Team:WashU StLouis/Notebook

Washington University - Penn State iGEM

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WashU and Penn State iGEM 2015

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Charlotte Bourg Washington University in St. Louis

Make CμLtures

C_exp2

The Moon Lab needed, strains JM109 and MG1655 for their experiments

Make overnight cμLtures from frozen stock to make electrocompetent cells the next day (in Moon lab):

from glycerol stock, set up two 50mL cμLtures in 250-mL flasks
grow overnight

Make Lb

C_exp4

The Moon Lab needed, strains JM109 and MG1655 for their experiments

Transformation (in Moon lab):

- Take MG1655 and WM1788 electrocompetent cells out of -80°C and place on ice
- Take PSL2397 (plasmid) out of -80°C and place on ice
- Add 2 μL of PSL2397 directly into MG1655 tube
- Set pipette over 40 μL and draw up the MG1655 and PSL2397 mixture and place into electroporation cuvette
- Tap cuvette to ensure cells are at bottom of cuvette—shoμLd check to see that there are no gaps—and place into electroporator; turn on
- Immediately add 500 μL of LB into the cuvette then pour into cμLture tube
- Place cμLture tubes of WM1788 and MG1655 into incubator for 1 hour

Make Lb

C_exp6

There are 10 genes in the Nif cluster that have unknown functions. Overexpressing these can shead light on what they do

Use SnapGene to design primers for the 14 sequences to be overexpressed

- Check for EcoRI and XhoI restriction sites within each of the sequences
- Check for directionality on the plasmid: direct or complementary
- If a restriction enzyme does not have sites within the sequence, add a site for that restriction enzyme to the appropriate primer and add 6 adenines beyond the site on the primer so that the restriction enzyme will work properly
- If the restriction enzyme does have a site within the sequence, end the primer at the end of the sequence to be amplified to leave the ends blunt
- If direct, add EcoRI to the 5’ end and XhoI to the 3’ end as appropriate
- If complementary, add XhoI to the 5’ end and EcoRI to the 3’ end as appropriate
- Maintain a Tm for each primer above 60°C and approximately match the Tm of paired primers
- Ensure that there is only 1 binding site on the plasmid for each primer

Run PCR

C_exp8

Run PCR on 3 of the 14 amplicons

Run PCR

C_exp9

Run PCR on remaining amplicons

Overnight cμLtures

C_exp14

Start an overnight cμLture in 5 mL of LB at 37°C for transformation of ligated plasmids

Plate Cells

C_exp18

- Take cells out of 37°C room and spin down all but the control for efficiency of transformation (PE5A) for 4 minutes
- Take 100 μL from PE5A control and add to 900 μL of LB
- -Plate 100 μL of that dilution on the amp plate
- -Add sterile glass beads and shake laterally to spread around the cμLture
- -Dump beads into nonsterile glass beads container
- from centrifuged cμLtures, pipette off the media to the 100 μL mark and resuspend the pellet in that amount of media
- -Add fμLl resuspended quantity to the correct labeled plate
- -Add beads and shake
- Place all plates in 37°C room

Store plates

C_exp19

Take plates out of 37°C room, check for colonies, and place those that grew into the 4°C room for the remainder of the weekend

Run Gel

C_exp25

- Run digested plasmid through the gel

- Saw no band for digested plasmid and a band in unexpected location for the undigested plasmid need to redo the digestion of the plasmid

Confirm Inserts

C_exp26

Make 20 μL PCR reactions for plasmids purified yesterday (to attempt to confirm correct inserts)

Digest

C_exp33

Digest the blunted cut plasmid with Xho1 in a 25 uL reaction

Add the following to the Klenow reaction mixture (without purifying first)
2 uL digested buffers
2 uL Xho1
1 uL water
Place at 37C for ~3 hours

Check plates

C_exp34

Carlos checked plated cells for growth

1 colony on control; ~30-50 colonies on other plates
Placed plates in 4C room

Pick colonies

C_exp35

Carlos picked 2 colonies per plate and started overnight cultures in LB

Transform

C_exp38

Transform plasmids from overnight

Purify plasmid from overnight cultures of colonies
Quantify cocentrations of plasmids
Set up 20 uL PCR reaction for the purified plasmids to confirm inserts
transform ligated plasmids
- Add the ligation reaction products produced to labled vials of competent cells
- Incubate cells with ligation products added on ice for 25 minutes
- Heat shock at 42C for 1 minue; replace on ice for ~2 minutes
- Add 900 uL of LB to each tube (in the hood)
- Place on shaker at 37C for 1 hour
Pour plates
- Melt LB agar in a secondary container of water in the microwave
- Wait for agar to cool before adding 1 uL of chloramphenicol per mL LB agar
- Pour plates with ~20mL agar with antibiotic
- Allow plates to dry in hood
Plate cells
- Centrifuge, pipette off the supernatant to 100 uL mark, then resuspend the pellet in that amount
- Add resuspended cells to labeled plates; spread with with sterile glass beads. Place plates in 37C room
Run a gel of PCR products (to confirm inserts in transformed cells)

Check colonies

C_exp39

Check for colonies from plated cells

Control grew colonies, as did the others
Pick a colony with a pipette tip and resuspend colony in the media
Add 50mL of LB to a conical tube; add 50 uL of chloramphenicol to that
Add 5 mL of that mix to each of the 10 labeled sterile culture tubes.
Pick a colony with a pipette tip and then either resuspend the cells in tube or drop the tip into the tube
Plate on shaker in 37C room at 250 rpm
Add 5 mL LB to another steril culture tube. Add 5 uL LB to the tube. Scrape frozen stock of PA2C-TesA with a pipette tip and place the tip in the Lb; place on shaker in 37C room

Miniprep PA2C-TesA

C_exp44

Miniprep PA2C-TesA (using 10X the amount of buffers as suggested by the kit)

Pour the 50 mL culture into a 50 mL conical and centrifuge to a pellet
Pour off the supernatant
Resuspend in 2500 uL of resuspension buffer and vortex or pipette in and out until no clumps remain
Add 2500 uL of lysis buffer and invert 10 times to mix (no vortexing). Incubate on ice for 5 minutes
Centrifuge at 13,000 rpm and 4C for 10 minutes
Pour the supernatant through a syringe with a cotton ball in it to filter
Transfer 800 uL of supernatant to a column in a collection tube and centrifuge at 13,000 rpm for 1 minute.
Discard the filtrete and replace in the same collection tube
Repeat previous steps with additional 800 uL of filtered supernatant until all supernatant has gone trhough column
Add 500 uL of wash buffer A and centrifuge at 13,000 rpm for 1 minute. Discard the flow through andreplace column in the tube
Centrifuge at 13,000 rpm for 1 minute
Place on thermomixer at 65C for 5 minutes
Place column in a new 1.7 mL microcentrifuge tube and add 50 uL of water warmed to 65C; let sit at room temperature for 1 minute
Centrifuge at 13,000 rpm and 1 minute
Quantify concentration on nanodrop
On 2% agarose gel, add: 2.5 uL 1kb pluss ladder to wells on either side. 8uL of each PCR product

Run gel

C_exp44

Run a gel to see if PA2C-TesA purified yesterday is okay

Pour a gel with 10 wells of ~35 mL 1% agarose with 2 uL EtBr
Run 100 ng of plasmid max -> 2.5 uL with .5 uL loading dye
2.5 uL ladder on each side

Repeat transformation

C_exp45

Purify digested PA2C-TesA with DNA clean & concentrator kit
Start a Klenow reaction
Digest the plasmid from the Klenow with Xho1
load the digested plasmid into a gel and gel purify the correctly sized band
Ligate the digested insert with digested plasmid
Transform the ligation products into chemically competant cells

Start overnight of more PA2C-TesA

C_exp46

Attempt to PCR the inserts from the HDK overexpression plasmid using the nifHDK F and R primers

C_exp48

Redo HDK PCR

C_exp50

Redo HDK PCR from nif cluster plasmid to attempt construction of the HDK overexpression plasmid again

Primer design for minimal nif plasmid

C_exp51

Redo PCR of HDK and cce_0550

C_exp53

Cut and purified band for 0550, but HDK band was incorrect

Ligate 0550 and HDK

C_exp54

Ligate genes into plasmid

Repurify 0550 PCR product (due to a bad peak on nanodrop)
Digest repurified 0550 with Xho1
Clean up digest with DNA clean & concentrator kit
Set up ligation for 0550 and HDK with PA2C-TesA

PCR genes for minimal nif plasmid-grouped by size into 3 sets

C_exp55

PCR for small and large size groups
Gradient PCR from 58C to 65C
100 uL rxn split into 4 tubes of 25 mL each, at different temperatures in gradient thermocycler
Run gels of a small amount of each of tube. Saw bands for very few of the expected products

PCr medium size group

C_exp56

PCR for minimal nif plasmids continued

C_exp57

PCR more 0550 and HDK for overexpression plasmids

C_exp58

EcoR1 digest of PA2C-TesA
Run 0550 PCR product in a gel and gel purify it
Digest 0550 with Xho1
Run HDK PCR product in a gel and gel purify it

Prepare 1 plasmid of nif cluster

C_exp59

PCR amplify tetracyclin resistance gene off of one plasmid and pB1a except for the amplicilin resistance gene, using primers designed for Gibson ligation
Blunt the ends of PA2C-TesA cut with EcoR1 using S1 nuclease
- Add at least 10 units of S1 nuclease per microgram of plasmid, but use 10 units if you use less than 1 microgram
- Use a 10x final concentration of buffer
- Add water to a 30 uL final reaction volume
- Place at room temperature for 45 minutes, then add 4 uL of .25M EDTA and incubate at 75C for 20 minutes to stop the reaction
After blunting with S1 nuclease, clean up with DNA clean & concentrator kit, then start an Xho1 digest
Set up Gibson assembly for backbone resistance change for one of the minimal plasmids
- Use 7.5 uL of Gibson mix premade by another member of the lab
- Dilute to a 10 uL final reaction volume
- Place at 50C for 1 hour to ligate
- Add 50 ng of each piece to be ligaged
  
  Based on the concentrations of DNA we had, we were unable to add 50ng of each piece, and maximized the amount of DNA added by not diluting with water
transform Gibson assembly products using chemically competent transformation protocol
Cut PA2C-TesA backbone band from gel and gel purify
Make new tetracyclin stocks for Gibson assembly products transformation
- Measure 50 mg of tetracycline hydrochloride on weighing paper
- Add 70% EtOH to a 10 mL final volume; mix
- Add 4 uL per mL to 20 mL Lb (80 uL of tetracycline)

Checked on 7/16/2015, no colonies from Gibson products

Transform ps8k plasmid into DH10B to make more

C_exp61

Golden Gate reactions

C_exp63

Set up a 10 uL Golden Gate reaction of all 8 pieces for the pS8K cysE2-hesB minimal plasmid, using a molar ratio that compares concentration/size(bp) of each piece. Inserts should all be in a 3 to 1 ratio to the backbone

mix reaction mixtures and DNA together
- DNA should be 7 uL
- Add: 1 uL T4 ligase buffer, .5 uL Bsa1, .5 uL T4 ligase, .3 uL Dpn1, .5 uL ATP at 10 uM, .3 uL BSA
Spin down and put in thermocycler
- 37C for 3 minutes
- 16C for 4 minutes
- Repeat first 2 steps 50 times
- 50C for 5 minutes
- 80C for 5 minutes
- Hold at 4C

Pick colonies

C_exp64

Pick 5 colonies from DH10B plate of golden gate reaction mixture transformation. Pick 1 colony of ps8k

Work on HDK & 0550

C_exp65

Digest PA2C-TesA with EcoR1, PCR more 0550 and HDK

Golden gate cysE2-hesB

C_exp67

Run a gel to check for cysE2-hesB PCR product
Run a gel to cut the backbone from the PA2C-TesA Xho1 digest
Digest ps8k minipreps with pst1 and Pme1 to check for proper sizes
Set up a golden gate of the seven inserts only, excluding the backbone from this reaction, using a 1 to 1 molar ratio of all Golden Gate inserts

Repeat Golden Gate

C_exp68

Set up a PCR using the 7 insert Golden Gate reaction as template: make 2 15 microliter reactions, one using 1 microliter of the golden gate as template and the other using 1 microliter of a 10x dilution of the template
Run a gel of a small amount of the ligation mix(template for the PCR)
Start digest of HDK and 0550 PCR products with Xho1
Clean and concentrate the digest
PCR of inserts with 10x dilution of template looks good -- start a 100 uL reaction scaled up
Run a small amount of cleaned up digest products on gel to check for proper bands -- got expected bands
PCR tetracyclin and pB1a for the backbone resistance change with the newly-arrived Golden Gate primers(rather than the Gibson primers)

More Golden Gate

C_exp70

Re PCR the ps8k backbone to ligate cysE2-hesB into
transform golden gate ligation of pB1a and tetracycline into DH10B chemically competent cells
Cut and purify the 7 gene ligation PCR from the gel
Set up a Golden Gate ligation of the 7 insert PCR product and the amplified pS8k backbone in a 10 uL reaction

Transform the Golden Gate product into chemically competent DH10B

C_exp71

Check for inserts

C_exp72

Miniprep the overnight of ps8k, ps8k cysE2-hesB 1 through 5 ( 5 colonies picked from the plate), and pB1a-tet
Start a Golden Gate reaction of the 7 inserts for the second plasmid
Digest pB1t with Spe1 and BamH1 (alone and together) to check for correct insert
Digest pS8k cysE2-hesB with Pme1 and Pst1 (alone and together) to check for insets

Send in sequencing primers

C_exp74

Dilute sequencing primers for ps8k cysE2-hesB
Sequence miniprep #2 of ps8k cysE2-hesB (clearest bands on the gel from digest check)
Sequencing PcR reactions
- 50 uL reactions using ps8k cysE2-hesB #2 as template
- Amplify ~1 kb per primer pair, with each read overlapping by about 100 bp (the way the sequencing primers were designed)

Check Plasmid

C_exp75

Run a gel of a small amount of nifh to nifn golden gate mixture and all of the pB1t PCR
Saw no visible band for the Golden Gate mixture where expected for full ligation
Cut bands of pB1t PCR
Miniprep the overnight clulture of PA2C-TesA
Digest PA2C-TesA with EcoR1
Clean and Concentrate the digest, then start a klenow reaction
Resuspend newly-arrived sequencing primers for the next minimal nif plasmid, using TE buffer

Repeat Golden Gate

C_exp77

Try inserts in groups of 3 and 4 rather than all 7
nifH, nifD, nifK and nifB, hesA, nifE, nifN

Do Goldan Gate 2 at a time

C_exp78

Do Golden gate 2 inserts at a time, in pairs as follows (leaving out nifK from the groupings for now): nifH & nifK, nifB & hesA, nifE & nifN

Run PCRs to confirm

C_exp79

EN did not work. Try PCR with varied MgCl concentration and annealing temperatures.
Cut HD and BA from gel and gel purify
Start a Golden Gate reaction of HD with K

Attempt to digest then ligate separately

C_exp81

Digest
- 1.5 hour digestion
- 20 uL reaction
- Bsa1, digest buffer, and 1 to 2 uL of insert to be digested
- Clean and Concentrate
Ligate cleaned up pieces
- T4 ligase
- T4 ligase buffer
- .5 uL ATP
- 10 uL reaction

More EN

C_exp83

Run PCRs of nifE and nifN ligation from separate digestion and ligation

Use E F and N R primers
Use 2 additional pairs of primers from the sequencing primers designed for this plasmid to amplify smaller portions of E and N that cross the junction, to check for appropriate ligation
Start overnight ligation of digested E and digested N at 4C

Check on ligation reactions

C_exp84

Ran gel of HDK BA, HDK BA E N, and overnight 4C E N ligations
Start PCRs from each, in addition to the room temperature EN ligation

Both amplifications of EN look good - cut and purify together
Neither HDKBA nor HDKBAEN worked
PCR more HDK from golden gate template
Start digest of BA and EN with Bsa1 for 2 step digestion then ligation

Blake Actkinson Washington University in St. Louis

Preparation of Electrocompetent Cells

B_exp1

These are the strains we put the nif cluster in this summer.

Prepare cultures of DH10B

25 ml of each culture were transferred into 500 mL of LB
-These flasks were then grown at 37 deg C, 250 rpm for 1 hour, 30 min in incubator
Measure absorbance
- Cuv1-.1055, Cuv2-.1084
After absorbances were measured, cultures were added to ice bucket and allowed to cool for 15 minutes.
-Centrifuge bottles were centrifuged for 25 min. at 4 deg.
After centrifuge, bottles were taken to cold room
- Chilled 10% glycerol was added, 55 ml each, into each bottle
  
  It’s worth noting that % glycerol in each bottle is roughly 4.4%
- 70 Ml of water were added to each bottle
-Bottles were placed back into centrifuge for another 256 minutes at 4 deg C-After centrifuge, bottles were taken to cold room once more
Supernatant was drained
- 10% glycerol was added to 4 tubes
- Roughly 15 mL were allotted to each tube
- Tubes were taken and centrifuged
After centrifuge, conical tubes were taken to cold room
- Supernatant was drained
- 1 mL of 10% glycerol was added to each tube, pellet allowed to resuspend
- Tubes were taken to centrifuge again, 3000 RPM @ 4deg C

Designed program to find sgRNAs in Java,

B_exp2

Need to find a place for dCAS9 to bind

Designed program to find sgRNAs in Java, key feature is finding the PAM sequence

More electrocompetent cell prep

B_exp3

EC cell prep for Strains: MG1655 and JM109

Two culture were incubated overnight
-In the morning, 26 mL of culture were placed into 500 mL of LB
-These flasks were incubated for an hour beginning at 10:34
-Both flasks were taken out around 11:35 P.M.
-Optical measurements were performed on both colonies using a 10x dilution factor
- i. The optical density of JM109 was 0.287 ii. The optical density of MG1655 was 0.39
-JM109 went back to be incubated at 11:56 A.M.
-Optical measurement performed on JM109 once again
- Optical density was 0.38
-Optical density for both strains was determined to be satisfactory -Thus, each flask was equally partitioned into two centrifuge bottles
-Centrifuge bottles were left to sit for 20 minutes
-After sitting for 30 minutes, bottles were centrifuged at 3000 RPM for 25 minutes
-Upon centrifugation, supernatant was drained, pellet was resuspended in 125 mL of water
-Bottles were centrifuged again for another 25 minutes
-Upon centrifugation, supernatant was drained and 55 mL of 10% glycerol were added to each bottles
-Pellet was resuspended in each bottles
-After resuspension, each bottles was partitioned into 4 15 mL conical tubes
-Conical tubes were centrifuged under same conditions
-From a combined conical tube, 40uL aliquots were created for JM109 and MG1655
-These aliquots were stored in -80 deg C conditions Electroporation
-We transformed MG1655 and WM1788
-To do this, we obtained copies of pSL2397 plasmids
-2.5 uL of this plamsmid DNA was collected and mixed with pipettor
-After this, the plasmid DNA was inserted into a cuvette
-The entirety of the aliquot containing the E.Coli strains was dumped into the cuvette
-The plasmid was shocked into the strain for 5.5s
-Strain was taken out and put into conical tube for an hour
-The same process was repeated with MG1655 strain

Picked Colonies

B_exp5

Picked colonies from yesterday's prep

Preparation of Frozen Stock

B_exp6

Store Cells from yesterday

-Cells were taken from picked colonies on June 4
-We took 500 uL of that culture, added it to 500 uL of 30% glycerol solution, then place din -80k deg C conditions.
-We then prepared 30% glycerol solution for later use
-Next, we prepared the CRIPSR/dCas9 plasmid for PCR
-To do this, we separated the plasmid from its host bacterium
-First, we took the bacterial culture and centrifuged it
-Then, we discarded LB growing solution that was supernatant
-Next, we resuspended bacterial cells in water
-Buffer to lyze the cell was added
-Immediately after, cold neutralization buffer was added to neutralize the process
-Then, tubes were centrifuged at 16000 g for 43 minutes
-Supernatant was transferred to a separation column
-These columns were then centrifuged for 30 seconds
-The flow through was discarded and the colonies were placed back into the collecting tubes
-200 uL of Endo-Wash Buffer were added to each tubes
-Each tubes was centrifuged under same conditions for 30 seconds
-Upon centrifugation, 400 uL of Zappy’s buffer were added to each tube
-Each tube was centrifuged for another 2 minutes
-Upon centrifugation, high pure molecular water was added to each column and each column sat for two minutes
-Each column was centrifuged, same condition, for another 2 minutes
-After this step, dissolved plasmid DNA had collected on bottom of column
-Each column was taken to spectrometer to determine concentration and purity

-Concentrations and purities, in order

Tube	Conc (ng/uL)	Purity
Tube 1

1.87 127.1 Tube 3 1.79 82.7

After that, each DNA sample was placed into freezer to be stored

Prepare Gel

B_exp9

We prepared a gel for DNA purification

-Mix 1g of Agarose with 100 mL of TAE buffer.
Note that you can cool the mixture with cold, running water
-Add 5 uL of cybersafe (5uL/100 mL of buffer) once mixture cools
-Put in combs
-Tape side of tray
-Pour agarose gel in tray
-Let gel cool for 1 hour
Run gels for 1 hour

Ran gels

B_exp13

6x loading dye, 10 uL, was added to each sample
Take yellow tape off of sides
Buffer was added until the top of the hardened gel plate was covered
Ladder was inserted

i. Make sure you have ladder for each row of running lanes
Then,inset DNA into each well

Make sure not to creat bubbles. Do not tap plate
125V, 30 min

i. Make sure there are bubbles forming on the negatively charged end

Another miniprep of plasmid

B_exp14

Grow stock overnight
Add 600 uL of culture to 1.5 mL mc tube
Centrifuge for 30 seconds to pellet cells

Do at max speed
Discard supernatant
Add 600 uL of deionized water to cell pellet and resuspend by vortexing the tubes
Add 100 uL of 7x lysis buffer and mix by inverting 46 times proceed to next step within 2 minutes
Solution should change from opaque to clear blue
Add 350 uL of cold neutralization buffer and shake hard to mix
Sample will turn yellow when neutralization is complete and a yellowish precipitate will form
Invert the sample an additional 23 times to ensure complete neutralization
Centrifuge at 16,000 rpm for 2 min 30 seconds
Transfer the supernatant into Zymo Spin column (small, without cups) by simply dumping it from microcentrifuge tube into column and avoiding the pellet
Place into a collection tube and centrifuge for 30 seconds
Discard the liquid, protect the filter and place the column back into the same collection tube
Add 20 uL of endowash buffer to the column and centrifuge for 30 seconds
Add 400 uL of Zyppy wash Buffer to the column and centrifuge for 30 seconds
Discard liquid and centrifuge for 2 minutes
Transfer column into clean 1.5 mL microcentrifuge tube and add 30 uL of highpure molecular water directly to column and let stand at room temp. for 2 min
Centrifuge for 30 seconds to elute plasmid
Boot up Nano2000 program and prepare a blank by place 1 uL of high pure molecule water on stand
Test each sample

Post ligation, transformation

B_exp20

Need: 500 uL of LB
NO MORE THAN 2 uL OF LIGATION PRODUCT (had to start over on 4)
DH10B competent cells, 2 aliquots
Sanity A flame
Obtain <2 uL of ligation product
Insert ligation product into DH10B cell tube
Put mixture into chilled cuvette
Ensure mixture @ bottom and no air bubbles
WIPE OFF CUVETTE WITH KIMWIPE
Insert into Eporator set @ 2500
While cuvette is being transformed, hover 400 uL pipette tip over flame then proceed to fill 500
Once transformation is complete immediately insert LB into cuvette
Dump contents of cuvette into falcon tube and let incubate for 1 hour

4 was a failute because too much buffer was present. Salts caused archs

Colony PCR

B_exp22

First, 12 PCR tubes obtained
Added 50 uL of HP water to each
Prepared a 1ml/uL LB:AMP mixture and used 60 mL of mixture total
Obtained 1F, 2F, 3F, 4F plates
Picked 3 colonies from each plate and put 1 into each PCR tube
Then, I pipetted 5 mL of LB/AMP mixture into a culture tube
After this was complete, dropped pipet tip corresponding to each culture tube and set them aside
Next, PCR reaction was prepped
Reaction was done with 7 uL of template (not entirely DNA)
292.5 uL of water
52 uL of DMSO
130 uL of Buffer
13 uL of dNTPs
42.5 uL of forward primer
42.5 uL of reverse primer
6.5 uL of Phusion polymerase
These were components of master mix
Each added to 7 uL of template DNA strand
Annealing temperature of PCR:60 degrees

Make Amp/Kan plates

B_exp23

Made Amp/KAN plates:
i. Protocol on pg. 7
ii. 12.5 g Miller LB
iii. 7.5 g Agar
iv. put magnetic stir bar into bottle
v. Pour UV deionized water, 500 mL, into bottle (25g/L miller LB, 15 g/L Agar)
vi. Took bottle to be autoclaved
vii. Bottle autoclaved on agar cycle, loose cap, for two hours
Grew culture of sgRNA plasmids for miniprep the following day

Went to Pakrasi lab to observe acetylene injection

B_exp25

Prepped M9 media

B_exp26

Prepped culture with aTc

B_exp27

Prepared for assay

B_exp28

Prepped for assay
Spun down cells @ 3200g @ 4 deg C for 15 minutes
Took out culture tubes, discarded supernatant
Added 4mL of M9 media to each tube and resuspended
Centrifuged cells 3200g @ 4 deg C for 20 minutes
Took out culture tubes, discarded supernatant
Added 4 mL of M9 media to each tube and resuspended
Centrigued cells 3200 g @ 4 deg C for 25 minutes
Toook out culture tubes, discarded superantant
Added 4 mL of M9 media to each tube and resuspend
Centrifuge cells 3200g @ 4 deg C for 30 min.
Took out culture tubes, discard supernatant

Prepped cultures of JM109 with pSL2397 for a miniprep

B_exp29

Miniprepped pSL2397

B_exp30

iGEM characterization of BBa_K577895

B_exp31

Transformed colonies of DH10B containing pSB1C3 with 577895, pAH34, and pAH35
Prepared cultures of these colonies

Started induction experiment

B_exp32

Started with induction experiment, but halfway through realized I forgot to make frozen stock, so the experiment was nixed and restarted for the following day

Started induction experiment

B_exp33

ODs of overnight cultures were measured in the TECAN
More culture tubes were made containing each overnight culture diluted to an OD of 0.2
Put these new, diluted culture tubes into incubator for two hours
While this was happening, prepared well plates for induction
Calculations were made such that I serially diluted aTc stock concentration 5x across each well
0.1 mL of each cell culture was added to its respective well
Well plates were taken and incubated for 6 hours
At the end of the 6 hours, fluorescence was measured using

Results showed that part expressed RFP constitutively

Another induction

B_ex34

Transformed iGEM part into another strain of DH10B

Prepped sgRNA cultures

B_exp36

Prepped cultures of sgRNA plasmid (pTetpConsgRNAT1p15adCas9I4), DH10B, pAH34, pAH35, 2 cultures of pSB1C3 (one was the first transformation, the other the second)

Induction Experiment

B_exp37

July 14 Cultures pAH34, pAH35, and pSB1C3 did not grow again last night, Ran an induction experiment with just the retransformed DH10B with pSB1C3(refer to pSB1C3 dd)

Results showed that iGEM part did not express RFP at all across any inducer concentrations

Induction experiment

B_exp38

Prepared new stocks of pAH34, pAH35, and pSB1C3. Did another induction experiment involving two colonies of pSB1C3 dd, pSB1C3, pAH34, pAH35, and DH10B

Results were consistent with earlier findings; pSB1C3 dd is colorless, pSB1C3 is red

Prepare more sgRNA

B_exp39

Checked samples

B_exp40

Did Dpn1 of 23 samples. Did chemical transformation for 33 samples. Did Eporation of 8 samples

All Failed

Ran PCR

B_exp41

Ran a PCR for 9 samples, Did gel recovery.

Ran PCR

B_exp42

Did DNA cleanup of 9 samples. Loaded 26 samples for colony PCR. Ran a PCR for 9 samples

Prepped gel for samples

B_exp42

Prepped gel for 9 samples. Prepped forzen stock from colony PCR for 17 samples

Prepped frozen stock of 7 samples

B_exp43

Dpn1 digested two samples, Prepped frozen stock of 7 samples

Dpn1/Gel recovered 8 samples

B_exp45

Dpn1/Gel recovered 8 samples. Retransformed 13 other samples

Did another colony PCR

B_exp46

Did another colony PCR for troublesome samples. Did another PCR for 15 other samples; concentrations were too low post clean-up

Vanderbilt Induction Exp

B_exp47

Want to run an induction experiment with troublesome Vanderbilt promoters transformed into two separate strains:BL21 and MG1655 Grew cultures of these two parts transformed into these two strains as well as wild type strains of MG1655 and BL21

Results obtained from Vanderbilt induction were nebulous at best

Grew more cultures for Vanderbilt induction experiment

B_exp48

Negative control didn't grow; experiment nixed

Grew more cultures for Vanderbilt induction experiment

B_exp49

Grew cultures for another round of induction experiments on the Vanderbilt part using OUR protocol.

Grew more cultures for Vanderbilt induction experiment

B_exp50

Induced with IPTG and put each plate into the incubator to grow for 24 hours, roughly

B_exp51

Prepared media

B_exp52

Obtain frozen stock
Grow overnight in LB and selected antibiotic
Spin down at 3000 RPM for 15 minutes
Discard supernatant
Resuspend in M9 media, 4 mL
Spin down at 3000 RPM for 20 minutes
Discard supernatant again
Resuspend in M9 media
Spin down at 3000g for 25 minutes
Discard supernatant
Resuspend in M9
Spin down at 3000g for 30 minutes
Discard supernatant
Take OD
Calculate how much you’ll need to have 0.1 OD in 5mL
Prepare all bottles by adding 10 uL of antibiotic
Place stopper in bottle
Place in 30 degree shaker
Grow overnight
Next morning, take OD again
Goal is to have it at 0.2
React away oxygen in anaerobic chamber
Put them back in 30 degree shaker
Next day, inject acetylene
Take zero point
Put back in shaker
Take 24 hour point

Jessica O'Callaghan Pennsylvania State University

Learn The Enviornment

J_exp1

This was my first week with the iGem team, even though I got to work on some segments of the project from home. My first two days consisted of getting acclimated with the computer programming (Komodo and Cyberduck), and specifically running python and gams files from excel data on the E. coli model. I had the opportunity to work with Laura, who was very helpful in explaining the scripts we were running. Tom was also able to send me information on python and unix commands, so navigating through these programs was easier than expected. I’ve worked on and off with MATLAB, but I actually found python and gams to be a lot more user friendly.

Biomass and ATP calculations

J_exp2

Once I generated my input files using a python code provided to me (I only had to personalize my directory information), I was able to run FBA scripts for both the WM1788 wild type and nitrogen fixation models. I was able to determine maximum biomass values for both, as well as maximum ATP values from these biomass outputs. For the WM1788 wild type model, I got a maximum biomass value of 2.97 and a maximum ATP production value of 547.03. For the WM1788 nitrogen fixation model I got a maximum biomass value of 2.52 and a maximum ATP production value of 391.274. These values checked with Laura’s. From there, I ran FVA on both the wild type and nitrogen fixation models, and generated spreadsheets for flux data on each reaction in the model.

model, I got a maximum biomass value of 2.97 and a maximum ATP production value of 547.03. For the WM1788 nitrogen fixation model I got a maximum biomass value of 2.52 and a maximum ATP production value of 391.274. These values checked with Laura’s.

The Galactose Pathway

J_exp3

On Wednesday, I started to look at the pathway taken by galactose as it enters the cell, thereafter initiating glycolysis. Given our model predicted higher cell growth rates for galactose as a sole energy source than for glucose, which contradicts most available literature, we were trying to determine if there were any reactions that would help explain this phenomena (for example if there is a reaction supplying additional energy in the galactose model). However, even though I was able to identify the series of reactions the take place immediately following the uptake of galactose, which include converting alpha Dgalactose to beta Dgalactose, and elementary reactions depending on enzymes such as 4epimerase and galactokinase, a greater amount of energy was required to initiate the conversion of galactose to glucose to G6P than was required to initiate the conversion of just glucose to G6P. We are still in the process of determining where these inconsistencies are coming from by looking into the first set of reactions that takes place when glucose is introduced to E. coli.

I was able to identify the series of reactions the take place immediately following the uptake of galactose, which include converting alpha Dgalactose to beta Dgalactose, and elementary reactions depending on enzymes such as 4epimerase and galactokinase, a greater amount of energy was required to initiate the conversion of galactose to glucose to G6P than was required to initiate the conversion of just glucose to G6P. We are still in the process of determining where these inconsistencies

Knocking out PDH and PFL

J_exp4

Lastly, Laura was working on finding an alternate path in the model that produced acetylcoA. However, the only reactions listed involve the conversion of pyruvate to acetylcoA. We are interested in determining which reactions can be potentially knocked out of the model to maximum the production of acetylcoA. Tom wanted me to run gams files that removed the PDH (pyruvate dehydrogenase) and PFL (pyruvate formate lyase) reactions from the model, which are directly involved in the production of acetylcoA, to determine if whether or not knocking one out will maximum flux through pyruvate synthase (POR5), which is the only other reaction involved in the production of acetylcoA. However, our findings show that knocking either PDH or PFL from the model has no effect on the flux through this reaction, or on one another. Considering the flux ranges remained the same (0 10,000) for the PDH and PFL reactions, these reactions can potentially be removed from the model. When both PDH and PFL were both knocked from the model, the flux ranges varied slightly through the pyruvate synthase reaction from 9,948.9478 444.251305 (for the lower and upper bounds respectively) to 4068.693 444.251. Even though the lower bound was less negative, its magnitude was still significantly larger than the upper bound. You can refer to the PDH, PFL, and POR5 reactions below.
Lastly, we identified that the main reaction converting glucose into G6P was glc_DASH_D_c + atp_c → g6p_c + h_c + adp_c (involves hexokinase) and not pep_c + glc_DASH_D_c → pyr_c + g6p_c ( 0 - 7155.54 to 0 - 20 respectfully)

Knocking out PDH and PFL

J_exp4

Lastly, Laura was working on finding an alternate path in the model that produced acetylcoA. However, the only reactions listed involve the conversion of pyruvate to acetylcoA. We are interested in determining which reactions can be potentially knocked out of the model to maximum the production of acetylcoA. Tom wanted me to run gams files that removed the PDH (pyruvate dehydrogenase) and PFL (pyruvate formate lyase) reactions from the model, which are directly involved in the production of acetylcoA, to determine if whether or not knocking one out will maximum flux through pyruvate synthase (POR5), which is the only other reaction involved in the production of acetylcoA. However, our findings show that knocking either PDH or PFL from the model has no effect on the flux through this reaction, or on one another. Considering the flux ranges remained the same (0 10,000) for the PDH and PFL reactions, these reactions can potentially be removed from the model. When both PDH and PFL were both knocked from the model, the flux ranges varied slightly through the pyruvate synthase reaction from 9,948.9478 444.251305 (for the lower and upper bounds respectively) to 4068.693 444.251. Even though the lower bound was less negative, its magnitude was still significantly larger than the upper bound. You can refer to the PDH, PFL, and POR5 reactions below.
Lastly, we identified that the main reaction converting glucose into G6P was glc_DASH_D_c + atp_c → g6p_c + h_c + adp_c (involves hexokinase) and not pep_c + glc_DASH_D_c → pyr_c + g6p_c ( 0 - 7155.54 to 0 - 20 respectfully)

Adding Flavodoxin to the Equation

J_exp6

At the end of last week, Laura added an additional reaction to the N2 WM1788 model that expends flavodoxin:

We found that the flux ranges changed for over 1,300 reactions. And as for the pyruvate synthase reaction, there was hardly any variation in the flux ranges ( 9948.95 to 443.698 and 9948.904 to 443.703 for max biomass and max flavodoxin respectively). We started to look at reactions whose flux ranges varied by more than a factor of 1.0 × 101.

Knockout PDH and PFL

J_exp7

After the meeting with iGem, I started researching potential growth implications of knocking out the pyruvate dehydrogenase and pyruvate formate lyase reactions from the model. Our data supports that knocking out either of these reactions individually has little to no effect on the flux through the corresponding reaction, or on the flux through the pyruvate synthase reaction. Research shows that pyruvate dehydrogenase deficiency has been associated with a buildup of lactic acid in the cell. The enzyme has also been identified for its essential role in initiating the series of reactions that make up the TCA cycle. According to a study conducted by Anderson ME, Marshall of RT JFood Safety in 1990, increased concentrations of organic acids have been shown to reduce cellular activity in E. coli, leading to higher levels of population reduction. As for the pyruvate formate lyase reaction, one study examined the effects of knocking out it’s associated enzyme on the yield of butanediol in a strain of E. coli known as Klebsiella pneumoniae. Even though the yield was 92.2% of it’ theoretical value, mutant growth was slightly reduced to the parental strain. Similar studies on other mutants of E. coli suggest that this growth deficiency is due to a redox imbalance rather than a reduced level of acetylcoA. These findings would eliminate the possibility of knocking out either of these reactions from the model.

After removing this reaction, we had to rerun all of our previous codes to update our figures.

Knockout Malate Oxidase

J_exp8

After knocking out malate oxidase, Laura and I reran FBA codes for the WT and N2WM1788 models. Our updated max biomass and ATP values for the wild type model were 0.821233 and 3.155221, respectively. And for the nitrogen fixation model, 0.454531 and 3.155221, respectively. Our updated ATPmreq constraint was then set from 10.0 to 3.15. We also reran the flavodoxin scripts, and found that our max biomass value, which we derived from maximizing flux through the flavodoxin reaction (0.010442), was 0.4545. I continued rerunning the pyruvate codes with the malate oxidase knockout however, I was having some difficulty with running the codes to completion several errors kept popping up. I later found out that these issues were arising from available storage space in my terminal, which was later resolved after I consulted Tom.

Original values (N2 outputs)	PDH	PFL	PDH and PFL
Max biomass: 0.454531	Flux range for PDH: 0 to 10,000	Flux range for PFL: 0 to 59.205	Flux range for POR5: 9986.136 to 9.202
Max biomass: 0.454531	Flux range for PDH: 26.0981627712 to 10,000	Flux range for POR5: 9964.701 to 9.202
Max biomass: 0.310129	Flux range for POR5: 24.575 to 24.588

Even though knocking out the PDH reaction seemed favorable when compared to the existing N2 WM1788 model, literature reveals that the removal of this reaction is associated with diminished cell growth, resulting from a buildup of organic acids. Therefore, we can not perform either of these knockouts.

Checking directionality

J_exp9

I began comparing the FVA outputs for maximizing biomass and maximizing flux through the flavodoxin reaction. I was interested in identifying which reactions changed directionality with the addition of this reaction. Compared to our original FVA outputs (before knocking out malate oxidase), which detailed some reactions with variations of up to 1.0 × 101 in flux ranges, these updated figures only had variations of up to 1.0 × 102. So even though almost 1,000 reactions in the model did have different bound values, these differences were incredibly slight. I was also to identify 14 reaction whose directionality changed upon the addition of the flavodoxin reaction I included reactions that had an upper/lower bound of zero that then went in the positive/negative direction.

These reactions include:

Maximizing ATP

J_exp11

Laura and I also began running FVA codes at max ATP, and then comparing those outputs to our existing max biomass outputs.

Even though we identified 976 reactions that changed directionality, 220 of those being nontransporter reactions (independent of ATP cell transport), the slight changes in flux ranges led up to the conclusion that the model is already ATPlimited.

Reporting to Igem

J_exp12

Overview: Laura and I finished up comparing the FVA outputs for the N2 WM1788 model at max biomass vs. max flavodoxin, but we had no conclusive feedback to report to iGem. Following my updates on the pyruvate knockouts, we reported to Cheryl that there is a potential to remove pyruvate dehydrogenase from the model. But we would need to counterattack any additional buildup of pyruvate from this knockout. We surmised that pushing flux through the pyruvate synthase reaction, and thus maximizing the production of flavodoxin, could account for any compromised cell growth.

The WashU branch was in the process of reworking the existing model to account for non recognition of certain promoters by E coli. These changes did not affect the WM1788 model that we are working with, given the nitrogen fixation reactions already account for any discrepancies. Laura and I completed all of our weekly tasks at the time, and were only responsible for summarizing our results and sending them to Cheryl. In addition to my reports, Tom, Laura, and I came to the conclusion that the pyruvate dehydrogenase reaction can potentially be removed from the model if we account for the buildup of pyruvate in the cell as a result. One way to counterattack this buildup of pyruvate, which will be converted into lactic acid and compromise cell growth/energy, is to push flux through the pyruvate synthase reaction and maximize the production of flavodoxin reductase. This will expend the excess pyruvate concentrations.

Try to maximize ATP

J_exp13

7/2 Laura and I began running FVA codes for maximizing ATP. We compared these outputs to the max biomass outputs, which we used from our previous flavodoxin trials.

We noticed that most of the reactions whose flux ranges changed by more than 0.001 also changed directionality. We also noticed that many reactions at max ATP had lower bounds that were more positive than their upper bounds. We treated these reactions as if their reaction bounds were reversed. For example, instead of reading the reaction bounds at max ATP for XYLI2 as 1.44E07 to 5.893E7, we read it as 5.893E7 to 1.44E07. A majority of reactions that changed directionality were transporter reactions. Because ATP is indirectly involved with transporting proteins and sugars into the cell, it would make sense as to why maximizing its production would potentially alter the directionality of transporter reactions. This is why we only reported nontransporter reactions that changed directionality, which still consisted of roughly 220 reactions. Of the nontransporter reactions we identified, we only took those reactions whose flux ranges varied by more than 0.005 into account. Reactions that varied by less than 0.005 we regarded as essentially having no change in flux. Analyzing a list this complex is incredibly tedious and would not reveal anything about the effects of maximizing ATP on the model. There are too many factors to take into account. However, our greater difference in flux ranges was 0.02, so there was no need to do a thorough analysis of the data comparisons.

Analysis of high delta flux reactions

J_exp14

Laura and I did a quick analysis of the 317 nonoverlapping reactions which change by more than 0.001 between the N2 fixing and WT WM1788 models. We each generated spreadsheets detailing the number of nonoverlapping reactions in each metabolic system/subsystem, what percentage of the total they make up, and what was the greatest change for each system. Overall, amino acid metabolism had the highest number of changing reactions (78), but the greatest change was only 0.637. Membrane transport, exchange, and inorganic ion transport only made up roughly 15% of changing reactions, but had the largest gap in flux ranges (14.6). We also started brainstorming about potential routes to go down, given we were wrapping up our flavodoxin and ATP analyses. Laura proposed looking more closely at dependence of the model on ATP, given flavodoxin, by contrast, is only involved in a handful of reactions. We started compiling a list of the top 10 reactions that produce/consume ATP. I personally was responsible for identifying consummation reactions. Initially I generated an excel file of reactions that only included ATP, and from there I identified which reactions had the most positive flux ranges, given ATP almost always appears as a reactant in the model.

Igem meeting consequences

J_exp15

In our meeting with iGem, Laura and I reported that the model is ATPlimited, a conclusion that we drew from our max biomass vs. max ATP analyses. We also shared some of the main reactions that either produce or consume ATP, and identified that a majority of these reactionsare apart of the carbon metabolism system. After consulting with Tom, and as a step moving forward, we agreed upon knocking out each individual gene from the model to observe its effect on FBA. Him and Maggie also suggested adding additional metabolites in the model, potentially aiding cell growth. Laura and I decided to divvy up these tasks, with her taking the former and me the latter. My experience with python and gams coding is very limited, so I took it upon myself to begin with reviewing the existing templates that Maggie provided at the beginning ofWeek 1. With the help of a python syntax guide, I was able to get a feel for how to generateoutputs files that can then be used to run FBA and FVA.

Figuring out Overexpression

J_exp16

I continued reviewing my existing python and gams to come up with a way to overexpress each of the metabolites in the model. Tom suggested created a “dummy” transporter reaction in my existing python code, which would carry each individual metabolite into the cytoplasm. I decided to take the list of metabolites already defined in the code and generate a new list which only included metabolites with cytoplasm compartments (Tom and I eventually realized that these metabolites are the only ones we are interested in given we are only concerned with “dummy” exchange reactions). With time and effort, I was finally able to generate a new list entitled metname. And from this list, after defining the new metabolite ids as exchange reactions, I was also to make new sij.txt, rxn.txt, and rxntype.txt output files.

Recalculating with new Metabolites

J_exp17

After compiling my new metabolite input files, I started tweaking with the max biomass code that Laura and I have been using to run FBA. Other than including these additional files in the code, I also included a loop statement, which iteratively runs through each metabolite. The data generated from each loop was then placed in an output file, which included max biomass, max ATP, and max flavodoxin.

Initially I set my max biomass constraint to the max biomass determined from running the code. However, setting it to this parameter overconstrained my output file. For example, only 9 of the metabolites had maximum ATP values other than the ATPmreq (3.15), and only 4 of them produced some amount of flavodoxin (all other metabolites produced none). Tom suggested setting the constraint to the original N2 WM1788 max biomass to see if I would get a wider range of figures. Sure enough, there was a much more diverse set of outputs, and almost all of the metabolites produced flavodoxin.

Carbon Constraints

J_exp18

In order to standardize my outputs, and make any viable recommendations to the UWash team for improving the model, Tom recommended that I constrain the amount of substrate in each exchange reaction. And to do so by the number of carbon molecules in each metabolite. We used glucose as a template, and set the supply at 60 (half the amount supplied by the 6 carbon atoms in glucose (20 per molecule)). I also compressed the list of exchange reactions to only include those metabolite who have at least one carbon atom. We planned on dividing the supply by the number of carbon atoms in each metabolite, so dividing by zero carbon atoms would result in computational errors. It took some time reformatting my input files to only include these carboncontaining metabolites, and I encountered several programming issues along the way. For example, I originally only updated the input file that regulates my substrates, and this resulted in flux outputs that were significantly larger than expected. I’m still in the process of readjusting the parameters in the code to account for the addition of these exchange reactions.

Carbon Overview

J_exp19

Last week, I started looking at the effects of adding supplemental metabolites to the WM1788 N2 fixing model. After creating a set of exchange reactions, and regulating the amount of substrate being imported by each reaction, I was able to generate max biomass, max ATP, and max flavodoxin outputs. We normalized our data by the number of carbons in each supplemented metabolite. This allowed the code to constrain our substrate by the number of additional carbon atoms supplied to the model. In my 7/10 journal, I mentioned that we used glucose as a template for regulating our substrate. I failed to mention why we used only 50%. I want to point out that we felt it was a good level to supplement growth. I was having some coding errors last Friday, and I realized that it was a formatting issue in one of my input files. I was still working through some technical issues by the end of the day. I also started working with Laura on our slides for the Monsanto powerpoint. Her and I wrote up a brief overview of FBA and FVA (refer to term sheet if unfamiliar with abbreviations), our general findings thus far, and current projects we are working on.

iGEM meeting and Monsanto Slides

J_exp20

In our meeting with iGem, Laura and I gave a brief summary on each of our projects, specifically what we’re doing and what our objectives are. After the meeting, we updated our Monsanto slides from Tom’s recommendations (mostly visual layout edits). I continued editing my GAMS code from the updates I made in my input files (only include carbon-containing metabolites).

Compile and Run?

J_exp21

After double checking my GAMS parameters, variables, and input files, I was finally able to run my code and generate flux outputs.

While I was reading through them, I realized that some of my carbon-number figures were off. I originally generated a carbon number input file from the iAF model. Specifically, I ran a python code that identifies the number of carbon atoms in each metabolites chemical formula. However, I realized that some of the metabolites were defined differently in the iAF model than in the WM1788 model. I had to manually go through the model and update any figures that were not matching up.

Finding Metabolites

J_exp22

After tediously updating my carbon number input file, I reran my GAMS code and collected my updated outputs in an excel file. Tom recommended I generate a scatter plot of max biomass vs. max ATP to see if I could identify any observable trends. Him and I noticed a portion of the plot that followed a rigid linear trend. Of the metabolites that were along this trend line, one was glucose, our models primary source of energy. Because our objective was to look into supplemental metabolites/energy sources, specifically those with higher flux values than glucose, we were only interested in those metabolite above glucose on the trend line. This consisted of 73 metabolites.

Given several metabolites in the model are either intermediates or impractical to isolate and provide the cell, I started doing a literature check on each one to determine its candidacy. Even though I only ruled out a handful of metabolites that are intermediates, all of the remaining metabolites were either phosphorylated or sugars, specifically glucose derivatives. We were only interested in these six sugars, which include maltotriose, maltotetraoise, maltose, maltopentaose, maltohexaose, and maltoheptaose.

Writing a summary

J_exp23

I completed my summary of metabolite additives for Cheryl, including a project overview, the general procedure I followed, constraints and reasons for such, and findings. We believe that the addition of these sugars, providing additional sources of energy, can promote cell growth and activity. I also added mine and Laura’s slides to the Monsanto powerpoint.

Meeting with iGEM and Succinate miracle?

J_exp24

Laura and I summarized the results of individual trials in our meeting with iGem. After mentioning the 6 sugars I identified from the addition of our metabolite exchange reactions, one member of the WashU team mentioned hearing of an outside branch having success with replacing glucose as a sole energy source with succinate. From this, I started transitioning on to a new topic, specifically other carbon-containing substrates that can replace glucose in our model. Tom also sat down with Dr. Maranas, who would orchestrate funding for our trip to the Jamboree. Even though Dr. Maranas oversees student research in our branch of the department, he does not play a direct role in the work Laura and I do. To verify that her and I are making productive use of our time here, he requested that each of us write a report detailing our project with iGem, along with our findings and future objectives.

Reporting Results

J_exp25

I began writing my report for Dr. Maranas, detailing the motive behind our work here and our ultimate goal of giving plants the ability to produce their own nitrogen. I also mentioned that our branch specifically oversees genome-scale modeling to help guide potential genetic interventions and facilitate nitrogen fixation in E. coli. From our model, we can use FBA to determine the flow of metabolites, or flux, through the network. And from gene knockouts/overexpressions, we can identify available pathways for producing/consuming a metabolite of interest, such as ATP.. As for our results, I focused primarily on how ATP and reduced flavodoxin are the limiting factors in our nitrogenase reactions. Specifically, I mentioned how we attempted to maximize reduced flavodoxin by manipulating pyruvate metabolism. I also focused heavily on my recent work with the metabolite exchange reactions, and how we are looking for ways to help aid cell growth/activity. From this, I have begun looking into other carbon-containing substrates that can replace glucose as the model’s source carbon source.

Checking out Succinate

J_exp27

I started editing my codes to begin running an FBA analysis on replacing glucose with succinate as our model’s sole carbon source. However, after normalizing our substrate to the number of carbon atoms in succinate, 30 mmol/g DW hr succinate vs. 20 mmol/g DW hr glucose, the model proved infeasible. After consulting with Tom, he suggested looking into studies on E. coli growth on succinate to determine if any there are any additional parameters we need to account for, like the presence of another carbon source.

Succinate + Fumarate < Glucose?

J_exp28

I stumbled upon a few online studies that detailed pairing succinate and fumarate as carbon sources for E. coli. Growth deficiency on succinate in our model, from my interpretation, has to do with the essentiality of two enzymes: succinate dehydrogenase and fumarate reductase. Fumarate is derived from the breakdown of glucose in the glycolysis pathway, so replacing glucose with succinate results in a deficiency of fumarate in the cell. I assumed that these two enzymes, which are responsible for promoting microbial metabolism and play an essential role in the citric acid cycle, can not catalyze cell activity if both succinate and fumarate are not at sufficient levels. When I added fumarate as an additional energy source, the model did experience some level of growth, but the max biomass value was only 0.269648 in comparison to 0.454531 for glucose. Tom recommended that I look into the pathway taken by succinate in E. coli determine if any transport reactions have been overlooked in our model. On a side note, I found one study that redirected the metabolism of glucose to succinate, and detailed increased succinate yields from increasing the expression of phosphoenolpyruvate carboxykinase and inactivating the glucose phosphoenolpyruvate- dependent phosphotransferase system. This resulted in increased ATP production and increased concentrations of phosphoenolpyruvate for carboxylation. Inactivating the pflB gene, which encodes pyruvate formate lyase, was also associated with increased succinate yields. However, the primary goal of this particular study was to increase the production of succinate, so I’m not yet sure if these same steps can be taken to maximize growth in our model.

OptKnock comes knocking

J_exp30

While Laura continued running her double gene knockout code, Tom and Maggie mentioned a code called OptKnock. Optknock suggests a list of potential reaction knockouts to couple growth production with the production of a desired product, in our case max biomass and reduced flavodoxin, respectively. Biomass values will vary depending on the percent biolevel we set in our code (refer to 7/29 for more details). Laura and I spent some time reviewing a chapter on OptKnock to become more familiar with it’s structure and objectives. The goal of running this code is to provide another route for identifying potential knockouts in our model.

Succinate could help Respiration

J_exp31

During our meeting with iGem, Charlotte clarified that using succinate as a carbon source is meant to promote respiration in the cell. I want to note that prior to the meeting, I also ran FBA on our N2 model with both glucose and succinate as carbon sources. I found that my max biomass and max ATP figures were identical to my original nitrogen fixing figures (with glucose as the sole carbon source). However, increased respiration would not appear in FBA, so Maggie suggested running FVA and comparing flux ranges for reactions in the citric acid cycle. The citric acid cycle provides a pool of chemical energy from the oxidation of pyruvate, so changes in flux ranges for reactions associated with this subsystem would imply changes in cellular respiration. After running FVA with the addition of succinate, I found that a majority of reactions in this subsystem had altered flux ranges in comparison to their original flux values. However, these changes were incredibly slight- i.e. less than a factor of 1*10^-2.

Start working with OptKnock

J_exp32

Laura and I began editing a OptKnock template that Maggie provided us. Some of these edits included our reaction input files and our ATP and percent biolevel constraints. We initially encountered some issues with getting our code to run, but we quickly identified them and were able to generate a list of potential double knockouts, which consisted of roughly 400 couples. At first glance, all of the double knockouts had the same max biomass and reduced flavodoxin levels. We set our percent biolevel to .9, so each double knockout had a max biomass value of 0.409 (.9 of 0.4545- i.e. our original max biomass value). We wanted to double check and see what effect these knockouts would have on max biomass and ATP levels in our model by running them through an iteration. Laura wrote a python code that splits a reaction input file into a series of coupled-knockouts, given our previous OptKnock output file only lists the couples without pairing them. She originally had to generate this code to run her gene knockouts, so all we really had to do was substitute in our new input file. While Laura edited her python code, I went ahead and ran OptKnock for triple reaction knockouts.

Knockouts did not have expected Results

J_exp33

After running our python code for both double and triple reaction knockouts, we found that none of the proposed knockouts from our OptKnock code were coupled with reduced flavodoxin. In other words, every knockout had the same max biomass and ATP level as our original nitrogen fixation model- i.e. without any knockouts. The only useful piece of information we drew from our outputs was that several reactions involved in pyruvate metabolism were included in the suggested knockouts. This would confirm some of our previous suggestions on increasing reduced flavodoxin production by manipulating pyruvate metabolism in the cell.

Summary for Website

J_exp35

While Laura resolved these issues in her code, I began our modeling summary for the website. Laura and I felt that an introduction, associated methods, formulation, and results section were appropriate. Because Laura is still working on her python code, I decided to cover the first two sections and my results from the metabolite exchange reactions.

Fixed another Bug

J_exp36

8/3 Laura regenerated outputs for her doublegene knockout code, but inconsistencies were still popping up in our data when we manually tested knockouts coupled with flavodoxin. We were finding that for noninfeasible knockouts, the maximum biomass, ATP, and reduced flavodoxin values were completely off from those in the iteration outputs. We finally were able to figure out that the code was coupling each gene with itself, resulting in several infeasible outputs. However, instead of skipping over these knockouts, the code outputted the previous set of output values, resulting in mismatched data. We resolved this issue but removing duplicate genes in our code.

Work on Website Summary

J_exp37

Following our meeting with iGem, Laura and I finalized our travels grants and began working on the modeling summary for our wiki page. We wanted to draw attention specifically to what genomescale modeling is and what we’re hoping to accomplish using methods such as FBA (navigating potential genetic interventions to optimize cofactors of nitrogenfixing in E. coli). Because most of our results were inconclusive, we wanted to focus on our discoveries with pyruvate metabolism, and how we can promote the production of reduced flavodoxin by redistributing flux through reactions involving pyruvate. We also included brief summaries of our recent work on metabolite exchange reactions and gene/reaction knockouts.

Work on Website summary

J_exp38

Laura and I finalized the first draft of our wiki page, after reworking it to include more visuals and minimal text. Even though computational modeling is extensive, we tried to summarize key points in flow charts and supplement them with brief descriptions. Laura also collected all of the outputs for the double gene knockout code. Even though our outputs were finally starting to match up, we found, unfortunately, that any knockout coupled with flavodoxin outputted an infeasible model.

Review First Draft

J_exp39

Laura and I sat down together and reviewed the completed first draft of our modeling summary. Considering Laura had to leave early, I finalized any remaining edits, such as redesigning our section on genomescale modeling. We wanted to break up any big chunks of texts. Before Laura left, her and I also wrote up our section on computational modeling for the abstract. Given the length constraint, we focused primarily on what our goal was in using computational modeling to promote nitrogenase activity.

Finished First draft

J_exp40

I reviewed the final first draft of our wiki modeling page, and any edits that the WashU team made to our abstract.

Mike Toomey Washington University in St. Louis

Preparation of Electrocompetent Cells

B_exp1

These are the strains we put the nif cluster in this summer.

Prepare cultures of DH10B

25 ml of each culture were transferred into 500 mL of LB
-These flasks were then grown at 37 deg C, 250 rpm for 1 hour, 30 min in incubator
Measure absorbance
- Cuv1-.1055, Cuv2-.1084
After absorbances were measured, cultures were added to ice bucket and allowed to cool for 15 minutes.
-Centrifuge bottles were centrifuged for 25 min. at 4 deg.
After centrifuge, bottles were taken to cold room
- Chilled 10% glycerol was added, 55 ml each, into each bottle
  
  It’s worth noting that % glycerol in each bottle is roughly 4.4%
- 70 Ml of water were added to each bottle
-Bottles were placed back into centrifuge for another 256 minutes at 4 deg C-After centrifuge, bottles were taken to cold room once more
Supernatant was drained
- 10% glycerol was added to 4 tubes
- Roughly 15 mL were allotted to each tube
- Tubes were taken and centrifuged
After centrifuge, conical tubes were taken to cold room
- Supernatant was drained
- 1 mL of 10% glycerol was added to each tube, pellet allowed to resuspend
- Tubes were taken to centrifuge again, 3000 RPM @ 4deg C

Designed program to find sgRNAs in Java,

B_exp2

Need to find a place for dCAS9 to bind

Designed program to find sgRNAs in Java, key feature is finding the PAM sequence

More electrocompetent cell prep

B_exp3

EC cell prep for Strains: MG1655 and JM109

Two culture were incubated overnight
-In the morning, 26 mL of culture were placed into 500 mL of LB
-These flasks were incubated for an hour beginning at 10:34
-Both flasks were taken out around 11:35 P.M.
-Optical measurements were performed on both colonies using a 10x dilution factor
- i. The optical density of JM109 was 0.287 ii. The optical density of MG1655 was 0.39
-JM109 went back to be incubated at 11:56 A.M.
-Optical measurement performed on JM109 once again
- Optical density was 0.38
-Optical density for both strains was determined to be satisfactory -Thus, each flask was equally partitioned into two centrifuge bottles
-Centrifuge bottles were left to sit for 20 minutes
-After sitting for 30 minutes, bottles were centrifuged at 3000 RPM for 25 minutes
-Upon centrifugation, supernatant was drained, pellet was resuspended in 125 mL of water
-Bottles were centrifuged again for another 25 minutes
-Upon centrifugation, supernatant was drained and 55 mL of 10% glycerol were added to each bottles
-Pellet was resuspended in each bottles
-After resuspension, each bottles was partitioned into 4 15 mL conical tubes
-Conical tubes were centrifuged under same conditions
-From a combined conical tube, 40uL aliquots were created for JM109 and MG1655
-These aliquots were stored in -80 deg C conditions Electroporation
-We transformed MG1655 and WM1788
-To do this, we obtained copies of pSL2397 plasmids
-2.5 uL of this plamsmid DNA was collected and mixed with pipettor
-After this, the plasmid DNA was inserted into a cuvette
-The entirety of the aliquot containing the E.Coli strains was dumped into the cuvette
-The plasmid was shocked into the strain for 5.5s
-Strain was taken out and put into conical tube for an hour
-The same process was repeated with MG1655 strain

-We finalized sgRNA sequences

B_exp4

-In picking sgRNA sequences, we determined that sequences close to the beginning of the strand would be the most effective -Using a computer program, we ran gene sequences through and found the most effective sgRNA sequences i. Java program that predicates itself on finding the PAM sequence

Preparation of Frozen Stock

B_exp6

Store Cells from yesterday

-Cells were taken from picked colonies on June 4
-We took 500 uL of that culture, added it to 500 uL of 30% glycerol solution, then place din -80k deg C conditions.
-We then prepared 30% glycerol solution for later use
-Next, we prepared the CRIPSR/dCas9 plasmid for PCR
-To do this, we separated the plasmid from its host bacterium
-First, we took the bacterial culture and centrifuged it
-Then, we discarded LB growing solution that was supernatant
-Next, we resuspended bacterial cells in water
-Buffer to lyze the cell was added
-Immediately after, cold neutralization buffer was added to neutralize the process
-Then, tubes were centrifuged at 16000 g for 43 minutes
-Supernatant was transferred to a separation column
-These columns were then centrifuged for 30 seconds
-The flow through was discarded and the colonies were placed back into the collecting tubes
-200 uL of Endo-Wash Buffer were added to each tubes
-Each tubes was centrifuged under same conditions for 30 seconds
-Upon centrifugation, 400 uL of Zappy’s buffer were added to each tube
-Each tube was centrifuged for another 2 minutes
-Upon centrifugation, high pure molecular water was added to each column and each column sat for two minutes
-Each column was centrifuged, same condition, for another 2 minutes
-After this step, dissolved plasmid DNA had collected on bottom of column
-Each column was taken to spectrometer to determine concentration and purity

-Concentrations and purities, in order

Tube	Conc (ng/uL)	Purity
Tube 1

1.87 127.1 Tube 3 1.79 82.7

After that, each DNA sample was placed into freezer to be stored

Prepare Gel

B_exp9

We prepared a gel for DNA purification

-Mix 1g of Agarose with 100 mL of TAE buffer.
Note that you can cool the mixture with cold, running water
-Add 5 uL of cybersafe (5uL/100 mL of buffer) once mixture cools
-Put in combs
-Tape side of tray
-Pour agarose gel in tray
-Let gel cool for 1 hour
Run gels for 1 hour

Made culture

M_exp10

Made culture for the transformed DH10B so that we could create frozen stock

Helped out in Zhangs lab

M_exp11

Helped Charlotte and Carlos in the Zhang lab as they ran PCR's, gels, DNA purification

Zhang RE Digest (30 microl)

M_exp12

PCR

M_exp13

Preparing samples
- Add HP molecular water 10x the amount of DNA in primer tube to get a 100 uM concentration after centrifuging
- Vortex each tube
Preparing solutions
- Create 10 uM solutions of DNA by transferring primer DNA from 100 uM solution to a microcentrifuge (10 microl) and add 90 microl water
- Make a mastermix for the PCR, which should have an amount of liquid n+ 1 times the number of rexn you have
- Per rxn
  - 1 microl template
  - .5 microl phusion polymerase (mastermix)
  - 2.5 microl forward primer
  - 2.5 microl reverse primer
  - 1 microl dNTP's (mastermix)
  - 4 microl DMSO (mastermix)
  - Fill rest of 50 microl with water (micromix)
- Get out 150 ml mcf tube per reaction and add mastermix plus DNA
Thermocycler
- Put tubes in top tray and close lid
- Tighten the dial on lid so that it's tight, but doesn't break off
- Choose the new protocol button
- Make sure to change the vol
- Make sure to change the polymerization time (72 C) to make sure phusion can extend whole DNA strand
  
  Phusion's rate is 1000 bp/15 sec
- Change primer annealing temp. to what you want
  
  Should be 4 degrees C less for DMSO solutions
Making an agarose gel
- Measure out 1 g of agarose and add to 100 ml of TAE in a 250 ml flask
- 2. Put in microwave for 1.5 min.
- 3. Let cool after microwaving and then add 7 microl Sybersafe
- 4. Make a gel plate by drying sides off and taping twice
- 5. Pour gel into plate until it reaches comb inserts level
- 6. Make sure no air bubbles are in gel by combing the gel with comb
- 7. Place comb into gel and let sit for 1.5 hrs.
- 8. Throw out all trash into gel bin
Loading a gel
- 1. Take comb out perpendicularly and place plate in clean buffer
- 2. Grab samples from Thermocycler on 50 microl tray
- 3. Dilute samples with loading dye from fridge to 1x to 6x
- a. If PCR products are 50 microl, this means adding 10 microl dye
- 4. Mix by drawing up ~60 microl from the bottom of tube, not middle
- 5. Put in well, approaching perpendicularly
- a. Do not go in so far as to poke gel!
- 6. Continuously push the product into the well and pull the pipetter out as
- soon as the first stop is hit
- 7. Load DNA ladder (5 microl) (1 kb)
- 8. Place black end of electrode onto side with DNA
- 9. Press mode on machine 3 times to find time, set to 30 min., let sit

PCR

		1X	9x
Template	1 microl	-
Pusion	.5 microl	4.5 microl
F Primer	2.5 microl	-
R Primer	2.5 microl	-
dNTP's	1 microl	9 microl
HF Buffer	10 microl	90 microl
DMSO	4 microl	36 microl
Water	28.5 microl	256.5 microl

DNA ligation

M_exp18

i. Want 100 ng backbone (bb) with 5x insert (in)
ii. (5)x(100 ng bb/(6819bp/bb))x((820 bp/in)/(46.7ng/microl)) = 1.28 microl of insert
iii. 100 ng bb/45.1 ng/microl = 2.22 microl bb
iv. Totals
- 8 microl
- 1 microl T4 ligase
- 1 microl buffer
- .2 microl PNK
let ligation reaction sit for 1-1.5 hrs.
transform cells with plasmid

David Ayeke Washington University in St. Louis

Make CμLtures

D_exp2

The Moon Lab needed, strains JM109 and MG1655 for their experiments

Make overnight cμLtures from frozen stock to make electrocompetent cells the next day (in Moon lab):

from glycerol stock, set up two 50mL cμLtures in 250-mL flasks
grow overnight

Make Lb

D_exp4

The Moon Lab needed, strains JM109 and MG1655 for their experiments

Transformation (in Moon lab):

- Take MG1655 and WM1788 electrocompetent cells out of -80°C and place on ice
- Take PSL2397 (plasmid) out of -80°C and place on ice
- Add 2 μL of PSL2397 directly into MG1655 tube
- Set pipette over 40 μL and draw up the MG1655 and PSL2397 mixture and place into electroporation cuvette
- Tap cuvette to ensure cells are at bottom of cuvette—shoμLd check to see that there are no gaps—and place into electroporator; turn on
- Immediately add 500 μL of LB into the cuvette then pour into cμLture tube
- Place cμLture tubes of WM1788 and MG1655 into incubator for 1 hour

Make Lb

D_exp6

There are 10 genes in the Nif cluster that have unknown functions. Overexpressing these can shead light on what they do

Use SnapGene to design primers for the 14 sequences to be overexpressed

- Check for EcoRI and XhoI restriction sites within each of the sequences
- Check for directionality on the plasmid: direct or complementary
- If a restriction enzyme does not have sites within the sequence, add a site for that restriction enzyme to the appropriate primer and add 6 adenines beyond the site on the primer so that the restriction enzyme will work properly
- If the restriction enzyme does have a site within the sequence, end the primer at the end of the sequence to be amplified to leave the ends blunt
- If direct, add EcoRI to the 5’ end and XhoI to the 3’ end as appropriate
- If complementary, add XhoI to the 5’ end and EcoRI to the 3’ end as appropriate
- Maintain a Tm for each primer above 60°C and approximately match the Tm of paired primers
- Ensure that there is only 1 binding site on the plasmid for each primer

Run PCR

D_exp8

Run PCR on 3 of the 14 amplicons

Run PCR

D_exp9

Run PCR on remaining amplicons

Overnight cμLtures

D_exp14

Start an overnight cμLture in 5 mL of LB at 37°C for transformation of ligated plasmids

Plate Cells

D_exp18

- Take cells out of 37°C room and spin down all but the control for efficiency of transformation (PE5A) for 4 minutes
- Take 100 μL from PE5A control and add to 900 μL of LB
- -Plate 100 μL of that dilution on the amp plate
- -Add sterile glass beads and shake laterally to spread around the cμLture
- -Dump beads into nonsterile glass beads container
- from centrifuged cμLtures, pipette off the media to the 100 μL mark and resuspend the pellet in that amount of media
- -Add fμLl resuspended quantity to the correct labeled plate
- -Add beads and shake
- Place all plates in 37°C room

Store plates

D_exp19

Take plates out of 37°C room, check for colonies, and place those that grew into the 4°C room for the remainder of the weekend

Run Gel

D_exp25

- Run digested plasmid through the gel

- Saw no band for digested plasmid and a band in unexpected location for the undigested plasmid need to redo the digestion of the plasmid

Confirm Inserts

D_exp26

Make 20 μL PCR reactions for plasmids purified yesterday (to attempt to confirm correct inserts)

Digest

D_exp33

Digest the blunted cut plasmid with Xho1 in a 25 uL reaction

Add the following to the Klenow reaction mixture (without purifying first)
2 uL digested buffers
2 uL Xho1
1 uL water
Place at 37C for ~3 hours

Check plates

D_exp34

Carlos checked plated cells for growth

1 colony on control; ~30-50 colonies on other plates
Placed plates in 4C room

Pick colonies

D_exp35

Carlos picked 2 colonies per plate and started overnight cultures in LB

Transform

D_exp38

Transform plasmids from overnight

Purify plasmid from overnight cultures of colonies
Quantify cocentrations of plasmids
Set up 20 uL PCR reaction for the purified plasmids to confirm inserts
transform ligated plasmids
- Add the ligation reaction products produced to labled vials of competent cells
- Incubate cells with ligation products added on ice for 25 minutes
- Heat shock at 42C for 1 minue; replace on ice for ~2 minutes
- Add 900 uL of LB to each tube (in the hood)
- Place on shaker at 37C for 1 hour
Pour plates
- Melt LB agar in a secondary container of water in the microwave
- Wait for agar to cool before adding 1 uL of chloramphenicol per mL LB agar
- Pour plates with ~20mL agar with antibiotic
- Allow plates to dry in hood
Plate cells
- Centrifuge, pipette off the supernatant to 100 uL mark, then resuspend the pellet in that amount
- Add resuspended cells to labeled plates; spread with with sterile glass beads. Place plates in 37C room
Run a gel of PCR products (to confirm inserts in transformed cells)

Check colonies

D_exp39

Check for colonies from plated cells

Control grew colonies, as did the others
Pick a colony with a pipette tip and resuspend colony in the media
Add 50mL of LB to a conical tube; add 50 uL of chloramphenicol to that
Add 5 mL of that mix to each of the 10 labeled sterile culture tubes.
Pick a colony with a pipette tip and then either resuspend the cells in tube or drop the tip into the tube
Plate on shaker in 37C room at 250 rpm
Add 5 mL LB to another steril culture tube. Add 5 uL LB to the tube. Scrape frozen stock of PA2C-TesA with a pipette tip and place the tip in the Lb; place on shaker in 37C room

Miniprep PA2C-TesA

D_exp44

Miniprep PA2C-TesA (using 10X the amount of buffers as suggested by the kit)

Pour the 50 mL culture into a 50 mL conical and centrifuge to a pellet
Pour off the supernatant
Resuspend in 2500 uL of resuspension buffer and vortex or pipette in and out until no clumps remain
Add 2500 uL of lysis buffer and invert 10 times to mix (no vortexing). Incubate on ice for 5 minutes
Centrifuge at 13,000 rpm and 4C for 10 minutes
Pour the supernatant through a syringe with a cotton ball in it to filter
Transfer 800 uL of supernatant to a column in a collection tube and centrifuge at 13,000 rpm for 1 minute.
Discard the filtrete and replace in the same collection tube
Repeat previous steps with additional 800 uL of filtered supernatant until all supernatant has gone trhough column
Add 500 uL of wash buffer A and centrifuge at 13,000 rpm for 1 minute. Discard the flow through andreplace column in the tube
Centrifuge at 13,000 rpm for 1 minute
Place on thermomixer at 65C for 5 minutes
Place column in a new 1.7 mL microcentrifuge tube and add 50 uL of water warmed to 65C; let sit at room temperature for 1 minute
Centrifuge at 13,000 rpm and 1 minute
Quantify concentration on nanodrop
On 2% agarose gel, add: 2.5 uL 1kb pluss ladder to wells on either side. 8uL of each PCR product

Run gel

D_exp44

Run a gel to see if PA2C-TesA purified yesterday is okay

Pour a gel with 10 wells of ~35 mL 1% agarose with 2 uL EtBr
Run 100 ng of plasmid max -> 2.5 uL with .5 uL loading dye
2.5 uL ladder on each side

Repeat transformation

D_exp45

Purify digested PA2C-TesA with DNA clean & concentrator kit
Start a Klenow reaction
Digest the plasmid from the Klenow with Xho1
load the digested plasmid into a gel and gel purify the correctly sized band
Ligate the digested insert with digested plasmid
Transform the ligation products into chemically competant cells

Start overnight of more PA2C-TesA

D_exp46

Attempt to PCR the inserts from the HDK overexpression plasmid using the nifHDK F and R primers

D_exp48

Redo HDK PCR

D_exp50

Redo HDK PCR from nif cluster plasmid to attempt construction of the HDK overexpression plasmid again

Primer design for minimal nif plasmid

D_exp51

Redo PCR of HDK and cce_0550

D_exp53

Cut and purified band for 0550, but HDK band was incorrect

Ligate 0550 and HDK

D_exp54

Ligate genes into plasmid

Repurify 0550 PCR product (due to a bad peak on nanodrop)
Digest repurified 0550 with Xho1
Clean up digest with DNA clean & concentrator kit
Set up ligation for 0550 and HDK with PA2C-TesA

PCR genes for minimal nif plasmid-grouped by size into 3 sets

D_exp55

PCR for small and large size groups
Gradient PCR from 58C to 65C
100 uL rxn split into 4 tubes of 25 mL each, at different temperatures in gradient thermocycler
Run gels of a small amount of each of tube. Saw bands for very few of the expected products

PCr medium size group

D_exp56

PCR for minimal nif plasmids continued

D_exp57

PCR more 0550 and HDK for overexpression plasmids

D_exp58

EcoR1 digest of PA2C-TesA
Run 0550 PCR product in a gel and gel purify it
Digest 0550 with Xho1
Run HDK PCR product in a gel and gel purify it

Prepare 1 plasmid of nif cluster

D_exp59

PCR amplify tetracyclin resistance gene off of one plasmid and pB1a except for the amplicilin resistance gene, using primers designed for Gibson ligation
Blunt the ends of PA2C-TesA cut with EcoR1 using S1 nuclease
- Add at least 10 units of S1 nuclease per microgram of plasmid, but use 10 units if you use less than 1 microgram
- Use a 10x final concentration of buffer
- Add water to a 30 uL final reaction volume
- Place at room temperature for 45 minutes, then add 4 uL of .25M EDTA and incubate at 75C for 20 minutes to stop the reaction
After blunting with S1 nuclease, clean up with DNA clean & concentrator kit, then start an Xho1 digest
Set up Gibson assembly for backbone resistance change for one of the minimal plasmids
- Use 7.5 uL of Gibson mix premade by another member of the lab
- Dilute to a 10 uL final reaction volume
- Place at 50C for 1 hour to ligate
- Add 50 ng of each piece to be ligaged
  
  Based on the concentrations of DNA we had, we were unable to add 50ng of each piece, and maximized the amount of DNA added by not diluting with water
transform Gibson assembly products using chemically competent transformation protocol
Cut PA2C-TesA backbone band from gel and gel purify
Make new tetracyclin stocks for Gibson assembly products transformation
- Measure 50 mg of tetracycline hydrochloride on weighing paper
- Add 70% EtOH to a 10 mL final volume; mix
- Add 4 uL per mL to 20 mL Lb (80 uL of tetracycline)

Checked on 7/16/2015, no colonies from Gibson products

Transform ps8k plasmid into DH10B to make more

D_exp61

Golden Gate reactions

D_exp63

Set up a 10 uL Golden Gate reaction of all 8 pieces for the pS8K cysE2-hesB minimal plasmid, using a molar ratio that compares concentration/size(bp) of each piece. Inserts should all be in a 3 to 1 ratio to the backbone

mix reaction mixtures and DNA together
- DNA should be 7 uL
- Add: 1 uL T4 ligase buffer, .5 uL Bsa1, .5 uL T4 ligase, .3 uL Dpn1, .5 uL ATP at 10 uM, .3 uL BSA
Spin down and put in thermocycler
- 37C for 3 minutes
- 16C for 4 minutes
- Repeat first 2 steps 50 times
- 50C for 5 minutes
- 80C for 5 minutes
- Hold at 4C

Pick colonies

D_exp64

Pick 5 colonies from DH10B plate of golden gate reaction mixture transformation. Pick 1 colony of ps8k

Work on HDK & 0550

D_exp65

Digest PA2C-TesA with EcoR1, PCR more 0550 and HDK

Golden gate cysE2-hesB

D_exp67

Run a gel to check for cysE2-hesB PCR product
Run a gel to cut the backbone from the PA2C-TesA Xho1 digest
Digest ps8k minipreps with pst1 and Pme1 to check for proper sizes
Set up a golden gate of the seven inserts only, excluding the backbone from this reaction, using a 1 to 1 molar ratio of all Golden Gate inserts

Repeat Golden Gate

D_exp68

Set up a PCR using the 7 insert Golden Gate reaction as template: make 2 15 microliter reactions, one using 1 microliter of the golden gate as template and the other using 1 microliter of a 10x dilution of the template
Run a gel of a small amount of the ligation mix(template for the PCR)
Start digest of HDK and 0550 PCR products with Xho1
Clean and concentrate the digest
PCR of inserts with 10x dilution of template looks good -- start a 100 uL reaction scaled up
Run a small amount of cleaned up digest products on gel to check for proper bands -- got expected bands
PCR tetracyclin and pB1a for the backbone resistance change with the newly-arrived Golden Gate primers(rather than the Gibson primers)

More Golden Gate

D_exp70

Re PCR the ps8k backbone to ligate cysE2-hesB into
transform golden gate ligation of pB1a and tetracycline into DH10B chemically competent cells
Cut and purify the 7 gene ligation PCR from the gel
Set up a Golden Gate ligation of the 7 insert PCR product and the amplified pS8k backbone in a 10 uL reaction

Transform the Golden Gate product into chemically competent DH10B

D_exp71

Check for inserts

D_exp72

Miniprep the overnight of ps8k, ps8k cysE2-hesB 1 through 5 ( 5 colonies picked from the plate), and pB1a-tet
Start a Golden Gate reaction of the 7 inserts for the second plasmid
Digest pB1t with Spe1 and BamH1 (alone and together) to check for correct insert
Digest pS8k cysE2-hesB with Pme1 and Pst1 (alone and together) to check for insets

Send in sequencing primers

D_exp74

Dilute sequencing primers for ps8k cysE2-hesB
Sequence miniprep #2 of ps8k cysE2-hesB (clearest bands on the gel from digest check)
Sequencing PcR reactions
- 50 uL reactions using ps8k cysE2-hesB #2 as template
- Amplify ~1 kb per primer pair, with each read overlapping by about 100 bp (the way the sequencing primers were designed)

Check Plasmid

D_exp75

Run a gel of a small amount of nifh to nifn golden gate mixture and all of the pB1t PCR
Saw no visible band for the Golden Gate mixture where expected for full ligation
Cut bands of pB1t PCR
Miniprep the overnight clulture of PA2C-TesA
Digest PA2C-TesA with EcoR1
Clean and Concentrate the digest, then start a klenow reaction
Resuspend newly-arrived sequencing primers for the next minimal nif plasmid, using TE buffer

Repeat Golden Gate

D_exp77

Try inserts in groups of 3 and 4 rather than all 7
nifH, nifD, nifK and nifB, hesA, nifE, nifN

Do Goldan Gate 2 at a time

D_exp78

Do Golden gate 2 inserts at a time, in pairs as follows (leaving out nifK from the groupings for now): nifH & nifK, nifB & hesA, nifE & nifN

Run PCRs to confirm

D_exp79

EN did not work. Try PCR with varied MgCl concentration and annealing temperatures.
Cut HD and BA from gel and gel purify
Start a Golden Gate reaction of HD with K

Attempt to digest then ligate separately

D_exp81

Digest
- 1.5 hour digestion
- 20 uL reaction
- Bsa1, digest buffer, and 1 to 2 uL of insert to be digested
- Clean and Concentrate
Ligate cleaned up pieces
- T4 ligase
- T4 ligase buffer
- .5 uL ATP
- 10 uL reaction

More EN

D_exp83

Run PCRs of nifE and nifN ligation from separate digestion and ligation

Use E F and N R primers
Use 2 additional pairs of primers from the sequencing primers designed for this plasmid to amplify smaller portions of E and N that cross the junction, to check for appropriate ligation
Start overnight ligation of digested E and digested N at 4C

Check on ligation reactions

D_exp84

Ran gel of HDK BA, HDK BA E N, and overnight 4C E N ligations
Start PCRs from each, in addition to the room temperature EN ligation

Both amplifications of EN look good - cut and purify together
Neither HDKBA nor HDKBAEN worked
PCR more HDK from golden gate template
Start digest of BA and EN with Bsa1 for 2 step digestion then ligation

Laura Beebe Pennsylvania State University

Find a method of calculating NAD/NADH

L_exp1

Searched literature for a method of calculating NAD/NADH reaction

found incorrect ratio according to literature. Need new method to calculate this

Find Relatives of JM109 and WM1788

L_exp2

Literature/database search for relatives of JM109 and WM1788 to construct models.

Learned they are K-12 derivatives.

Researched K-12 model

L_exp3

Researched which reactions to remove from K-12 model in order to construct WM1788 and JM109 models using genotypes provided by Wash U

Checked ATP production

L_exp4

Made final WM1788 model, found max biomass and ATP production. Made K-12 model from Monk paper, found max biomass and ATP production. Made additional corrections to DH10B model, found new max biomass and ATP. Had lower biomass (growth) than K-12 derivatives. Continued work on JM109 model with help from Jess.

found new max biomass and ATP. Had lower biomass (growth) than K-12 derivatives. Continued work on JM109 model with help from Jess.

Compared Carbon Sources

L_exp5

Compared strain growth and ATP production on various carbon sources.

Noticed high biomass/high ATP when using galactose as a carbon source. Need a literature search to explore this finding.

Compared Carbon Sources

L_exp6

Continued comparing strains based on nutrient differences. Started researching differences in E. coli metabolism for glucose vs. galactose.

Added N2 fixation to WM1788

L_exp7

Added N2 fixation reactions to WM1788 model, using flavodoxin as electron donor. Created a set of nutrients for the N2 fixing strain (contains free nitrogen and lacks fixed nitrogen). Found max biomass (2.52) and max ATP (391.3) for N2 fixing WM1788.

Normalizing wild type biomass indicates a difference in ATP production of 202 mmol ATP/(g cell dry weight x hr) between WT and N2 fixing cells. This value makes sense according to nitrogenase flux range.

Ran FVA on WT WM1788

L_exp8

Ran FVA on WT WM1788 and N2 fixing WM1788. Starting comparing the flux range overlaps between the two outputs using Excel.

Strange result

L_exp9

Observed CO2 and H2O export fixed for N2 fixing WM1788. Started checking pathways (glycolysis, PPP, TCA cycle, pyruvate metabolism, reactions involving flavodoxin) to find out why.

Compared flux of N2 fixing of WM1788

L_exp10

Compared flux ranges of N2 fixing and wild type WM1788 within main metabolic pathways (glycolysis, PPP, TCA cycle, pyruvate metabolism) as well as within reactions that included oxidized or reduced flavodoxin.

Main result- pyruvate synthase (produces acetyl coA from pyruvate) in the N2 fixing strain has a higher flux than in wild type. Additionally, many reactions within TCA cycle have a lower flux in the N2 fixing strain than in wild type (this is expected- N2 fixing WM1788 should have a depressed metabolism), except for malate dehydrogenase (produces oxaloacetate from malate).

Continued analysis of WM1788

L_exp11

Continued analysis of differences between wild type and N2 fixing WM1788.

Determined neither strain is using the glyoxylate shunt. Performed comparisons of all reactions involving malate, oxaloacetate, acetyl coA, and pyruvate. Malate oxidase (reaction at equilibrium) has a much more negative flux in N2 fixing WM1788 than in wild type. Seems that oxaloacetate from malate dehydrogenase (higher flux in N2) feeds malate oxidase reaction to produce more malate and oxygen in the N2 fixing strain.

Exploring relationship between pyruvate and acetyl coA in N2 fixing strain versus wild type

L_exp12

Continued analysis of differences between wild type and N2 fixing WM1788.

Identified two other reactions containing both acetyl coA and pyruvate (besides pyruvate synthase), however both have the same flux in both N2 fixing and wild type. The flux is 0-10,000, meaning they are cycling. Jess explored deletion of these two reactions to see if removing them would help to push flux through pyruvate synthase and aid nitrogen fixation. Also looked at other ways to produce acetyl coA and other ways to spend pyruvate; however, no result yet for viable knockouts.

Exploring relationship between pyruvate and acetyl coA in N2 fixing strain versus wild type

L_exp13

Continued identifying reactions that involve either the production of acetyl coA or the expenditure of pyruvate. Looked for other ways to make products in reactions that spend pyruvate, indicating the potential for a knockout. No conclusive result.

Created a flavodoxin expenditure reaction

L_exp14

Created a flavodoxin expenditure reaction (analogous to ATP maintenance reaction) in order to determine maximum possible flavodoxin production in the cell. Compared FVA outputs for N2 fixing WM1788 at fixed max biomass producing maximum flavodoxin and regular N2 fixing WM1788 at fixed max biomass.

Created a flavodoxin expenditure reaction (analogous to ATP maintenance reaction) in order to determine maximum possible flavodoxin production in the cell. Compared FVA outputs for N2 fixing WM1788 at fixed max biomass producing maximum flavodoxin and regular N2 fixing WM1788 at fixed max biomass.

Made comparisons between N2 fixing WM1788 at fixed biomass and N2 fixing WM1788 with maximized flavodoxin at fixed biomass.

L_exp15

Made comparisons between N2 fixing WM1788 at fixed biomass and N2 fixing WM1788 with maximized flavodoxin at fixed biomass. Had to spend some time troubleshooting when the comparison didn’t yield values that we expected.

Did not notice any large differences between values at max flavodoxin and values without max flavodoxin-- ie. can conclude that maximizing flavodoxin as much as possible is already a way for the cell to maximize biomass.

Research malate dehydrogenase and malate oxidase

L_exp16

Based on information that malate dehydrogenase and malate oxidase reactions had a much higher flux in the N2 fixing cells than in wild type, did a database search to find information about ∆G values for these reactions in order to potentially change their directionality in the model.

Malate dehydrogenase directionality could not be changed. After some searching, found malate oxidase reaction had been deleted from major E. coli databases in the past year. Determined that the reaction should be removed from the model.

New maximum biomass and maximum ATP

L_exp17

Obtained new maximum biomass and maximum ATP production values for all strains after removing the malate oxidase reaction from the model.

Values were significantly smaller than before, but are more logically consistent with the model. Strain DH10B now had the highest biomass and ATP production, but only by .0002 mmol/(g cell dry weight X hour).

Ran FVA and began comparing flux ranges

L_exp18

Ran FVA and began comparing flux ranges at maximum biomass for N2 fixing WM1788 and WT WM1788. Looked for differences in pathways between the N2 fixing and wild type strains. Also ran FVA for N2 fixing WM1788 at fixed biomass with maximum flavodoxin production, and N2 fixing WM1788 at mixed biomass without maximum flavodoxin (did not find many differences in flux ranges, as before).

Compared flux ranges between N2 fixing and WT WM1788 at a fixed max biomass

L_exp19

Compared flux ranges between N2 fixing and WT WM1788 at a fixed max biomass, looking particularly for reactions whose directionality changes between the N2 fixing and wild type strains. Assembled a list of these reactions, how much they changed by, and the metabolic systems they belong to. Next week will need to perform the same comparisons with both strains set at maximum biomass, in order to eliminate wide flux variations in the wild type WM1788 for reactions that contribute directly to biomass.

Continuing flux range comparisons

L_exp20

Compared directionality of fluxes for N2 fixing and wild type WM1788 at maximum biomass. This eliminates potentially large changes in reactions which are not a component of biomass.

Major result

Finish flux range comparisons

L_exp21

Finished up comparisons. Looked through fluxes of transporter reactions in search of an explanation for ATP synthase directionality change in N2 fixing WM1788 vs. wild type. Did not find any conclusive result.

Finished flux range comparisons

L_exp22

Did a flux range comparison of N2 fixing WM1788 at maximum ATP production vs normal N2 fixing WM1788. Many reactions changed their directionalities, but the largest change was .02. Can determine from this that the N2 fixing strain is already ATP limiting-- ie. there are no major differences when ATP is maximized.

Performed an analysis on all reactions with non-overlapping flux ranges between N2 fixing WM1788 and the wild type

L_exp23

Performed an analysis on all reactions with non-overlapping flux ranges between N2 fixing WM1788 and the wild type. Determined which metabolic system each reaction was in and what percentage of the non-overlaps each system made up.

Amino acid metabolism was the system with the most non-overlaps, but it had the smallest changes. Ion transport, exchange, and inner/outer membrane transport made up a little over 15% of the non-overlaps together, but had the highest changes.

Analyzed ATP production/consumption in N2 fixing WM1788.

L_exp24

Analyzed ATP production/consumption in N2 fixing WM1788. Compiled a ranked list of reactions which produce ATP. Jess compiled a list of the top 10 reactions in the model which consume ATP.

Started working on the Python script

L_exp25

Started working on the Python script for knocking out all of the genes in the N2 fixing model. Started by writing a code which takes the GPRs (gene protein relationships) present in the model and creates a list of all the unique genes.

Continued working on the Python script

L_exp26

Continued working on the Python script

L_exp27

Continued working on the Python script.

L_exp28

At this point it can generate a list of reactions that will be deleted for each gene knockout.

Monsanto Slides

L_exp30

Made a rough draft of slides for the team’s presentation to Monsanto next week.

Continued working on Python script

L_exp31

Continued working on the Python script and accompanying GAMS code. Modified the GAMS code so that it will output the values for biomass, ATP, and flavodoxin in one file, instead of requiring multiple GAMS codes for each value. Continued working on the slides for Monsanto.

Continued working on the Python script

L_exp32

Fixed various errors in the GAMS code and continued working on the Python script. Ran the full code for the first time. It gives output in correct formatting, but values are incorrect. Finished the slides for Monsanto presentation and added notes.

Worked on debugging the final code.

L_exp33

Fixed all remaining errors

L_exp34

Fixed all remaining errors in the single gene knockout code, ran the code, and received the results, which are summarized as follows:

Single Gene Knockout Results:

For 8 genes, max flavodoxin was 2.07

All other genes

Report for Dr. Maranas and KO code

L_exp35

Continued working on the report and code.

L_exp36

Continued working on the report and code.

L_exp37

Finished both the report and code.

L_exp37

Worked on code

L_exp38

Tried running my gene double knockouts code but it timed out.Broke up the code into smaller pieces that will be run simultaneously. Also started reading about OptKnock.

Worked on code

L_exp39

Started setting up and debugging the OptKnock code with Jess. We want to try to couple flavodoxin production with biomass.

Worked on code

L_exp40

Ran OptKnock for double reaction knockouts and continued to debug. Wrote a Python code that will determine if the OptKnock suggested knockouts couple flavodoxin with biomass or not. Jess ran an OptKnock code for triple reaction knockouts.

Worked on code

L_exp41

Wrote a new Python code to determine of the OptKnock suggested triple reaction knockouts couple flavodoxin production to biomass.

First OptKnock results

L_exp42

Analyzed OptKnock results; however, none couple flavodoxin to biomass. Note that OptKnock suggests reaction, not gene knockouts.

Second OptKnock results

L_exp43

In my final week, I fixed the issues with my double gene knockout codes (so there are no longer discrepancies and the outputs are correct). Unfortunately, we did not find any double knockouts that coupled flavodoxin production to biomass. Future research could look into why this is the case. Additionally, Jess and I worked on getting travel grants and funding in order for our trip to the iGEM Giant Jamboree in September. compiled our wiki modeling summary and worked on making edits for it.

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Team:WashU StLouis/Notebook

- Experiments From Jun 02

Make CμLtures

- Experiments From Jun 03

Make Lb

- Experiments From Jun 04

Make Lb

- Experiments From Jun 09

Run PCR

- Experiments From Jun 10

Run PCR

- Experiments From Jun 11

Overnight cμLtures

- Experiments From Jun 12

Plate Cells

- Experiments From Jun 13

Store plates

- Experiments From Jun 15

Run Gel

- Experiments From Jun 17

Confirm Inserts

- Experiments From Jun 18

Digest

- Experiments From Jun 19

Check plates

- Experiments From Jun 20

Pick colonies

- Experiments From Jun 22

Transform

- Experiments From Jun 23

Check colonies

- Experiments From Jun 24

Miniprep PA2C-TesA

- Experiments From Jun 25

Run gel

- Experiments From Jun 26

Repeat transformation

- Experiments From Jun 30

Start overnight of more PA2C-TesA

- Experiments From Jul 01

Attempt to PCR the inserts from the HDK overexpression plasmid using the nifHDK F and R primers

- Experiments From Jul 02

Redo HDK PCR

- Experiments From Jul 03

Primer design for minimal nif plasmid

- Experiments From Jul 06

Redo PCR of HDK and cce_0550

- Experiments From Jul 07

Ligate 0550 and HDK

- Experiments From Jul 08

PCR genes for minimal nif plasmid-grouped by size into 3 sets

- Experiments From Jul 09

PCr medium size group

- Experiments From Jul 10

PCR for minimal nif plasmids continued

- Experiments From Jul 13

PCR more 0550 and HDK for overexpression plasmids

- Experiments From Jul 14

Prepare 1 plasmid of nif cluster

- Experiments From Jul 16

Transform ps8k plasmid into DH10B to make more

- Experiments From Jul 17

Golden Gate reactions

- Experiments From Jul 20

Pick colonies

- Experiments From Jul 21

Work on HDK & 0550

- Experiments From Jul 22

Golden gate cysE2-hesB

- Experiments From Jul 23

Repeat Golden Gate

- Experiments From Jul 24

More Golden Gate

- Experiments From Jul 25

Transform the Golden Gate product into chemically competent DH10B

- Experiments From Jul 27

Check for inserts

- Experiments From Jul 28

Send in sequencing primers

- Experiments From Jul 29

Went to Pakrasi lab to observe acetylene injection

Prepped M9 media

Prepped culture with aTc

Prepared for assay

Prepped cultures of JM109 with pSL2397 for a miniprep

Miniprepped pSL2397