)

![](https://static.igem.org/mediawiki/2017/e/ef/Virginia--MnSod2-2.png)

**Figure 2:** Results from the trial performed over 10/22/17

Again, the observation is made that as the duration spent opened increases, the level of relative fluorescence picked up by the optical reader does as well. It is also evident in **Figure 2** that as the dissolved oxygen content increases, the relative fluorescence decreases.These results both contradict and support our original hypothesis and indicate that an unmeasured effect is causing the DO content to decrease in cultures that spend more time exposed to atmospheric oxygen.

![](https://static.igem.org/mediawiki/2017/a/af/Virginia--MnSod3-1.png)

![](https://static.igem.org/mediawiki/2017/a/a4/Virginia--MnSod3-2.png)

**Figure 3:** Aggregated data for Mn-SOD in *Pc. denitrificans* across both trials

Combining both trials yields stronger evidence for the trends described above, as a positive linear trend is also observed when plotting relative fluorescence against time spent exposed to atmospheric oxygen. In addition, the plot of RFU versus DO shows weaker linear relationship with coefficient of determination and correlation coefficient of only 0.085 and 0.292 when both trials are aggregated into a single dataset. This is weaker than the observed relationship between RFU value and time spent open, which had corresponding R2 and R values of 0.341 and 0.584. This indicates a moderate linear relationship between RFU value and time spent open.

In addition, Mn-SOD was tested in *Pc. denitrificans* and the results corroborated claims in the literature and results found by experimentation. The R and R2 values for the relationship between RFU value and time spent open were 0.198 and 0.445. The relationship between DO content and RFU value had R and R2 values of 0 and 0. These results were mildly positive and indicate to us that Mn-SOD generally behaves the same way in *Pc. denitrificans* as it is reported to in *E. coli*.

![](https://static.igem.org/mediawiki/2017/0/05/Virginia--MnSod4-1.png)

![](https://static.igem.org/mediawiki/2017/f/fa/Virginia--MnSod4-2.png)

**Figure 4:** Scatterplots of relative fluorescence versus time and dissolved oxygen content

##### VhB

VhB has been described in literature and previous iGEM projects as being maximally active in hypoxic conditions, ideally at 2% dissolved oxygen saturation. When tested with the above describe protocol, results are inconclusive. The first trial was conducted on 9/25/17 and yielded positive data that corroborated previous evidence of maximal induction under hypoxic conditions. As time the tube spent open increased, measurements of RFU decreased, indicating inhibition of GFP expression. This followed a linear relationship with R and R2 values of 0.044 and -0.210. There was almost no observed relationship between relative fluorescence and dissolved oxygen content, which had R and R2 values of 0.022 and 0.148. Both relationships were very weak and indicate little relationship between RFU value and either DO or time.

![](https://static.igem.org/mediawiki/2017/1/10/Virginia--MnSod5-1.png)

![](https://static.igem.org/mediawiki/2017/6/6f/Virginia--MnSod5-2.png)

**Figure 5:** Scatterplots of relative fluorescence versus time and dissolved oxygen content DH5a E. coli transformed with the VhB reporter biobrick.

When tested on 10/22/17, there is a clear positive relationship between time spent exposed to atmospheric oxygen and the intensity of the relative fluorescence. These data are not only contradictory to the other data collected on 9/25/17, but it is also contradictory to the results found in literature and by other iGEM teams. Because of this, it is reasonable to conclude that there may be a bias in the measurement of fluorescence emitted by cells expressing the VhB reporter biobrick. The relationship between time and RFU value had R and R2 values of 0.369 and 0.607. This is a moderately strong relationship. The relationship between DO and RFU was almost negligible and its R and R2 values were only 0.007 and -0.0837.

![](https://static.igem.org/mediawiki/2017/8/8a/Virginia--MnSod6-1.png)

![](https://static.igem.org/mediawiki/2017/4/4f/Virginia--MnSod6-2.png)

**Figure 6:** Scatterplots of relative fluorescence versus time and dissolved oxygen content DH5a E. coli transformed with the VhB reporter biobrick.

When all data is aggregated between trials, the R and R2 values are much stronger. For the relationship between RFU and time, they are 0.393 and 0.627. For RFU and DO they are 0.236 and 0.486. It must be stressed, however, that these results are contradictory to previous findings and there is strong likelihood of bias in the results. One potential source may be due to the actual versus observed DO level. Volumes used for testing were smaller than the volume recommended for use by the Vernier DO probe. There was also the issue of the time taken to complete all required measurements. The total time between removal of samples from the shaking incubator and the start of the qPCR protocol to measure relative fluorescence was between one and one and a half hours. In that time, gene transcription and translation could have changed significantly.

##### DH5a Untransformed

In order to compare expression against a control baseline, we also measured the fluorescence of an untransformed culture of DH5a E. coli cells. When a line of best fit was calculated for the data, there was almost no significant relationship between observed relative fluorescence and either the dissolved oxygen content and the time spent open and exposed to the atmosphere. This was most evident when the data from both replications were aggregated. The coefficient of determination for relative fluorescence versus dissolved oxygen content was 0.008. The coefficient of determination for relative fluorescence versus time spent open is 0.041. These values correspond to correlation coefficients of 0.0894 and 0.202 respectively. These are both very weak linear relationships.

![](https://static.igem.org/mediawiki/2017/7/79/Virginia--MnSod7-1.png)

![](https://static.igem.org/mediawiki/2017/5/5f/Virginia--MnSod7-2.png)

**Figure 7:** Aggregated data from both trials for fluorescence measurements of untransformed DH5a E. coli.

When the data was split by trial run, however, there were also contradictory findings. Data from the first trial conducted on 9/25/17 suggest a positive relationship between relative fluorescence and hours spent open and exposed to atmospheric oxygen, while the relationship between dissolved oxygen and relative fluorescence was close to nonexistent. The coefficients of determination for each were 0.198 and 0.094. These correspond to correlation coefficients of 0.444 and 0.306 respectively. This means the relationship between relative fluorescence and hours spent open to the atmosphere is moderately strong while the relationship between relative fluorescence and dissolved oxygen content is weak.

![](https://static.igem.org/mediawiki/2017/a/a1/Virginia--MnSod8-1.png)

![](https://static.igem.org/mediawiki/2017/8/8c/Virginia--MnSod8-2.png)

**Figure 8:** Scatterplots with trends fit to the data for untransformed DH5a bacteria.

Results from the second trial conducted on 10/22/17 indicate that time spent exposed to atmospheric oxygen has an opposite effect on relative fluorescence. There is a moderate to weak relationship between time and relative fluorescence. The relationship between relative fluorescence and dissolved oxygen content, however, is non-existent just as it is in the first trial. The coefficients of determination were found to be 0.207 and 0.017. These correspond to correlation coefficients of 0.455 and 0.130. This means a weak to moderate negative relationship between dissolved oxygen and relative fluorescence and a weak relationship between relative fluorescence and dissolved oxygen.

![](https://static.igem.org/mediawiki/2017/5/5c/Virginia--MnSod9-1.png)

![](https://static.igem.org/mediawiki/2017/7/7f/Virginia--MnSod9-2.png)

**Figure 9:** Scatterplots with trends fit to the data for untransformed DH5a bacteria.

#### Testing Device Efficacy by Monitoring Ammonium Removal and Nitrate Production

---

Results from ammonium flux indicate that the Paracoccus denitrificans (PD) transformed with HAO/cytochrome oxidizes ammonium more effectively than untransformed PD, but less effectively than Nitrosomonas europaea (NE), which is a native nitrifier [NE -2.79 <-1.86 PD (transformed) < -1.33 PD (untransformed)].

![](https://static.igem.org/mediawiki/2017/8/82/Ammonium_Concentration_over_Time.png)

**Figure 10:** Illustrates change in ammonium concentration following spike to 50mM ammonium over a 60 minute interval. Depicts average change for three trials of each bacteria, PD, NE, and PD (transformed). Data supports the hypothesis that the integration of HAO/cyt gene complexes into PD increases its nitrification capacity.

![](https://static.igem.org/mediawiki/2017/7/76/Nitrate_Production_Over_Time.png)

**Figure 11:** Illustrates change in nitrate concentration following spike to 50mM ammonium over a 60 minute interval. Noticeable increase in nitrate production by transformed PD with HAO/cyt gene complexes indicates that the HAO/cyt oxidize hydroxylamine and nitrite effectively within the Paracoccus chassis.

These results were anticipated due to the kinematic properties of the reaction sequence.The first step in nitrification is oxidation of ammonium to hydroxylamine. While the monooxygenase enzyme is not present in the PD (transformed), Paracoccus does contain limited nitrifying capacity, as outlined in project design. The spike in ammonium, in addition to the high ammonium culturing conditions, is expected to induce this forward oxidation. Once hydroxylamine forms, the Hydroxylamine oxidoreductase (HAO) and associated cytochromes present in the PD (transformed) oxidize hydroxylamine to nitrate.

This brick is maximally induced in the presence of 1mM arabinose, so we expect any hydroxylamine formed to be further oxidized to nitrate. This result is demonstrated in **Figure 11**. Due to Le Chatelier equilibrium principles, we anticipate that the immediate removal of the hydroxylamine intermediate increases the rate of ammonium oxidation. This prediction is reflected in **Figure 10**, as the rate of ammonium oxidation is greater in the PD (transformed) than that of the PD (untransformed).

The observed increase in nitrate production observed in **Figure 11** for the PD (transformed) indicates that the HAO/cyt BioBrick is functioning in the cell. As compared to PD (untransformed) and NE (untransformed), the PD (transformed) is expected to produce more nitrates because it is being maximally induced in the presence of arabinose.

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