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Manufacturing of Vibrio natriegens cell lysates for cell-free protein synthesis

Marco André Pereira Gomes MSc. in Biological Engineering

October 2020

Abstract CFPS is becoming increasingly more popular due to the advantages of protein production

when compared to the in vivo method. The process is considerably faster because it does not require the maintenance of cell cultures or an intricate downstream purification. This work aims to understand the best way to cultivate Vibrio natriegens cells and how different growth media (LB, LB-V2 and BHI) can affect the quality of lysates prepared thereof for the purpose of CFPS. The growth rates (0.98 h-1, 0.42 h-1, and 1.11 h-1) and doubling times (42.5 min, 100.3 min, and 37.4 min) for each situation were determined to see how the cell reacts to different growth conditions. Following cell recovery, lysates were obtained by sonication. An analysis of the lysates obtained was performed to evaluate their quality in what concerns to the protein concentration and the relative quantity of ribosomes. For that, a Pierce™ BCA Protein Assay was performed to determine the protein concentration, a monolith chromatography and an extraction of the total RNA were done to obtain information regarding the presence of ribosomes. The concentration of total proteins obtained in the lysates varied in the range 5.18-10.25 mg/mL and a high concentration of RNA was achieved (169.2 mg/mL), which can indicate a high presence of ribosomes.

Keywords: Cell-free system, Vibrio natriegens; lysates of Vibrio natriegens, lysate quality, in-vitro protein synthesis.

1. Introduction Cell-free protein synthesis (CFPS)

technology was first described and used by Nirenberg and Matthaei while studying the genetic code. A few years later, the technology was used to investigate the regulatory mechanisms of the tryptophan and lactose operons of E. coli. Subsequent developments were made that resulted in various CFPS platforms that make it possible nowadays to express proteins in a fast and relatively inexpensive way (Carlson et al., 2012).

CFPS platforms bring several advantages to the field of production of proteins. For example, the open reaction environment characteristic of CFPS makes it possible to add or exclude the required substrates and to expedite the optimization of the ambient conditions necessary for protein production. Generally, CFPS technology is compatible with short timelines and increased flexibility in regards to the removal or addition of compounds when compared to in vivo methods (Perez et al., 2016).

When compared to in vivo methods (figure 1.1), CFPS is considerably faster since it doesn’t require the maintenance of cell cultures or an intricate downstream purification.

CFPS systems can also produce several proteins that are difficult to synthesize in cells.

Additionally, the system can include non-natural or chemically modified amino acids into the defined positions throughout the translation (He, 2008).

1.1. CFPS Platforms CFPS platforms can be separated into

two categories, which are the bottom-up and top-down approaches to protein synthesis. The

Figure 1.1: Comparison of in vivo protein expression with cell-free protein synthesis. CFPS systems provide a faster way to product development (taken from Carlson et al., 2012).

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PURE (purified recombinant elements) systems build the protein synthesis from the bottom up (Shimizu et al., 2014). In regard to the top-down approach, these are attributed to the cell extracts. The cell extracts are obtained by clarifying the whole cell lysates through centrifugation in order to remove all the unwanted material (Pratt, 1984). The final crude extracts have all the biological components required for translation, protein folding, and energy regeneration (Perez et al., 2016).

1.1.1. Microbial crude extract systems

E. coli Crude extracts from E. coli (ECE) are

one of the most studied systems for CFPS use. This platform is commercially available and is already used in large scale applications. The main reasons for the adoption of the ECE platform are its high yields (up to 2.3 g/L of GFP (Caschera & Noireaux, 2014)), and optimized extract preparation process (Swartz, 2006). This platform can scale linearly, which is ideal for protein synthesis at the industrial level.

Vibrio natriegens CFPS platforms based upon Vibrio

natriegens have been developed over the last years. This platform is potentially an ideal candidate for CFPS since V. natriegens has one of the shortest doubling times of all known organisms, displays a high rate of protein synthesis, and is metabolically highly efficient (Wiegand et al., 2018). The highest translation efficiency in the V. natriegens CFPS platform is usually obtained when extracts are prepared from cells collected in a tight window during the mid-exponential phase (Gregorio et al., 2019). Another advantage of this platform in terms of extract preparation is its high resistance to damage due to over-lysis (Gregorio et al., 2019). This fact makes it possible for inexperienced CFPS users to easily obtain a good quality extract (Des Soye et al., 2018).

1.2. Methodological Differences between Platforms The selection of a certain CFPS

platform will be, at first, determined by the applications associated with a given platform, keeping in mind that several platforms can be used for the same application. The choice of platform can take into account the accessibility and complexity of

the methods used to produce the cell extracts, as well as the productivity of the platform. However, most of the platforms follow a similar workflow (figure 1.2)

Productivity Matching the application with platform

productivity is essential for success because different platforms can be better suited for the manufacture of different proteins and not all of the applications need an optimization of the protein yield (Gregorio et al., 2019). The industrial level of protein production is associated with E. coli or wheat germ platforms because of simplified workflow and high yields. However, for small-scale assays, most of the platforms are suitable, even with low yields (Gregorio et al., 2019).

Growth The growth media is one of the most

important aspects to be considered in order to obtain a rich lysate for CFPS. It is highly variable between platforms and different media can reach different results. The vessels where growth is performed are also an important variable, with a great variety being used, from fermenters to baffled flasks (Gregorio et al., 2019). After the growth, the cells are recovered typically by centrifugation.

Extract Preparation Extract preparation involves several

steps, which are pre-lysis preparation, lysis, and post-lysis processing. Several lysis methods can be applied to one platform, but the lysis method can vary from platform to platform. Centrifugation is performed, after lysis, to remove cell membrane fragments and other debris. The extracts are then frozen at -80 ºC if CFPS is not performed immediately (Gregorio et al., 2019).

1.3. Future Outlook

The renewed scientific interest in CFPS has resulted in drastically increased batch yields, active reaction duration, and reaction volumes

Figure 1.2: General workflow for preparation of cell-free extract and set up of CFPS reactions, representing the essential steps from the cell growth to the CFSP reaction (taken from Gregorio et al., 2019).

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(Carlson et al., 2012). CFPS will be vital in the near future as protein production factory. CFPS technology will also have an increasing role in supplementing in vivo synthetic biology efforts. Because of its flexibility and high-throughput potential, cell-free systems are perfectly suited to synthesize large libraries, not only for genetic networks but, also, for evolutionary or proteomics studies (Takemori et al., 2015). In conclusion, CFPS has a lot of potential as a new way to rapidly and easily synthesize proteins and the use of this technology in the synthetic biology field is expected to grow in the future (Perez et al., 2016).

2. Materials and Methods 2.1. Media

Three different types of media were used in this work: LB-V2, LB and brain-heart infusion (BHI). Table 2.1 summarizes the composition of each medium. Each medium was sterilized before use, in order to avoid any contaminations. Table 2. 1: Summarized composition for each medium used to grow V. natriegens cells. Quantities indicated are for the preparation of 1 L of medium (Failmezger et al., 2018; Wiegand et al., 2019)

LB-V2

Component Quantity (g) Final

Concentration (mM)

Final Volume

(L) LB Broth 25 -

1 NaCl 11.69 200 MgCl2 2.20 23.1 KCl 0.31 4.2 LB

Component Quantity (g) Final

Concentration (mM)

Final Volume

(L) LB Broth 25 - 1

BHI

Component Quantity (g) Final

Concentration (mM)

Final Volume

(L) Brain Heart

Broth 37 - 1

2.2. Cell bank

A cell bank was prepared from Vibrio natriegens (ATCC® 14048™). The original pellet was re-hydrated with 1 mL of LB-V2 medium and then 0.8 mL were transferred to 5 mL of LB-V2 in a falcon tube. Overnight incubation was performed at a temperature of 30oC and under orbital shaking (225 rpm). Negative control was performed to assure that no contaminations were present in the medium.

After the overnight incubation, an adequate amount of the cells was transferred to a 100 mL Erlenmeyer containing 25 mL of

LB-V2. The volume of the overnight culture transferred should be enough to ensure an optical density of around 0.1. If the volume needed is superior to 20% of the total volume, the inoculum culture should be centrifuged (3 min, 4 oC, 6000 g) in order to remove the excess medium and then resuspend the pellet in 1 mL of LB-V2 from the Erlenmeyer. Following inoculation, the culture is incubated at 30oC and 225 rpm and optical density (OD600nm) is monitored until a value of approximately 1 is reached. At this point, cell growth is halted.

Two types of cell banks were constructed, one master bank and a working bank. For the master bank, 0.65 mL of the cell culture are added to 0.35 mL of glycerol 50% (v/v) in an Eppendorf tube (10 Eppendorf tubes in total). For the working bank, 65 µL of the cell culture were added to 35 µL of glycerol 50% (v/v) in an Eppendorf tube (15 Eppendorf tubes in total).

2.3. Construction of the growth curve

Pre-inoculum were prepared by adding 10 µL of cell suspension from the working bank to a falcon with 5 mL of the medium. An overnight incubation, in an orbital shaker, was started at 30 ºC and 225 rpm. After that, an adequate amount of the cell culture was added to an Erlenmeyer containing a volume of 500 mL of medium. The volume of inoculum transferred should be enough to ensure a starting optical density of around 0.1. If the volume needed is superior to 20% of the total volume, the inoculum culture should be centrifuged (3 min, 4 oC, 6000 g) in order to remove the excess medium and the resulting pellet resuspended in 1mL of the medium from the Erlenmeyer. Following inoculation, the culture was incubated at 30 oC under orbital shaking (225 rpm). The OD600nm was monitored in increments of time of 15 or 30 minutes in order to obtain the growth curve. This step was performed for every medium used in this work.

2.4. Lysate production 2.4.1. Cell Growth

The pre-inoculum was made by adding 10 µL of cell suspension from the working bank to a 15 mL Falcon containing the desired medium (LB, LB-V2, and BHI). The incubation occurred overnight, in an orbital shaker, at a temperature of 30ºC and 225 rpm. Negative control was used in order to ascertain that the medium was free from contaminations.

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After the overnight growth, an adequate volume of the pre-inoculum was added to an Erlenmeyer containing 125 mL of medium. The volume of inoculum added was calculated in order to obtain an OD600nm of approximately 0.1. If the volume needed is superior to 20% of the total volume, the inoculum culture should be centrifuged (3 min, 4 oC, 6000 g) in order to remove the excess medium and the resulting pellet resuspended in 1 mL of the medium from the Erlenmeyer. Following inoculation, the culture was incubated in an orbital shaker at 30 oC and 225 rpm and optical density (OD600nm) was monitored. The cell growth was stopped when the OD600nm reached a value around 1. After the cell growth was complete, centrifugation was performed (20 min, 4 ºC, 3500 g) in order to separate the cells from the medium. The supernatant was discarded, and the pellet was stored at -80ºC.

2.4.2. Preparation for Lysis The S30A lysis buffer was prepared

following the composition presented below, in table 2.2 (Wiegand et al., 2018). Once prepared, this buffer should be conserved and used at 4 ºC. Table 2. 2: Composition of the S30A lysis buffer. Mg-acetate and K-acetate were used in this case, whereas in the literature Mg-glutamate and K-glutamate are typically used (Wiegand et al., 2018).

The pellet of V. natriegens cells was

defrosted in ice, resuspended in 10 mL of the lysis buffer (4 ºC) and transferred to one or more Falcons. Centrifugation was performed (10 min, 4 ºC, 3500 g) in order to separate cells from the supernatant. The supernatant was then discarded. This procedure should be done two times to ensure that cells are thoroughly washed with the lysis buffer. Following the last centrifugation step, 500µL of lysis buffer was used to resuspend the cell pellet and then transferred to 2 mL Eppendorf tubes (1.5 mL is the maximum volume that each Eppendorf tube should contain). If not used immediately, the cells should be conserved at -80 ºC.

2.4.3. Lysis of the cells The lysis of the cells was performed by

sonication. For that, the 2 mL Eppendorf tubes containing the cell suspension were vortexed and put on ice inside a beaker with the caps open. The sonication probe was placed on the surface of the liquid. The parameters of the sonicator (Bandelin Sonoplus HD3200) used were: probe with 3.175 mm of tip diameter; frequency of 20 kHz; 50 % amplitude; pulse ON time of 10s; pulse OFF time of 60s. Three cycles were performed if the volume was lower than 500 µL, and 6 cycles were performed if the volume was higher than 500 µL. After the sonication step was concluded, centrifugation (30 - 45 min, 4 ºC, 16000 g) was performed in order to remove cell debris. Without disturbing the pellet, the supernatant was removed and transferred to Eppendorf tubes. The lysate should be kept at -80 ºC until use.

2.5. Analysis of the lysate content 2.5.1. Protein Concentration

The lysates’ protein concentration was determined using a Pierce™ BCA Protein Assay Kit. The calibration curve was prepared using S30A lysis buffer as a diluent for the lysates. Bovine serum albine (BSA) was used as the protein standard. After preparation, samples were measured at 562 nm in a plate reader.

2.5.2. SDS-PAGE Sodium dodecyl sulfate-polyacrylamide

gel electrophoresis (SDS-PAGE) was performed to analyze and verify the presence and diversity of proteins in the lysates. Some of the samples were previously precipitated using acetone (5 µL of lysate sample with 10, 20 or 30 µL of acetone). All samples were denatured in reducing conditions with 100 mM (DTT) at 100 ºC for 5-10 minutes. The resolving gel was composed by 11.68 % acrylamide/bis-acrylamide 40 % solution, 1x resolving buffer (0.375 M Tris-HCl (pH 8.8), 0.1 % SDS), 0.05 % TEMED and APS (Ammonium Persulfate). The stacking gel was composed by 3.89 % acrylamide/bisacrylamide 40 % solution, 1x stacking buffer (0.125 M Tris-HCl (pH 6.9), 0.1% SDS), 0.10% TEMED and 0.05% APS. For the electrophoresis run, each gel lane was loaded with 20 µL of the mixture containing the protein samples and 25 µL Laemmli sample buffer (Bio-Rad). The molecular weight marker (Precision Plus

S30A lysis Buffer

Component Quantity Final

Concentration (mM)

Final Volume

(L) Tris Solution (pH 8.0) -1

M 25 mL 50

0.5 Mg-acetate 1.50 g 14 K-acetate 2.94 g 60

DTT 1 mL 2

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Protein Standards 250-10 kDa, Bio-Rad) lane was loaded with 3 µL. The separation of the proteins occurred at 90 V until the dye front reached the end of the gel.

When the electrophoresis ends, the gel must be stained in order to observe the bands. For this, the gel is submersed in the staining solution (Blue comassie (R 250) 0,25 % (p/v), methanol 50 % (v/v) and acetic acid 10% (v/v)) for 15 minutes, under agitation. When the bands are revealed, the solution can be discarded, and the gel can be kept in water.

When the intensity of the bands was unsatisfying, the gels were silver stained. The silver staining of the gels started with the fixation step with a solution containing 30 % (v/v) ethanol and 10 % (v/v) acetic acid during 2 h followed by 3 washing steps. The first washing step was done with 30 % (v/v) ethanol for 10 min followed by two washing steps of 10 min each with deionized water. The sensibilization step was performed with 0.02 % (w/v) sodium thiosulfate for 1 min followed by 3 washing steps of 30 s each with deionized water. The next step was staining with 0.15 % (m/v) silver nitrate for 30 min followed by a washing step with deionized water for 1 min. The development step was done with a solution containing 3 % (m/v) sodium carbonate and 0.05 % (v/v) formaldehyde. This solution was applied until the appearance of brown bands in the gel. The stopping reaction occurred by the addition of 5 % (v/v) acetic acid for 15 min. At the end of the staining, the gel was scanned in a densitometer (GS800 Calibrated Densitometer, Bio-Rad).

2.5.3. Monolith chromatography Lysates were analyzed by monolith

anion-exchange chromatography in order to fractionate the mixture and hopefully quantify ribosomes. For this procedure, an Äkta Purifier 10 (GE Healthcare) system and a quaternary amine Convective Interaction Media (QA) monolithic disc were used. The lysis buffer S30A (A) and lysis buffer S30A + 1 M NaCl (B) were used as the mobile phases. The software used to define the chromatographic methods and record the chromatograms was Unicorn 5.11 (GE Healthcare). For all the assays, the absorbance at 280 nm and the conductivity of buffer solutions exiting the column were recorded. Various fractions were collected in order to proceed to further analysis.

2.5.4. RNA extraction

Total RNA was extracted from the most promising fractions obtained in the monolith chromatography using the NZY Total RNA Isolation kit (Nzytech). While this procedure can be performed at room temperature, the isolated RNA should be treated with care because of its sensitivity. To ensure RNA stability, samples should be stored at -20 ºC for the short-term or at -70 ºC for the long-term. The digestion mix was prepared before the starting of the RNA isolation protocol. For each isolation, the exact amount of digestion mixture required was prepared: 10 µL of DNase I (reconstituted) and 90 µL of digestion buffer. The lysate was applied to the NZYSpin Homogenization column placed in a 2 mL collection tube and centrifuged for 1 min at 11000 g. The flow-through was saved, transferred to a new 1.5 mL microcentrifuge tube, and mixed immediately with 350 µL of 70 % ethanol by pipetting up and down. The lysate was pipetted into an NZYSpin Binding column. Another centrifugation was performed (11000 g, 30 s), the flow-through was discarded and the column was placed in a new collection tube. 350 µL of Buffer NI were added and another centrifugation was performed with the same settings; the flow-through was again discarded. For each isolation, 95 µL of Digestion mix were added into the center of the silica membrane of the NZYSpin Binding column and incubated at room temperature for 15 minutes. After incubation, 200 µL of Buffer NWR1 were added and another centrifugation was performed at 11000 g for 1 minute. The flow-through was discarded and the column was placed in a new collection tube. 600 µL of Buffer NWR2 were added and centrifugation was initiated at 11000 g for 1 minute. The flow-through was discarded. A wash with 250 µL of Buffer NWR2 and consequential centrifuged (11000 g, 2 x 1 min) was performed in order to dry the column membrane. The flow-through was discarded.

Lastly, the NZYSpin Binding Column was placed in a clean 1.5 mL RNase-free microcentrifuge tube and 40-60 µL of RNase-free water were added directly to the column membrane. After that, centrifugation was performed at 11000 g for 1 minute in order to elute the RNA. The concentration of RNA obtained in the eluate was measured using a Nanodrop (Thermo Fisher Scientific).

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2.5.5. Transmission electron microscopy TEM was used to try to observe ribosomes from the lysates. Fractions 28-30 obtained after fractionation of a lysate obtained from cells grown in BHI medium by monolith chromatography were used as sample material. The procedure involved the adsorption of 5 µL of sample onto flow discharged cooper 200 mesh grids coated with carbon for 5 minutes. Samples were then washed five times with distilled water and stained with 2 % uranyl acetate for 5 minutes. All grids were dried using No. 1 Whatman filter paper and the images were obtained with a Hitachi 8100 200 kV transmission electron microscope, equipped with a CCD Megaview II bottom-mounted camera

3. Results and discussion 3.1 Growth curves, growth rates and

doubling times V. natriegens (ATCC® 14048™) cells were grown in three different media: LB, LB-V2 and BHI. For every media, the growth was performed under the same conditions (30 ºC and 225 rpm) and using the same equipment. Measures of the OD600nm were taken with increments of time of around 20-30 minutes in order to get a real representation of the growth of the cells. As seen in figure 3.1, it was possible to follow the growth of the cells throughout the three typical growth phases: lag phase, exponential phase, and stationary phase.

The growth rates obtained were equal to 0.98 h-1, 0.42 h-1, and 1.11 h-1 for the LB, LB-V2, and BHI media, respectively, which corresponds to doubling times of 42.5 min, 100.3min, and 37.4 min. The final OD obtained was 2.74, 1.49, and 2.62 for the LB, LB-V2, and BHI media, respectively. This data indicates that BHI is the medium more effective in what concerns to the growth rate of V. natriegens cells under the operational

conditions used. BHI is a very rich medium that can provide all the nutrients needed for the proliferation of the V. natriegens. There is no surprise regarding this result. However, it is surprising to verify that the LB-V2 medium had such a low value of growth rate and doubling time, even more when compared to the LB medium without supplementation. It is possible that the introduction of salts in the medium can, in some way, inhibit the growth of the cell culture. However, as important as the growth rate is, it is more important to understand if the salts introduced in the LB-V2 medium implicate something in the ability of the cells to produce proteins and other compounds while growing. The obtained results contradicts values found in the literature that state that V. natriegens has a doubling time of around 10 minutes (Wiegand et al., 2018). Even with the best medium used, the doubling times obtained here were 3.7-fold larger.

3.2. Cell lysate production Starting from 125 mL of the cell culture

broth obtained with the three media tested, around 1.2-1.4 mL of cell lysates were achieved. However, the method used to separate the lysate from the cell debris was not most adequate - a pipette was used to carefully remove the supernatant lysate, which inevitably resulted in a fraction of the lysate being lost alongside with the debris-containing pellets. This separation of the lysate from the debris must be done very carefully because any debris that is transferred accidentally will drastically reduce the extract’s capacity for high yielding protein synthesis. Having this in mind, it is preferable to lose some amount of lysate than contaminate it with debris.

The volume of lysate that was possible to obtain was very positive. Most of the literature describes the production of lysates from higher volumes of cell culture (around 250 mL in 1 L Erlenmeyer) and the production of the lysates are in the same line as our results, with higher production from higher volumes of cell culture (Wiegand et al., 2018).

3.3. Analyses of lysate content 3.3.1. Protein Concentration

The concentration of total protein in the lysates obtained from the three different productions of the V. natriegens lysate, with three different media, was measured using a Pierce™ BCA Protein Assay Kit. The following

0

0,5

1

1,5

2

2,5

3

0 0,5 1 1,25 1,5 1,75 2 2,25 3 3,25 3,5

OD6

00nm

Time (h)

LB

BHI

LB-V2

Figure 3. 1: Growth of Vibrio natriegens cells in LB/LB-V2/BHI medium. Cells were grown in a shake flask and incubation was performed in an orbital shaker at 225 rpm and at 30 oC.

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table (table 3.1) summarizes the concentration of total protein for each growth medium. Table 3.1: Concentration of total protein present in V. natriegens lysates obtained from cells grown with LB, LB-V2, and BHI media. Results were obtained using the Pierce™ BCA Protein Assay Kit.

The lysate obtained from cells grown with BHI, and taking into account that all three lysates start the cell culture with an OD600nm of around 0.1 (0.11 for the LB medium and 0.08 for the LB-V2 and BHI media), has a clear higher production of total protein when compared with cells grown in LB and LB-V2.

This could be because of some stress imposed by the medium in the cell culture that contributes to the need to produce a higher amount of proteins. In what concerns the LB and LB-V2 media, LB-V2 resulted in a higher concentration of total protein in the lysate, although the growth rate was significantly lower than the LB. The reason for that could be the addition of salts in the medium LB-V2 and, has a response, the cells tend to produce more protein in response to the presence of salts.

None of the lysates produced here had a concentration of total protein comparable to that found in the literature, which is around 20 mg/mL (Wiegand et al., 2018). However, the starting volume of culture used in the studies by Wiegand et al was, in general, around 250 mL, whereas here a volume of 125 mL was used. This can justify the difference because when doubling the starting volume, it is expected to have double the concentration of protein. Nevertheless, ineffective lysis of the cells can cause a decrease in the final protein concentration.

3.3.2. SDS-PAGE An SDS-PAGE analysis of the lysates was performed to characterize the diversity of proteins present in the lysates. Figure 3.2 shows the SDS-PAGE obtained when analyzing the lysate obtained from cells grown in LB-V2 medium.

It is possible to verify that there is a vast diversity of proteins and a high concentration of them as well. However, the SDS-PAGE alone does not give us information regarding the concentration of ribosomes, which are essential for the success of a cell-free system. It should be mentioned that unfortunately, and due to the closure of the labs, there wasn’t

enough time to do a successful SDS-PAGE comparing all the lysates from the three different media. That would have been a good way to identify some differences in the diversity of proteins in each lysate. However, major differences in protein composition between the three lysates are not expected.

3.3.3. Monolith chromatography Monolith chromatography is presented in the literature (Trauner et al., 2011) as a fast and efficient way to separate ribosomes from a crude extract of cells. In this work, the lysates obtained from cells grown in the three media under analysis were injected into a quaternary amine Convective Interaction Media (QA) monolithic column and eluted with a linear gradient of NaCl (Figures 3.3-3.5). Fractions of the eluate were collected to proceed to further analysis.

LB LB-V2 BHI 5.18 ± 0.75

(mg/ml) 6.02 ± 0.75

(mg/ml) 10.25 ± 1.92

(mg/ml)

Figure 3.2: SDS-PAGE analysis of the lysate obtained from cells grown in LB-V2 medium. Each column represents the following: 1 - original lysate without any treatment; 2 - lysate five times diluted; 3 - lysate 10 times diluted. The letter M represents the Precision Plus Protein Standards Duo Color (Bio Rad).

Figure 3.3: Anion-exchange chromatography analysis of a V. natriegens cell lysate obtained from cells grown in LB medium. The chromatogram was obtained following the injection of 1 mL of lysate in a quaternary amine Convective Interaction Media (QA) monolithic column. Lysis buffer S30A (A) and lysis buffer S30A + 1M NaCl (B) were used as the mobile phases. The blue line represents the measures obtained by 280 nm UV, that correlates with the presence of proteins. Fractions 2-4, 5-8, 10-12, 19-20, 21-26, 28-30 and 31 were collected for further analysis. The procedure was performed in an Äkta Purifier 10 equipped with the Unicorn 5.11 software (GE Healthcare).

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The chromatograms corresponding to lysates LB (Figure 3.3) and BHI (Figure 3.5) display a tall peak eluting at medium NaCl concentration. The absorbance signal of these peaks is clearly saturated. This suggests that a dilution should have been performed in order to have smaller peaks whose areas could then be used for quantitative purposes. Nevertheless, the peaks obtained clearly correspond to a high concentration of that specific protein or group of proteins. Other notable features of the chromatograms include a flowthrough peak and smaller peaks eluting right before and after the start of the gradient elution. The LB-V2 lysate produced a

chromatogram with considerably smaller peaks (Figure 3.4), most probably due to the loss of some of the cell extract when injecting the sample in the column. However, the results are in general qualitatively similar to the results displayed in the other chromatograms, in particular in what concerns the presence of a peak eluting at medium NaCl concentration. Interestingly, in this case it is clearly visible that the material in this peak has a higher absorbance at 254 nm, which corresponds to maximum of absorbance of nucleic acids, than at 280 nm, which corresponds to the maximum of absorbance of proteins. This strongly suggests that ribosomes could be present in this peak. Further analysis is necessary in order to understand if the chromatography was successful in the separation of the ribosomes. If the tall peaks effectively represent the ribosomes, it’s possible to conclude that the separation occurred and that there is a high concentration of ribosomes.

3.3.4. SDS-PAGE of the fractions from chromatography

An SDS-PAGE analysis of the fractions obtained after chromatography was performed (Figure 3.6). Only fractions from the separation of the BHI lysate were analyzed here. Ideally, this SDS-PAGE should have been done to every fraction collected after separating the three lysates, but, unfortunately, that was not possible in this case.

This SDS-PAGE gel can give an idea of the chromatographic separation obtained. When compared to the original lysate, all the peaks represent that some kind of separation occurred because they don’t present the same number of bands of the original lysate.

Figure 3.4: Anion-exchange chromatography analysis of a V. natriegens cell lysate obtained from cells grown in LB-V2 medium. The chromatogram was obtained following the injection of around 0,5 mL of lysate in a quaternary amine Convective Interaction Media (QA) monolithic column. Lysis buffer S30A (A) and lysis buffer S30A + 1M NaCl (B) were used as the mobile phases. The blue line represents the measures obtained by 280 nm UV that correlates with the presence of proteins. Fractions 2-4, 6-8, 13-15, 17-18, 23-24 and 25-26 were collected for further analysis. The procedure was performed in an Äkta Purifier 10 equipped with the Unicorn 5.11 software (GE Healthcare).

Figure 3.5: Anion-exchange chromatography analysis of a V. natriegens cell lysate obtained from cells grown in BHI medium. Chromatogram The chromatogram was obtained following rom the injection of 1 mL the of lysate in a from the quaternary amine Convective Interaction Media (QA) monolithic column LB medium. It was used lysis buffer S30A (A) and lysis buffer S30A + 1M NaCl (B) were used as the mobile phases. The blue line represents the measures obtained by 280 nm UV, that correlates with the presence of proteins. Fractions 2-4, 5-7, 10-12, 13-14, 15, 16-19 and 22-25 were collected for further analysis. The procedure was performed in an Äkta Purifier 10 equipped with the Unicorn 5.11 software (GE Healthcare).

Figure 3.6: SDS-PAGE and consequently silver staining of the fractions obtained after chromatographic separation of the lysate derived from cells cultured in BHI media. The numbers at the top of the figure represent the following: 1 – original lysate diluted 1:10; 2 – flowthrough 5-7; 3 – flowthrough 2-4; 4 – fractions 10-12; 5 – fractions 13-14; 6- fractions 15; 7- fractions 16-19; 8 – fractions 22-25. The letter M represents the Precision Plus Protein Standards Duo Color (Bio Rad).

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However, with this analysis it was not possible to confirm if ribosomes were successfully separated. In order to determine the concentration of ribosomes in the lysate, some added analytical steps are necessary. This further analysis should also give some information in regards of the efficiency of the chromatography.

3.3.5. Quantification of total RNA The quantification of the total RNA was

performed in the fraction corresponding to the largest peak obtained from the chromatographic separation of the BHI lysate (fractions 22-25). This quantification was preceded by the extraction of the total RNA. This extraction was performed as indicated in the Materials and Methods section. A concentration of total RNA of 169.2 g/L was obtained. Since the ribosomal RNA represents about 80 % of the total RNA present in a cell and assuming that the same proportion is maintained in the lysate, the result corresponds to a concentration of ribosomal RNA of 135.36 g/L. The result obtained is very promising in what concerns to the viability of the monolith chromatography as an effective tool to separate ribosomes. Additionally, this result shows a high content in ribosomes and that is a good indication that this lysate is suitable to support a cell-free protein synthesis system.

3.3.6. Transmission electron microscopy In order to have a visual notion of the

results obtained, material collected in fractions 22-25 obtained after fractionation of a lysate of cells grown in BHI medium by monolith chromatography was observed with a Hitachi 8100 200 kV transmission electron microscope, equipped with a CCD Megaview II bottom-mounted camera (Figure 3.7).

In figure 3.7 it is possible to see some dark structures with an approximately round shape that are, most likely, ribosomes. This

conclusion can be made taking into account that the average size of a procaryotic ribosome is around 21 nm (Kaczanowska & Rydén-Aulin, 2007), which corresponds to the approximate size of the dark structures.

4. Conclusions Cell-free protein systems are an effective

alternative to cell-based expression systems. One of the biggest advantages of the use of CFPS is the possibility to monitor the complex reaction network directly because it is easy to collect samples and enables on-line monitoring. This makes these systems attractive to several applications, like protein production, biotechnology, biotherapeutics, biomaterials, and synthetic biology (Whittaker, 2013). However, there are some obstacles to overcome, such as the ability to reliably synthesize any biologically active protein in a universal platform, the lack of a cost effective and scalable CFPS platform (Carlson et al., 2012).

The focus was placed on the very first step needed to implement a successful cell-free system, the production of the cell lysates. Various parameters should be taken into account when producing such cell lysates, and in this work, the emphasis was placed on the impact that the media used to grow the Vibrio natriegens cells has on the quality of the resulting lysates.

The first step of this analysis was to try to understand if the growth rate and doubling time of Vibrio natriegens are affected by changes in the composition of the media. The best results were obtained when using the BHI medium, which resulted in a growth rate of 1.11h-1 with the fastest doubling time (0.62 h).

The next step was to evaluate the quality of the lysates obtained by sonication. The quality was inferred from the concentration of proteins and, more precisely, from the number of ribosomes present in the lysate. BHI was, once again, the medium that provided the best results with the highest concentration of proteins from all the media tested. The high content in proteins is a promising sign of a suitable lysate for a cell-free system

Ribosomes, being the protein factories of cells, are essential for the success of CFPS. With that in mind, it was important to try to understand if ribosomes are present in the lysates and their concentration. To do that, an anion exchange monolith chromatography was performed to try to separate and estimate the

Figure 3.7: Image obtained from Hitachi 8100 200 kV transmission electron microscope, equipped with a CCD Megaview II bottom-mounted camera. Visual notion of the fraction 22-25 of the BHI medium, obtained from the monolith chromatography.

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quantity of ribosomes in the lysate. With that, it was possible to analyze the concentration of total RNA of one of the fractions obtained from the chromatography to the BHI lysate. This analysis resulted in a very promising result, demonstrating a high content in RNA and, consequently, a high content in ribosomal RNA. This indicates the presence of a considerable number of ribosomes.

Future work Unfortunately, this dissertation was

affected by the COVID-19 pandemic and it wasn’t possible to perform several tests that were scheduled to happen. For example, this works lacks a comparison of the results obtained with the BHI medium with the results from the other media used (LB and LB-V2). This lack of comparison was particularly harmful in what concerns the analyses of the fractions obtained from the monolith chromatography because it wasn’t possible to understand the ribosome content from the lysates from the LB and LB-V2 media. The efficiency of the disruption of the cells should also be tested in detail. An ineffective disruption can promote a lower concentration of proteins in the lysates. For that, lysate samples should be plated in cell media to confirm if all cells initially present were effectively disrupted. Additionally, it would be interesting to cultivate cells in a controlled bioreactor in order to understand the impact that scale-up could have on the quality of the lysates. Lastly, differences in the content of the lysates should be tested when using cells collected at different stages of cell growth, like the stationary phase and the death phase.

Acknowledgements I would like to thank Professor Miguel

Prazeres for the opportunity to work in this subject and for the support given, specially taking into account that this work ended earlier than expected due to the COVID-19 pandemic. I also would like to thank to all my colleges, of 7th and 8th floor, for all the help, advice, and support. Your help was really precious to me.

I would like to thank to my family and friends for all the support throughout this journey.

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