15-09-01 MDH Assay (in vitro)

Do MDH Assay in vitro like in papaer from Zürich.

Used strains are #IGEM# as MDG strain and K12 wild type as control.

We use the supernatant with NAD+ for spectiometric measurement (340 nm).

Expected: In #IGEM# lysat the absorbance should increase because more NADH should get created. The level NADH of #IGEM# should be higher than in wild type.

15-09-02

check if the MDH is present in supernatant or cell pellet via SDS-PAGE.

Result: The SDS-PAGE has no useful information, even the MDH in the pellet of whole cells is not visible.

Do Overnightculture of #IGEM# & #OHES# (as control: glgC in A30 in BL21 GOLD DE3) for next try.

15-09-03

Do 25 ml maincultures from overnights. Induction with IPTG was done at 11:48 OD of #IGEM# 0.565; OD of #OHES# 0.698.

Prepare the two cultures after 6 h for MDH in vitro assay (IGEM OD 3.83; OHES OD 4.04). => pellet 5.5 ml of culture; beadbeated with 0.1 mm beads;

Prepare Stocks for assays:

• 1mlStock= 50 mM KPi, 5mM MgSO4, 500µM NAD, 1M MeOH
• Nash reagent: Nash reagent (1 L) 2 ml acetylaceton 300 g/L ammonium acetate 6 ml acidic acid fill up to 1 L with deionized water. (add a sample in the ratio of 1:1; measure at 412 nm)

Do SDS-PAGE with whole cells, cell-lysat pellet and supernatant from both clones.

• #IGEM# Cellpellett => OD 10
• #OHES# Cellpellett => OD 10
• #IGEM# Cellfragmentpellett => OD 9
• #OHES# Cellfragmentpellett => OD 9
• #IGEM# Supernatant => no dilution
• #OHES# Supernatant => no dilution

'

Result of SDS-PAGE:

See SDS gel "15-09-03 MDH Assay expression check + new constitutive MDH + Poly.png"

In #IGEM# cell fragment pellet is a big band of MDH size => we assume that the MDH is not solvable behind a T7 promoter.

We assume the same fact for glgC in #OHES#.

Nash test (pretest)

Made each measurement in triplicates in plate reader (blank, IGEM, OHES).

• F1-3 100 µl Stock sol. + 100 µl Nash reagent
• G1-3 95 µl Stock sol. + 5 µl #IGEM# supernatant + 100 µl Nash reagent
• H1-3 95 µl Stock sol. + 5 µl #OHES# supernatant + 100 µl Nash reagent

Incubation of microtiterplate started at 21:26 at 37 °C and 100 rpm

1. Measurement at 22:35 t=1 h 9 min
2. Measurement at 00:36 t=3 h 10 min

• Measure last measurement with pathlength correction & once again without airbubbles

Nash pretest results

After 3 h small tendency towards postive formaldehyde production of #IGEM# supernatant sample. But this contradict our assumptions from SDS.

Improvements for Nash

• Do more samples for measurement
• Measure at 412 for Nash reaction and a second wavelength at 340 nm for NADH production
• Do calibration with formaldehyde
• use fragment pellets for measurement too

in vitro Assay

measure absorbance at 340 nm in platereader with microtiterplate

for each well is 190 µl stock sol. used and 10 µl sample

• A1 OHES A2 IGEM; suprnatant as sample
• B1 OHES B2 IGEM; suprnatant as sample
• C1 OHES C2 IGEM; suprnatant as sample; pipett both sample at once from one person
• D1 OHES D2 IGEM; whole cells as sample (10 µl too)
• E1 OHES E2 IGEM; pipett first the supernatant then the stock sol.
• E5 OHES E7 IGEM; supernatant as sample; check if position next to each other has an influence.
• F1 OHES F2 IGEM; cellfragments but the liquid part of it (really no beads!!!)

Results of in vitro assay

Improvements for in vitro assay

• after cell distubtion do not centrifuge. centrifuge the liquide without the beads separately to get a bead free cellfragment pellet.
• try the cuvetts measurement additionally

Preparations for next day

Overnightculture of #IGEM# & #OHES#

freeze pellets of todays maincultures 13h after IPTG induction (Pellets made of #IGEM# 15 ml OD 4.21; #OHES# 13.5 ml OD 4.37)

To Do:

• write ETH Zürich our problem and our results
• analyse our data from platereader
• new aliquotted NAD (try to do everything on ice because NAD is not that stable in water!!!) (done)
• do new mainculture (25-50 ml) out of the overnights at 30 °C to reduce the risk of inclusion bodies. (done)
• check if MDH is in supernatant of lysat or as inclusion body in the fragments (use the 13h samples) via SDS (done)
• check the same for 30 °C main cultures (6 h after induction with IPTG) (samples are taken)
• try to put cell fragment into refolding buffer, maybe the MDH in the inclusion body can get the native shape again. (canceled)

15-09-04

SDS-PAGE

Do SDS-PAGE of 13h samples after induction.

See gel picture: 15-09-04 MDH Assay expression check 13 h samples.png

30 °C expression test

Do 50 ml LB mainculture of overnights from #IGEM# & #OHES#. Induction with IPTG (only 2/3 as usual (33.3 µl)) at OD #IGEM# 2.8; OD #OHES# 1.7.

Take 6 h sample (15 ml). (OD #IGEM# 5.87; OD #OHES# 4.99.) Pellet and freeze them.

Take 18 h samples (15 ml) date (5.9.). (OD #IGEM# 6.91; OD #OHES# 5.41.) Pellet and freeze them.

Nash Assay

A1-A5: IGEM Cells

B1-B5: OHES Cells

C1-C5: IGEM Lysate Pellet

D1-D5: OHES Lysate Pellet

E1-E5: IGEM Lysate Supernatant

F1-F5: OHES Lysate Supernatant

G1-G5: Blank

A10-A12: 100 µg Formaldehyd

B10-B12: 80 µg Formaldehyd

C10-C12: 60 µg Formaldehyd

D10-D12: 40 µg Formaldehyd

E10-E12: 20 µg Formaldehyd

F10-F12: 10 µg Formaldehyd

Measurements were done like described above. Additionally, fluorescence was measured at an excitation wavelength of 410 nm and an Emission wavelength of 510 nm. In the blank no cell/lysate solution was added. The lysate pellet was resuspended in 200 µl water.

Results

• really fast reaction (even without incubation) in the wells with formaldehyde standards
  • low upper detection limit
  • use lower concentrations for the calibration next time
• higher adsorption and fluorescence in all IGEM samples compared to OHES
• highest adsorption and fluorescence in the IGEM lysate pellet as well as very low adsorption and fluorescence in IGEM lysate supernatant
  • strengthens the assumption of the MDH ending up in inclusion bodies

Trafo of Poly(K-RDP) and MDH(A30) plasmids in strain C43 & SHuffle T7 express

SHuffle T7 Express [http://www.lucigen.com/OverExpress-C41-DE3-and-C43-DE3-Competent-Cells/ C43]

This is the first part of new Masterplan!!!

Transformation of #XULU# in competent C43 cells & into SHuffle T7 express cells.

Transformation of #BZBQ# in competent C43 cells & into SHuffle T7 express cells.

Put 30 µl on one plate and rest (225 µl) on a separate plate.

Trafoplates incubation started at 18:45.

TO DO in Morning

• Masterplates of each trafo and overday cultures (15x each).
• overday culture (5x each) of #AW9K# & #IGEM# from plate.

To Do in general

• Analysing Nash experiment of today
• do Nash calibration with formaldehyde

new Masterplan to find expression strain for labeling experiment

Aim

We assume that the activity of MDH is the bottleneck of MCC pathway.

Find a clone that has less inclusion bodies of MDH and more activity in cell lysat supernatant in "in vitro Nash Assay". Compare BL21 GOLD DE3, C43 and Shuffle which have hopefully better solvable protein expression for labeling emperiment in Juelich. we check them all with MDH plasmid and Poly plasmid.

• less inclusion bodies at 30 °C cultivation
• (less inclusion bodies at 37 °C cultivation)
• can they all grow on M9 media?
• check wheather they carry the rigth plasmid
• optional additional info: can the strain grow on M9 with MeOH

Schedule

15-09-04:

• First step is to transform Poly plasmid and MDH in A30 into C43 cells and Shuffle T7 express cells. (done)

15-09-05

• Do Masterplates and overdays of all transformants

evening/night:

• Do cryo of selected overdays
• Do overnight cultures of C43 and Shuffle clones for next day plasmid prep
• plate the selected clones separately for labeling experiment in Juelich
• Do Maincultures out of the overdays in LB and M9 media (50 ml) (6xLB + 6xM9) Poly in BL21 (#AW9K#)/ C43 (#LNWY#)/ Shuffle (#FMLB#) & MDH A30 in BL21 (#IGEM#)/ C43 (#RHFS#)/ Shuffle (#M6L4#)
  • • the interesting cultures for following experiments are the M9 cultures if they are able to grow
  • 30°C or 37°C?? (iTS prefers 30 °C) (Tip from Dr. Ruff: try incubation and expression at 18 °C, 25 °C and 30 °C)
  • induce MDH (in A30) clones at OD 0.6-0.8 with IPTG
  • take samples after 6h & xh of induction => take 15ml, pellet it and freeze it.

15-09-06

• Plasmid prep of Overnight cultures to check the carried plasmids. (check via test PCR of Poly in K-RDP and testdigest of MDH in A30)
• optional: Inocculate new shake flask cultures for growth experiment with MeOH in progress see Methanol/Physiology#15-09-06 Physiology
• Cell lysis method has big influence for solvability of our enzymes (Dr. Ruff)
• Disrupt all pellets and prepare SDS samples from whole cells, supernatant & fragment pellet. (usually 12-20 % of proteins in inclusion bodies are solvable)
  • Do Nash Assay with all strains from all temperatures and all fragments (cell/fragment pellet/lysat supernatant)

Decision of clones/strains for labeling experiment

For the labeling experiment in Juelich we will use a Strain that carries "Poly" in K-RDP and a second strain that carries MDH in pSB1A30.

The strain has to fulfill these criteria:

1. carry the rigth plasmid
2. be able to grow on M9 media
3. the strain that expresses less inculsion bodies and has the most MDH activity in the supernatant in Nash Assay.

15-09-05

• Masterplates of all four transformations are done (18 clones picked each) incubation started at 12:00
• overdays of all four transformations are done (15 cloes each) & overdays of #IGEM# & #AW9K# (5 each) are done (start 12:00)

Night

• Do cryo of selected clones #RHFS# #LNWY# #M6L4# #FMLB#
• restart new overnights from masterplates of selected clones & plate them separately for labeling experiment (Done at 01:00). #RHFS# #LNWY# #M6L4# #FMLB#

• Prepare 50ml (M9) maincultures of:

• • OD of Overdays at inoculation-time: #RHFS# 1.84; #LNWY# 1.39; #M6L4# 1.84; #FMLB# 1.37; #IGEM# 2.68; #AW9K# 1.93
  • Inoculate 50 ml cutures with 1800 µl & 25 ml culture with 700 µl
  • calculated start OD of 50 ml M9 cultures: #RHFS# 0.07; #LNWY# 0.05; #M6L4# 0,07; #FMLB# 0.05; #IGEM# 0.1; #AW9K# 0.07
  • calculated start OB of 25 ml LB cultures: #RHFS# 0.05; #LNWY# 0.04; #M6L4# 0,05; #FMLB# 0.04; #IGEM# 0.08; #AW9K# 0.05

30 °C M9 Cultures: (Start: 00:20)

1. BL21 with MDH in A30 (#IGEM#)
2. C43 with MDH in A30 (#RHFS#)
3. SHuffle with MDH in A30 (#M6L4#)
4. BL21 with "Poly" in K-RDP (#AW9K#)
5. C43 with "Poly" in K-RDP (#LNWY#)
6. SHuffle with "Poly" in K-RDP (#FMLB#)

37 °C M9 Cultures: (Start 00:34)

1. BL21 with MDH in A30 (#IGEM#)
2. C43 with MDH in A30 (#RHFS#)
3. SHuffle with MDH in A30 (#M6L4#)
4. BL21 with "Poly" in K-RDP (#AW9K#)
5. C43 with "Poly" in K-RDP (#LNWY#)
6. SHuffle with "Poly" in K-RDP (#FMLB#)

Backup Cultures 37 °C (25 ml): (Start 00:34)

1. BL21 with MDH in A30 (#IGEM#) on LB media
2. C43 with MDH in A30 on LB media
3. SHuffle with MDH in A30 on LB media
4. BL21 with "Poly" in K-RDP (#AW9K#) on LB media
5. C43 with "Poly" in K-RDP on LB media
6. SHuffle with "Poly" in K-RDP on LB media

(we use M9 media with 40 mM Glucose)

Induction time was 4:14!!! of all MDH clones

OD measurement before induction:

37 °C 50 ml M9:

• #RHFS# 0,795
• #LNWY# 0,294
• #M6L4# 0,82
• #FMLB# 0,296
• #IGEM# 0,85
• #AW9K# 0,369

30 °C 50 ml M9:

• #RHFS# 0,538
• #LNWY# 0,223
• #M6L4# 0,499
• #FMLB# 0,22
• #IGEM# 0,722
• #AW9K# 0,294

37 °C 25 ml LB (Backups): Because strains are able to grow on M9 we use not all of these for sampling.

• #RHFS# ---
• #LNWY# 0.7
• #M6L4# ---
• #FMLB# 0.6
• #IGEM# ---
• #AW9K# 0.7

=> FMLB (Shuffle Poly) was used for Aquila shake flask experiment in Physiology

15-09-06

Samples that has to be taken

take 15 ml sample in falkon tube und freeze pellet! (please measure the OD of the culture when the sample is taken and note down.)

Take samples of all MDH carrying clones first to make them compareable between each other. Afterward take samples of all Poly clones.

37 °C 50 ml M9 cultures

• #RHFS# 6 h at 10:15
• #LNWY# 6 h at 10:15
• #M6L4# 6 h at 10:15
• #FMLB# 6 h at 10:15
• #IGEM# 6 h at 10:15
• #AW9K# 6 h at 10:15

37 °C LB cultures: only MDH clones for sampling

• #RHFS# 6 h at 10:15
• #M6L4# 6 h at 10:15
• #IGEM# 6 h at 10:15

30 °C 50 ml M9 cultures

• #RHFS# 6 h at 10:15
• #LNWY# 6 h at 10:15
• #M6L4# 6 h at 10:15
• #FMLB# 6 h at 10:15
• #IGEM# 6 h at 10:15
• #AW9K# 6 h at 10:15

OD measurement at first sampling (10:15):

C-M 30: 4.25 C-M 37: 3.79 C-M LB: 4.34

C-P 30: 1.56 C-P 37: 1.48

S-M 30: 4.22 S-M 37: 3.48 S-M LB: 4.41

S-P 30: 1.6 S-P 37: 1.5

B-M 30: 3.56 B-M 37: 3.70 B-M LB: 3.59

B-P 30: 1.62 B-P 37: 1.86

OD measurement at 20 h sampling (00:15 15-09-07):

C-M 30: 3.74 C-M 37: 3.76 C-M LB: 4.07

C-P 30: 4.30 C-P 37: 3.36

S-M 30: 3.80 S-M 37: 3.74 S-M LB: 3.58

S-P 30: 4.36 S-P 37: 3.39

B-M 30: 5.73 B-M 37: 4.94 B-M LB: 2.89

B-P 30: 5.57 B-P 37: 4.53

Procedure

MDH cloes and Poly clones have to be treated independently of each other!!

SDS-PAGE will be done at last because we have to figure out which samples to load according to the Nash results!!!

We analyse the MDH activity via Nash Assay (in vivo+in vitro) think about good platelayout for all samples of one sampling time. Use every sample in all 3 variations (cell/fragment pellet/supernatant) from 6h samples. Do whole cell Nash assay of all samples.

• We have to compare all MDH clones of one temperature and media-type between each other => figure out the strain specifications and influences
• We have to compare all Poly clones of one temperature between each other => figure out the strain specifications and influences

• after plotting all activities we compare the activities between the different expressiontemperatures of one clone-construct. => figure out temperatur influence.

• in Vitro Assay can be done in paralell but is not as important as Nash.

OD adjustments of 6h samples

• Polys: OD 19.9
• MDH in M9: OD 44.1
• MDH in LB: OD 30.1

Do disruption again 5-times bead beaten (0,1 mm) and place lysis liquid in new 1,5ml tube for cooling centrifugation to get glas free fragment pellet.

Supernatant Protein absorbance (280 nm) of 6 h samples

• Measured with 1:100 dilution.

(This is to analyse the measurements agains each other via their relative values)

C-M 30: 42.7 C-M 37: 30.6 C-M LB: 26.9

C-P 30: 23.0 C-P 37: 18.5

S-M 30: 31.2 S-M 37: 38.9 S-M LB: 25.9

S-P 30: 22.4 S-P 37: 18.4

B-M 30: 34.3 B-M 37: 33.9 B-M LB: 22.3

B-P 30: 18.9 B-P 37: 15.8

Plate layout: of Nash Assay Nr.3 15-09-06

Plate 1)

Plate 2)

Legend:

C= C43 (DE3); S= T7SHuffle Express (DE3); B= BL21 GOLD (DE3)

M= MDH in pSB1A30; P= Poly in K-RDP

30 °C= 30 °C in M9; 37 °C= 37 °C in M9; LB= 37 °C in LB

Z= Zelle/Cell; SN= Supernatant of lysat; P= cellfragment pellet

15-09-07

Nash Nr.4

Do Nash Assay with whole cells of 6 h samples and 20 h samples. Do the Assay at 30 °C and at 37 °C. Measure Absorbance at 412 nm and flourescence with exitation of 400 nm and extinction of 500 nm.

OD adjustment 20 h samples

• MDHs in LB: OD 14.3
• MDHs and Polys in M9: OD 32.5

Layout of 30°C Assay

Layout of 37 °C Assay

Legend:

C= C43 (DE3); S= T7SHuffle Express (DE3); B= BL21 GOLD (DE3)

M= MDH in pSB1A30; P= Poly in K-RDP

30 °C= 30 °C in M9; 37 °C= 37 °C in M9; LB= 37 °C in LB

6=6 h sample; 20=20 h sample

Z= Zelle/Cell;

Results

• General
  • 6 h samples show stronger response if positive
  • Mdh expressing strains show stronger responses than Polys
  • strains cultivated at 37 °C show higher response than the ones cultivated at 30 °C
  • the assay works better and faster at 37 °C
• BL21 with the mdh plasmid are the strongest positives leading to the conclusion that they express functional Mdh
• the strongest response is observed in BM 37 °C 6 h

this influences the selected strain for the Methanol/C13-Labeling Experiment

SDS-PAGE of 6 h samples

• whole cells, supernatant and cell fragments of 6 h samples were prepared for the SDS-PAGE

See SDS gel pictures at:

Conclusion

• all poly strains show bands at expected protein length showing that all 4 enzymes of the cascade are expressed
• the most distinct bands can be observed at lanes containing SDS samples of BL21 Gold poly strains

Discussion and Decision for 13C labeling experiment

Decision from Nash Assay and SDS-PAGE for 13C experiment in Jülich:

• For MDH expression use Bl21 GOLD at 37 °C => #IGEM# => IPTG induction at exact OD 0.8.
• Poly no decision from Nash possible
• SDS shows expression of all enzymes in all poly versions
  • BL21 at 37 °C shows clearest and partially most intensive bands
  • => Use BL21 in marked C experiment due to SDS results, comparability and more experience with the strain

Cell vs supernatant vs cell fragments

• The strongest responses can be observed in the Nash assays using the whole cells, some activity occurs in the supernatant, little in the fragments
• This is supported by the SDS-PAGEs showing the most distinct bands in the lanes containing SDS samples with whole cells
  • the Mdh most likely does not end up in inclusion bodies
  • the cell membrane might protect the Mdh from the harsh environment in the assay solution explaining the higher activity in whole cells

Post Test of Mdh strain from biorector 15-09-09

Nash assay with samples from the 13C experiment in Jülich

• comparison with GlgC expressing strains (#OHES#) as negative control in a high number of replicates to prove the functionality of the Mdh expressed by BL21 Gold (#IGEM#)
• only whole cells were used
• Nash assay at 37 °C
• #IGEM# sample was 4.5 h induced

Plate layout

Data:

Flourescence Values at Gain 75 Nash Assay after 7,5 hours of incubation at 37°C with excitation wavelength of 410 nm and an emission wavelength of 510 nm. C5 is an empty well, rest of column 5 is Blank, 1-4 & 6 is control strain with GlgC & column 7-12 is the strain with Mdh.

Results

• #IGEM# shows significantly more activity than #OHES#

Nash Test of IGEM and OHES #IGEM# expresses Mdh and #OHES# expresses glgC as a negative control (4.5 h after induction). Both constructs were incorporated in vector pSB1A30 and the BL21 strains were cultivated at 37°C. Each value is made from at least 35 replicates of each construct. Fluorescence measured at a gain of 100. IGEM shows a significantly higher activity of the MDH in comparison to a blank and OHES (glgC in BL21).

15-09-11 media test from reactors

A centrifuged sample from each bioreactor was used to check if formaldehyde is present in the media of the 13C experiment.

=> No significant difference between the start OD(412) or fluorescence data can be observed.

References