Protocols

Experimental Techniques of Molecular

Biology and Protein Expression

Summer term 2017

Cloning and expression of recombinant proteins in E. coli for the development of a

multifunctional membrane for water purification

Student project MultiBrane

Prof. Dr. Budisa

Supervisor: Saba Nojoumi, Franz-Josef Schmitt

1

Microbial cultivations

In this project we only worked with the coliform bacterium Escherichia coli. E. coli is one of

the best studied microorganisms and is commonly used in molecular biology. Naturally, E. coli

occurs in the intestine of mammals as an important part of the gut microbiome. Regularly, E.

coli laboratory strains are regarded as S1 organism, meaning that they are non-pathogenic

and require no special safety measures. Nevertheless, there are also harmful, toxin-producing

E. coli strains and as a scientist working with genetically modified organism we always took

care of an appropriate and sterile working manner.

Figure 1| E. coli cell schematic, source: Aqua di Cutinase project TU Berlin

2

For the cultivation of our E. coli strains we normally used LB medium (liquid broth medium)

that was supplemented with Kanamycin (50 mg/ml). Kanamycin is an antibiotic that is used to

select microorganism. We selected E. coli cells which had uptaken our plasmid and therefore

gathered a kanamycin resistance.

Table 1| LB medium recipe

component [g/L]

Trypton or Pepton 10

Yeast extract 5

NaCl 10

Kanamycin 0.05

dest. water

(adjust pH 7)

add to the desired volume

Agar (for solid media only) 1,5%

Liquid medium was prepared for cultivations of E. coli for transformations or protein

expression. Such cultivations were carried out in small cultivation tubes (10 ml) or in shake

flasks up to 1 L, which were incubated on a shaker platform at 200 rpm and 37 °C for regular

cultivations but 30 °C for protein expression.

Solid medium was prepared in agar plates to streak out E. coli or plating cells

subsequently after transformation to have the ability to select single clones. Incubation was

performed at 37 °C in a static incubator.

For all cultivations, it was taken care to take sterile materials and also work sterile, whenever

possible such work was done under a sterile bench.

3

OD measurement

To monitor growth and culture density we used OD600 measurements. Thereby, the density of

a cell suspension is determined at 600 nm in a spectrometer with cell-free medium as a

reference. The result is an absorbance value that should never exceed 1. If so, the measured

solution must be diluted. We usually measured in a 1 ml cuvette.

4

Competent cells

For the transformation of foreign DNA to E. coli the cells need the ability to take this DNA up.

Such cells are called competent and they need special treatment to increase their membrane

permeability for the uptake.

We applied two different protocols to generate competent cells:

● CaCl2 competent cells (chemically competent)

A 4 ml overnight culture of E. coli cells is used to inoculate 400 ml of fresh LB medium. The

cells are subsequently incubated until an OD600 of maximal 0.7 is reached. The culture is

portioned then into 50 ml reaction tubes and cooled on ice. Next the cells are harvested by

centrifugation at 4 °C for 10 min at a speed of 5000 x g. Cell pellets are cooled on ice and

resuspended in 10 ml of precooled MgCl2 solution (100 mM). The resuspension is chilled on

ice for approx. 30 minutes. Thereafter two aliquots are unified and centrifuged at 4 °C for 10

min at a speed of 4000 rpm. The pellets are resuspended in 2 mL sterile ice-cold CaCl2 solution

(100 mM CaCl2, 15% glycerol) each. The cells are now assumed to be competent.

Competent cells are aliquoted in portions of 50 or 100 μL, frozen in liquid nitrogen and stored

at -80 °C.

● electrocompetent cells

A 2-4 ml overnight culture of E. coli cells is used to inoculate 200 ml of fresh LB medium.

The culture is incubated at 37 °C and 200 rpm until the OD600 reaches 0.3 - 0.5. The cells are

subsequently harvested by centrifugation for 5 min at 4 °C and 3000 x g). The pellet is washed

twice with ice-cold 10% glycerol and afterwards resuspended in ice-cold 10% glycerol to a

final OD600 around 60 – 70. This cell solution is aliquoted in portions of 50 -100 µl, frozen in

liquid nitrogen and stored at -80 °C.

5

DNA amplification with Polymerase chain reaction (PCR)

Polymerase chain reaction, short PCR, has become the common procedure to amplify DNA

in molecular biology. All needed is a DNA template, which contains the desired DNA

sequence, primer - small DNA oligos that match the beginning and the end of the desired DNA

sequence perfectly and a DNA polymerase - an enzyme that can produce copies of DNA.

There are basically 5 steps:

● STEP 1: initial denaturation of the template DNA - to get single strand DNA (otherwise

the DNA polymerase cannot copy the DNA)

● STEP 2: denaturation of the template DNA

● STEP 3: Annealing of the primer

● STEP 4: extension of the primer along the DNA template (DNA copying by DNA

polymerase)

● STEP 5: final elongation - DNA polymerase finishes the copying process for all

templates

These steps are performed at specific temperatures and step 2 - 4 are repeated for several

times which leads to n2 -DNA fragments in each cycle. Optional the reaction can be stopped

and stored at 4 °C for a longer time period in the end.

Table 2| PCR programm

Step Temperature

[°C]

Time [min] Cycle nb.

1 Initial denaturation 95 3:00 1

2 Denaturation 95 0:30 30

3 Annealing 0:30 30

4 Extension 72 30

5 End elongation 72 10:00 1

6 Hold 4 infinity infinity

The annealing temperature depends on the melting temperature of the primers and the

reaction mixture components. It has to be calculated for every PCR which can be done online.

6

The timespan for the extension depends on the size of the DNA fragment and the synthesis

velocity of the applied polymerase.

We worked with the Taq-Polymerase (1 kb/min) and the Q5® High-Fidelity DNA Polymerase

by NEB (2 kb/min). the difference between both polymerases is the synthesis accuracy, which

is higher for the Q5®. Therefore, control PCRs were performed with Taq DNA Polymerase and

amplification of genes for further cloning was performed with Q5® DNA Polymerase.

We prepared a PCR mix freshly for every amplification after the following scheme:

Table 3| PCR mix pipetting scheme

amount comments

DNA template < 1 ng DNA amount needs to be

calculated

polymerase buffer

(10x)

2 µl specific for each polymerase

polymerase 0.125 - 0.25 µl

depends on the polymerase

protocol

primer #1 (10 µM) 0.5 - 1.25 µl

primer #2 (10 µM) 0.5 - 1.25 µl

dNTPs (10 mM) 0.5 µl

nuclease-free water up to 25 µl

Note: The primers were ordered from Sigma-Aldrich after in silico design. After arrival, they

were diluted to 10 µM aliquots and stored at -20 °C.

The result of the PCR is evaluated via gel electrophoresis.

7

Gel electrophoresis

To separate and analyse DNA fragments we used 1% agarose gels. Agarose was boiled with

the running buffer (50x TAE buffer: Tris, acetic acid, 0.5M EDTA) until the solution becomes

completely clear. This solution can be stored at 60 °C. Each gel is prepared with an appropriate

comb and a staining reagent is added. For staining we used either GelRedTM or ethidium

bromide and to determine the DNA fragment size 8 µl of DNA ladder (1 kb, GeneRulerTM

Thermo Fisher). The samples were diluted with nuclease-free water and loading buffer (6x,

Thermo Fisher) to a final volume of 6 µl and loaded to the gel. To run the gel electrophoresis

voltage is applied: we regularly applied 90 V for a 1 h run. However, sufficient separation must

be assured. There are loading buffer systems that include pre-stained DNA fragments (100

bp) which can be monitored during the run for their movement.

The run is stopped by disconnecting the voltage and transferring the gel to a photo-

documentation station. This instrument is used to read out DNA gels as it illuminates the

stained DNA. GelRedTM and ethidium bromide are both fluorescent when exposed to

ultraviolet light. The exposure can be monitored and captured by a camera. The gel images

can then be analysed.

Note: Ethidium bromide makes DNA visible by intercalation. This is could lead to mutations

even in living organism (mutagenic). The handling of ethidium bromide therefore need special

safety measures. GelRedTM makes DNA visible by binding to the sugar-phosphate backbone

and is thus assumed to be non-mutagenic.

8

Gel extraction

To separate DNA fragments of different size and utilization of these in further cloning steps

gel extraction was conducted.

Therefore, the mix that contains the DNA is conditioned with loading buffer and loaded to the

gel. The gel in this case has much larger gel bags thus a large volume can be loaded.

The gel is prestained with either GelRedTM or ethidium bromide and to determine the DNA

fragment size 8 µl of DNA ladder (1 kb, GeneRulerTM Thermo Fisher) is loaded to a small gel

bag. To run the gel electrophoresis voltage is applied: we regularly applied 90 V for a 1 h run.

However, sufficient separation must be assured. There are loading buffer systems that include

pre-stained DNA fragments (100 bp) which can be monitored during the run for their

movement.

The run is stopped by disconnecting the voltage and transferring the gel to a photo-

documentation station. This instrument is used to read out DNA gels as it illuminates the

stained DNA. GelRedTM and ethidium bromide are both fluorescent when exposed to

ultraviolet light. The exposure can be monitored and captured by a camera. The gel images

can then be analysed.

The exposure of DNA by ultraviolet light induces DNA damage. Since we want to utilize the

DNA from the gel afterwards, the exposure should be short with a low UV light intensity.

Furthermore, this also protects the person working directly at the illumination desk. This

person has to wear UV-protecting glasses and skin protection. The desired DNA fragment is

then cut out of the illuminated gel with a scalpel and transferred to a 1.5 ml reaction tube.

Depending on the applied kit, the reaction tube must be weighed without and with gel pieces

in to determine the weight of these. We used two different Gel extraction kits: Roti®-Prep Gel

Extraction, Roth and GeneJET Gel Extraction Kit, Thermo Scientific. The gel extraction was

performed according to the manufacturers manual. The eluted DNA concentration was

determined by UV - spectroscopy.

Note: Ethidium bromide makes DNA visible by intercalation. This is could lead to mutations

even in living organism (mutagenic). The handling of ethidium bromide therefore need special

safety measures. GelRedTM makes DNA visible by binding to the sugar-phosphate backbone

and is thus assumed to be non-mutagenic.

9

DNA concentration measurement

DNA concentration was measured after each step of our cloning protocol. The DNA solution

is measured with a UV-spectrometer in reference to an appropriate buffer blank. In order to

detect protein impurifications and solvent residues, the measurement includes four

wavelengths.

260 nm - DNA

280 nm - proteins

230 nm - organic impurifications

340 nm - ethanol

10

DNA purification

After enzymatic treatment of DNA with either polymerases or restriction enzymes the DNA

needs to be purified from theses enzymes and their buffers. Regularly, we used DNA

purification Kits for that (GeneJET PCR Purification Kit, Thermo Scientific). The purification

was performed according to the manufacturer’s manual.

In case we wanted to monitor the DNA between cloning steps we did a gel purification or gel

extraction.

11

Restriction digest

Restriction enzymes are part of the bacterial immune system and cut DNA specifically. For

this these enzymes need a specific recognition sequence called restriction site. Restriction

sites are mostly palindromic DNA sequences. When such as sequences is cut by a specific

enzyme this can either result in blunt ends without overhangs or sticky ends with overhang.

The enzymes that we applied all create a 5`prime overhang.

To clone our gene of interest we need to cut open the plasmid backbone and make the insert

fit into that frame. For each cloning procedure, we apply two different restriction enzymes to

generate overhangs with different base pairs. Thus, the plasmid cannot repair itself (auto-

ligation).

We prepared a digestion mix freshly for every restriction digest after the following scheme on

ice:

Table 4| Restriction digestion mix pipetting scheme

Volume [µl] comments

DNA up to 1 µg

restriction enzyme buffer 2 (10x buffer) buffer adequate for enzyme

enzyme #1 1 – 1.5 µl When both enzymes are

compatible with the same buffer enzyme #2 1 – 1.5 µl

pure water up to 20 µl

12

The table below presents the restriction sites of each enzyme that we applied. the choice of

enzymes was of course determined by the restriction sites on our plasmid.

Table 5| Restriction enzymes

enzyme restriction site

AgeI (BshTI) 5’...A’CCGGT...3’

3’...TGGCC’A...5’

BamHI 5’...G’GATCC...3’

3’...CCTAG’G...5’

BglII 5’...A’GATCT...3’

3’...TCTAG’A...5’

HindIII 5’...A’AGCTT...3’

3’...TTCGA’A...5’

NdeI 5’...CA’TATG...3’

3’...GTAT’AC...5’

SalI 5’...G’TCGAC...3’

3’...CAGCT’G...5’

XhoI 5’...C’TCGAG...3’

3’...GAGCT’C...5’

13

Sticky end ligation

Since we just apply restriction enzymes that cut the DNA double strand with a 5’ prime

overhang, we can perform sticky end ligation directly to the cut DNA fragments.

The T4 ligase is an enzyme that can join DNA strands. With this reaction, we insert our gene

of interest to the plasmid pET30b_FliC. Besides the avoidance of auto-ligation, the usage of

two different restriction enzymes for the restriction digestion will guarantee the correct

orientation of our GOI in the plasmid.

Figure 2| sticky end ligation scheme

The reaction shall be performed with an excess of insert, to promote the insertion. Therefore,

the equation below is used, it calculates the amount of insert and backbone on a mole bases.

The input of plasmid backbone is always 50 ng.

Equation 1|ligation ratio for insert

To calculate the needed volume of insert one needs to know the concentration of the insert

(after restriction), the excess factor (x) and the length of both DNA fragments (bp).

Note: A ligation control was always prepared. The ligation control monitors the auto-ligation

of the plasmid; thus, no insert is added to this ligation mix.

14

We prepared a ligation mix freshly for every ligation reaction after the following scheme on

ice:

Table 6| Ligation mix pipetting scheme

amount comments

Insert x µl as calculated

Backbone

(pET30b_FliC_MCS)

50 ng volume must be calculated

T4 Ligase buffer (10x)

2 µl

T4 Ligase 0.5 µl

pure water To 20 µl

15

Transformation of E. coli

Given the fact that we had two different types of competent cells the transformation was

performed according to each protocol. A transformation was conducted with 50 µl of

competent cells.

● CaCl2 competent cells (chemically competent)

A 50 µl aliquot is thawed on ice and subsequently maximal 5 µl of the ligation mix is added.

The cells are then chilled for 20 minutes on ice followed by a heat shock of 42 °C for exactly

45 seconds. Afterwards the cells are chilled on ice for three minutes and 750 µl LB medium

(w/o Kanamycin) is added. The cells recover for approx. 30 min at 37 °C and 450 rpm. Finally,

the culture is centrifuged for 1 min at room temperature and 7000 rpm speed, the supernatant

is briefly discarded and the residue volume is used to plate all cells to LB agar plates (with

Kanamycin). These plates are incubated overnight at 37 °C.

Note: In case a mini-prepped plasmid was transformed, only 80 µl of the recovered cells are

plated without any centrifugation.

● electrocompetent cells

A 50 µl aliquot is thawed on ice and pipetted into an electroporation cuvette. Afterwards,

maximal 2 µl of the ligation mix is added. The cuvettes are then transferred to the

electroporator and 1.8 kV are applied to the cell solution. 950 μL LB medium is added to the

transformed cells and the suspension is transferred to a sterile 1.5 mL microfuge tube. For cell

recovery, the culture is incubated at 37 °C and 4500 rpm for approx. 1 h. Finally, the

cells are plated on LB medium agar plates (with Kanamycin) and incubated overnight at 37.

Note: In case a mini-prepped plasmid was transformed, only 80 µl of the recovered cells are

plated without any centrifugation.

The plates were monitored the next day and the cfu (colony forming units) were determined.

Additionally, some clones were picked to perform colony PCR and to produce a sorted

backup plate of these clones.

16

Colony-PCR

The success of transformation and ligation was evaluated by colony-PCR. This method is

designed for high-throughput analysis of multiple clones.

The cells are picked from the transformation plate with a sterile pipette tip or a sterile

toothpick and each transferred to 50 µl TE-buffer or pure water. This solution is then boiled

for 10 minutes at 99 °C to lyse the cells and access the DNA (in a heat block).

Meanwhile the PCR reaction mix can be prepared according to the pipetting scheme without

DNA - this is called mastermix. The applied primers are chosen to specifically bind the

plasmid outside the cloning site. Therefore, the size of the amplific

ate will increase if the GOI is inserted.

The variables of the PCR program are then adapted to the chosen primer and the maximal

amplification length (with GOI inserted).

Table 7| PCR mix pipetting scheme for Colony-PCR

amount comments

DNA template 1 µl from boiled cell solution

Taq polymerase buffer

(10x)

2 µl for Taq Polymerase

Taq Polymerase 0.6 µl

according to Taq Polymerase

protocol

primer #1 (10 µM) 0.5 µl

primer #2 (10 µM) 0.5 µl

dNTPs (10 mM) 0.5 µl

nuclease-free water up to 20 µl

The reaction mix can be upscaled to the number of clones that should be analyzed and

prepared without the DNA template (master mix). This reaction mix can be distributed to PCR

reaction tubes and subsequently 1 µl of the boiled cell solution is added.

17

Table 8| PCR program for Colony-PCR

Step Temperature

[°C]

Time [min] Cycle nb.

1 Initial denaturation 95 3:00 1

2 Denaturation 95 0:30 30

3 Annealing 0:30 30

4 Extension 72 30

5 End elongation 72 10:00 1

6 Hold 4 infinity infinity

The annealing temperature depends on the melting temperature of the primers and the

reaction mixture components. It has to be calculated for every PCR which can be done online.

The timespan for the extension depends on the size of the DNA fragment. The synthesis

velocity of Taq Polymerase is 1 kb/min.

18

Miniprep

To isolate plasmid DNA from E. coli cells, when they were approved by colony-PCR or needed

to be transformed to another strain, was performed with the Thermo Fisher Miniprep Kit. All

steps are conducted as described in the manufacturers manual.

The amount of DNA eluted was evaluated always by UV - spectroscopy.

19

Sequencing

To identify or verify correct sequences of plasmid DNA we used DNA sequencing. First,

DNA concentration is determined by UV spectrophotometry. 12 μL of approx. 70 ng

of plasmid DNA are mixed with 3 μL of sequencing primer at a final primer

concentration of 2 μM. The primers are chosen as such that read lengths of 1 kb is not

exceeded. Plasmid DNA with higher concentration is diluted with

nuclease free water before to prepare approx. 70 ng sequencing sample. The

premixed samples were sent to Seqlab for sequencing.

20

Part II

Protein expression and purification

21

Expression systems background

T7-polymerase dependent expression

For protein expression the fusion-protein constructs are transformed to a different E. coli

strain: E. coli BL21gold. Since the recombinant protein should be produced in a high

amount, this protein expression strain is equipped with the T7-polymerase controlled by the

Lac-Operon.

This DNA dependent RNA-polymerase originally comes from the T7 bacteriophage. A phage

specifically attacks bacteria and replicates itself within the bacterial cells. This works as such

that the phage brings its own gene transcription system to specifically transcribe only its

genes and none from the the host cell, which will then be translated to proteins. This system

is a useful tool of genetic engineering as is guarantees the cells to produce predominantly

the desired protein when the T7-promoter-terminator systems is als present. Since we do not

want protein expression before we checked the plasmid, the cloning strain (E. coli DH10B)

was not equipped with the T7-polymerase. When our plasmid with the T7-promoter and T7-

terminator is transformed to our expression strain, expression would occur immediately.

However, recombinant protein expression is a huge burden for the cells and can induce

rapid cell death. To generate biomass before protein expression the production of proteins is

inducible. This is realized with the Lac-Operon. The Lac-Operon can inhibit and activate

gene expression dependent on Lactose or Lactose-analogues such as IPTG (Isopropyl-β-D-

thiogalactopyranosid).

When IPTG is added to a culture of E. coli BL21gold the T7-polymerase gene can be

transcribed and subsequently transcribe multiple copies of our gene of interest (Fig. 3).

22

Figure 3| T7-expression systems with IPTG induction, Patent Host-vector system for cloning and expressing genes. US

8623652 B2, 2014.

23

Protein Expression with selective pressure incorporation (SPI)

For protein expression in combination with selective pressure incorporation the cloned

plasmids are transformed to a different E. coli. strain: E. coli. B834. Since the recombinant

protein should be produced in a high amount, this protein expression strain is equipped with

the T7-polymerase controlled by the Lac-Operon. This system is similar to the expression of

fusion protein and is explained in detail in the fusion protein expression section.

Selective pressure incorporation is a method that enables scientists to incorporate non-

canonical amino acids (ncAA) in the amino acid sequence of protein. This method is used

predominantly in the field of structural biology but also in Xenobiology and facilitates

research regarding protein structure, stability and function (Budisa et al., 1998). However, it

also enables to incorporate amino acids which are capable of forming a covalent bond

besides the peptide bond enabling biomolecular click-chemistry (Kolb et al., 2001; Rösner et

al., 2015). SPI can take place because there is a naturally “relaxed” substrate specificity of

the Aminoacyl tRNA synthetase (aaRS) to the corresponding amino acid. That means the

affinity of the aaRS to the amino acid is under kinetic control and there is the possibility to

incorporate ncAAs.

Figure 4| Schematic description of the reprogramming of the protein translational machinery; Budisa.2005.

Engineering the Genetic Code. Wiley Weinheim, 1st Edition

Since the affinities of the canonical amino acids (cAA) are way higher than of any ncAA, SPI

doesn’t occur besides the E. coli strain is a knockout strain for the particular AA one wants to

replace (Rubini, 2004). Those strains were modified to have lost the capability of producing

24

one or more amino acids themselves, thus enabling them to take up an artificial analogue and

load it to the respective t-RNA (Rubini, 2004; Budisa et al., 1998). Since, it is desired to

maintain the protein’s functionality it is very promising to use methionine-auxotrophic strains

as methionine does not mediate any functional properties and is a relatively uncommon AA in

comparison to the other canonical AA (Rubini, 2004; Budisa et al., 1997). Thus, abundance

plays a crucial role too.

E. coli B834 also is a methionine-auxotrophic strain and incorporates azidohomoalanine (Aha)

or homopropargylglycine (Hpg) which have structural similarities to methionine.

The different affinity and therefore kinetic rates for the incorporation of ncAAs are depictured

in figure 5.

Figure 5| Lecture: Biotransformation und Synthetische Biologie, SoSe 2016, Technical University of Berlin, 2016.

25

For selective pressure incorporation this methionine-auxotrophic strain is cultured in

absence of methionine but with either Aha or Hpg (Fig. 6).

Both artificial amino acids have a reactive side chain, Aha - azide, Hpg - alkyne, to form a

covalent azide-alkyne bond via click chemistry. That will be used for coupling the root

flagellins to our cellulose backbone.

Figure 6| Incorporation of Aha while protein synthesis, Rösner et al. 2015.Click chemistry for targeted protein ubiquitylation and

ubiquitin chain formation. Nature Protocols 10, 1594–1611 (2015)

26

Induced Expression with E. coli

The basis for each expression is an overnight culture in adequate LB-medium (selective

pressure towards the plasmid by e.g. Kanamycin, see microbial cultivation section). The next

day a novel culture flask with fresh appropriate media is inoculated with 1 ml of the overnight

culture. This culture is monitored by OD600 determination at intervals until an OD600 of 0.5 –

0.7. The culture is then ready for production since the cells are in the exponential growth phase

and we have a sufficient number of cells. Production of recombinant proteins is an additional

burden to the host cells; thus, the culture is induced for production after some time of unlimited

growth. Before induction a SDS-Page sample was taken (see SDS-Page section).

Induction is performed with 0.8 mM IPTG (Isopropyl-β-D-thiogalactopyranosid, 1 M stock).

Furthermore, we occasionally tested expression with the addition of 0.2% lactose to the

culture.

The induced culture is then cultivated for 16 h at 20 °C and 180 rpm. These conditions were

found to be desirable to avoid the formation of inclusion bodies in E. coli. E. coli is unable to

secret proteins into the medium and if a critical level of proteins accumulates in the cells they

form aggregates referred to as inclusion bodies. Recovery functional proteins from inclusion

bodies is laborious and often not very successful.

After 16 h the OD600 is measured again, another SDS-Page sample is taken, and the culture

is subsequently harvested by centrifugation (4 °C, 8000 x g, 10 min).

A SDS-Page sample was taken from the supernatant, which was then discarded. The cells

were resuspended 15 ml NA-buffer (see purification section) and taken to cell lysis. When not

processed immediately, the cells can also be stored at – 20 °C.

When selective pressure incorporation (SPI) is performed alongside with the protein

expression the conditions are different.

The basis still is a culture, of a methionine-auxotrophic strain (E. c. B834) respectively, grown

in LB (with Kanamycin) at 37 °C,200 rpm for approx. 8 h. The main culture is performed in

NMM media (methionine free, with Kanamycin, see recipe below). Only a very low amount of

methionine is yet added to first induce the protein synthesis machinery of the cells and this

culture is incubated overnight at 30 °C,200 rpm. The next day the culture is checked for an

OD600 to be around 0.5 – 0.7. The SDS-Page sample is taken, and the culture subsequently

induced with 500 µl/l IPTG. Additionally, the methionine analogue is now added to the medium:

Homopropargylglycine 50 mg/l or Azidohomoalanine 100 mg/l). Expression is conducted for

3-4 h at 28 °C. The following steps are similar to the main protocol above.

27

Table 9| Composition of NMM medium from stock to final concentration for each component for 1 – 10 l. Ampicillin

is just a representative for antibiotics and is replaced for Kanamycin in our experiments.

28

UV-spectroscopy

For the determination of the concentration of our proteins we used UV-spectroscopy since

proteins absorb specifically at 280 nm. For the measurement a 1:10 solution in the

respective protein buffer is made and this sample is measures against a blank which only

contains the similar buffer but no protein. Classically, the protein concentration is then

measured by the correlation of Lambert-Beers Law (Eq. 2). Since the molecular extinction

coefficient is essential in that context and can vary from buffer to buffer we regularly used

the software tool ProtParam (Gesteiger et al., 2005) which calculates another absorbance

value in silico A0.1%. This value can be used to calculate the protein concentration [mg/ml] by

simple division of A / A0.1%.

𝐴 = ℰ ∗ 𝑑 ∗ 𝑐

Equation 2| Lambert-Beers-Law, A = absorbance value, Ɛ = molar extinction coefficient [l/cm*mol or g](depends

on sample and wavelength) , d = thickness of cuvette [cm] (often 1 cm), c = concentration of analyte [mol/l or g/l]

29

Affinity chromatography

Since all our proteins were cloned with a His-Tag, they can be separated from the other

proteins of E. coli by affinity chromatography. Nickel has a high affinity for Histidine and is

therefore applied as immobilized ligand in the column (IMAC = immobilizes metal ion affinity

chromatography). We used Ni-NTA columns from GE Healthcare (HisTrap™) for either 1 ml

or 5 ml.

For the purification we used different buffers, which are listed in the table below (table 10).

The workflow is also outlined in table format (table 11). The parts of sample treatment are to

be repeated for each sample and the elution is supervised by a PDA-detector (photodiode

array detector) that measures the flow as it passes for its absorbance at 280 nm.

Table 10| Composition of buffers required for affinity chromatography and related steps.

Buffer Amount Component

NA buffer (pH 8)

50 mM NaH2PO4

300 mM NaCl

20 mM Imidazole

5% Glycerol (optional)

NB buffer (pH 8)

50 mM NaH2PO4

300 mM NaCl

40 mM Imidazole

5% Glycerol (optional)

NC buffer (pH 8)

50 mM NaH2PO4

300 mM NaCl

300 mM Imidazole

5% Glycerol (optional)

30

Lysis buffer (pH 8) 50 mM NaH2PO4

500 mM NaCl

25 mM Imidazole

0.05% Tween 20

Wash buffer (pH 8)

50 mM NaH2PO4

500 mM NaCl

500 mM Imidazole

0.05% Tween 20

Wash buffer (pH 8)

50 mM NaH2PO4

500 mM NaCl

10 mM Imidazole

0.05% Tween 20

TEV reaction buffer (pH 8)

50 mM NaH2PO4

0.5 mM EDTA

1 mM DTT

Storage buffer (pH 8)

50 mM NaH2PO4

100 mM NaCl

5% Glycerol

31

Table 11| Workflow at LC-system (liquid chromatography) for His-tagged proteins.

Step In column Column

volumes

Out of column

Pretreatment Wash dH2O 5 Waste

Wash NC buffer 5 waste

Sample

treatment

Conditioning NA buffer 5 Waste

Load sample All cell lysate Collect flow

Wash 1 NB buffer 3 Collect wash 1

Wash 2 NC buffer Until elution

starts

Collect wash 2

Elution NC buffer Until elution

ends

Eluate

Cleaning NC buffer 2-3 Waste

Column

recovery

Wash dH2O 5 Waste

Storage 5 cv 20 5-10 waste

Prior to every purification we lysed the cells by adding lysis buffer and using a homogenizer

for > 10 pump steps with 60 Psi.

v

32

Dialysis

To change the buffer of our purified enzymes we used dialysis. This method in performed with

semipermeable membranes that allow molecules of a certain size to pass and therefore

change the buffer conditions of the proteins inside the membrane. The driving force for this

exchange is diffusion to equilibrate the concentrations of buffer components.

We apply dialysis membranes form Roth with a molecular cut off of 12- 14 kDa (Membra-

Cel™, Roth). The protein in buffer is filled into the closed membrane bag, which is placed into

a much higher volume (1 – 500fold) of the buffer that the protein should be in afterwards.

Dialysis is usually performed overnight or just a couple of hours, that depends on the initial

volume of the sample.

Regularly, we exchanged Nc-buffer and TEV buffer (4 °C, overnight) as well as TEV buffer and

NA buffer (4 °C, overnight) or NA buffer and storage buffer (4 °C, overnight) as.

33

TEV digest

After the initial purification of the His-tagged enzymes the His-tag needs to be cleaved off,

since, it hinders protein folding and functionality. Therefore, we use the TEV protease

(Tobacco Etch Virus protease) an endopeptidase that specifically cleaves ENLYFQ\S (amino

acid sequence) and releases the proteins from their His-tag.

The buffer needs to be exchanged from Nc-buffer to TEV buffer by dialysis (see dialysis

section). When the proteins are in the appropriate buffer, the concentration is measured by

VIS-spectroscopy (see VIS-spectroscopy section).

The protease is then added to the protein solution according to the following equation (Eq. 2)

and the sample is incubated at room temperature overnight.

𝑉𝑇𝐸𝑉 = 1

𝑐𝑇𝐸𝑉∗

𝑚𝑝𝑟𝑜𝑡𝑒𝑖𝑛

100

Equation 3: Formula to take a volume of OD600 = 1, dependent on the measured OD600 value. This makes the SDS-

Page analysis more comparative.

After TEV digestion, the desired enzymes are separated from the cut-off His-tags by IMAC

affinity chromatography. The protocol is basically the same, but now the proteins are within

the flow through when the column is loaded (see modified table below). That can be seen

also with the PDA-detector (280 nm).

34

Table 12| Workflow at LC-system (liquid chromatography) for protein purification from His-tag.

Step In column Column

volumes

Out of column

Pretreatment Wash dH2O 5 Waste

Wash NC buffer 5 waste

Sample

treatment

Conditioning NA buffer 5 Waste

Load sample All cell lysate Collect flow

(protein of

interest)

Wash NA buffer Until no signal

by detector

Collect flow

(protein of

interest)

Cleaning/Elution NC buffer 2-3 Waste

Column

recovery

Wash dH2O 5 Waste

Storage 5 cv 20 5-10 waste

35

SDS-Page

SDS-Page stands for sodiumdodecylsulfate polyacrylamid gel electrophoresis and represents

the counterpart for proteins of agarose gel electrophoresis. It is a simple and fast method to

pretest our expression for the desired protein. The identification is, similar to agarose gel

electrophoresis, by size only since the proteins are linearized by SDS.

Furthermore, we used this method to monitor the efficiency and success of our protein

expression strength, recovery and purification steps. Therefore, we took the following samples

via the process of each protein production:

• sample of culture before induction (S1, volume calculated)

• sample of culture at the end of induced expression (S2, volume calculated)

• sample of supernatant after cell harvesting (S3 - optional)

• sample of supernatant after cell lysis (S4, 50 µl)

• sample of cells debris after cell lysis (S5, 50 µl)

• sample of the column flow through (S6, 50 µl)

• sample of the washing steps (S7, 50 µl)

• sample of the eluate (S8, 15 µl)

Those samples taken directly from culture broth were calculated to equal an OD600 of 1 by the

following formula:

𝑉𝑠𝑎𝑚𝑝𝑙𝑒𝑠 =1000

𝑐𝑢𝑙𝑡𝑢𝑟𝑒′𝑠 𝑂𝐷600

Equation 4| Formula to take a volume of OD600 = 1, dependent on the measured OD600 value. This makes the SDS-

Page analysis more comparative.

The samples were all centrifuges at maximum speed of a desktop centrifuge for approx. 1 min

and the supernatant was discarded. Cells were resuspended in 60 µl pure water and 15 µl of

SDS-buffer. The buffer composition is given below. This solution was boiled than at 96 °C for

10 min and could then be loaded to the gel.

36

Table 13| Components of the 5x SDS-buffer

Amount Component

5x SDS buffer

(ready to boil and

subsequently load samples)

80 mM Tris pH 6.8

10% SDS

12.5% Glycerin

4% (v/v) 2-Mercaptoethanol

0.2% (w/v) Bromophenol blue

We prepared the SDS-gels, containing stacking and separation gel, ourselves according to

the following protocol:

Table 14| Recipe for preparation of SDS-Page gels and running buffer.

Gel type Amount Component

Stacking gel 125 mM stacking gel buffer

(1.5 M Tris-HCl pH 8.8)

5% Acrylamide

0.1% SDS

0.05% APS

0.17% TEMED

Separation gel 380 mM separation gel buffer

(0.25 M Tris-HCl pH 6.8)

12% Acrylamide

0.1% SDS

0.05% APS

0.05% TEMED

37

SDS-running buffer (1L) 30 g Tris-base

144 g Glycine

10 g SDS

Fill to 1000 ml

adjust pH 8.3

water

Before the samples were loaded to the gel, the gels were arranged in the gel chamber with

the running buffer and all bags were rinsed with a syringe. For the analysis of the gel a protein

ladder is added which must be chosen properly for the mass range the analyte is in. We usually

used the PageRuler™ (Thermo Scientific). Then, 10 µl of each sample is loaded and the gel

is run initially at 80 V for 30 minutes to compile all molecules to the running front. Afterwards,

the voltage in increased to 180 V for approx. 60 min.

The gel is subsequently stained with Coomassie staining, covered by the solution for 30 min

at room temperature. Thereafter, the gels are destained in water overnight while being slowly

shaked. The analysis is performed with a photo-dokumentation system.

38

Click reaction of membrane linker with root flagellin

For the functionalization of the membrane, the membrane first has is provided with a root

flagellin. Functionalized flagella fusion proteins can subsequently assemble onto the root to

form a flagella polymer. Both the membrane and the root flagellin have either an alkine or an

azide functional group from the non-canonic amino acids that were incorporated (Aha, Hpg).

For the click reaction, the following solutions were prepared:

Table 15| Recipe of click reaction buffer. Please note that the given concentrations refer to the amount of the

compound that should be added to the solution and does not mean a final concentration.

amount to be added

Potassium phosphate buffer (pH 7.6)

Potassium phosphate (K2HPO4, KH2PO4) 100 mM

NaCl 100 mM

Further components

amino guanidine hydrochloride 100 mM

THPTA 50 mM

CuSO4 20 mM

1:2 CuSO4:THPTA

sodium ascorbate 100 mM

The chemicals were diluted in potassium phosphate buffer. Sodium ascorbate is unstable in

aqueous solutions thus, it can only be diluted right before the reaction starts.

In a 50 ml round bottom flask, 10 ml of potassium phosphate buffer is mixed with a piece of

cellulose acetate with the respective linker, 1 mg FliC_NtermAHA/ HPG (the counterpart to

the linker on the membrane), aminoguanidine hydrochloride, sodium ascorbate, 1:2

CuSO4:THPTA. The air in the flask was replaced by N2 and the flask sealed with a septum. A

balloon was also filled with N2 and connected to the flask by a cannula through the septum to

maintain the N2 saturation in the flask.

Under a fume hood, the reaction mixture is stirred over night at room temperature.

When finished, the membrane was washed tree times in potassium phosphate buffer.

39

Flagella assembly

The flagellin monomers can be assembled either on root flagellin linked by click chemistry to

the cellulose membrane, or in solution. We performed the assembly on the membrane in a

24-Well plate. The flagellin (fusion) proteins have a start concentration of 4 mg/ml and are

diluted in assembly buffer. Finally, the solution has to cover the whole membrane (~ 450 µl

in a 24-Well plate).

To start the flagella assembly, 0.4M of (NH4)2SO4 is added and the sample is incubated at

28°C for 4 hours.

Table 16| Recipe of assembly buffer for flagella assembly

concentration

assembly buffer (pH 6.5)

NaH2PO4 40 mM

NaCl 30 mM