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Investigating the function of GroES

with hard-to-fold proteins

​in vivo

Authors

Oliver Hild Walett*1​, Emmanuel Berlin*2​, Johan Larsson*3, Sofie Arvidsson*2​, Filip Fors*4​, Marianne

Gavelius*5​, Filip Genander*6​, Johanna Granqvist*5, Philip Lifwergren*6​, Alexandra Sandéhn*7​, Martin

Viksten*, Irma Wenhov*​1​, Per Hammarström8​, Lars-Göran Mårtensson8​.

1​Undergraduate program of Chemical Biology, Linköping University 2​Undergraduate program of Medical Biology, Linköping University 3​Graduate program of Experimental and Medical Biosciences, Linköping University 4​Undergraduate program of Biology, Linköping University 5​Graduate program of Industrial Biotechnology, Linköping University 6​Graduate program of Devices and Materials in Biomedicine, Linköping University 7​Undergraduate program of Biomedical Sciences, Linköping University ​8​IFM, Department of Physics, Chemistry and Biology, Linköping University

*These authors contributed equally

Abstract

The use of molecular chaperones can increase the yield of correctly folded proteins. This is especially needed in the expression of proteins non-native to the host organism. This study set out to investigate the function of the chaperone GroES; a component in the GroE-system. The function of this chaperone has only been studied alone ​in vitro. Here we lay ground to further studies on GroES and its ability to act alone in vivo. ​GroES was expressed from a plasmid and characterized through its potential to increase the amount of correctly folded proteins. Characterization was mainly done by fluorescence spectroscopy with hard-to-fold proteins linked to fluorescent probes. Results show a very clear increase in fluorescence for most of the substrate proteins tested, indicating that GroES has a significant role in the GroE-system and perhaps outside of it.

Keywords

Molecular chaperone; Protein folding; GroE/GroEL/ES; Chaperonin 10; Chaperonin 60; Hard-to-fold proteins; Aggregation-prone proteins; Protein production

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Financial Disclosure

IFM, Department of Physics, Chemistry and Biology, Linköping University

Cenova, Unionen Östergötland, Ebba Biotech, BioArctic, Eppendorf, IDT Integrated DNA Technologies, NEB New England Biolabs.

Ethics statement

All proteins used in this report are non-toxic to humans. The bacterial strains used are classed as low-risk, and non-pathogenic to humans. The antibiotics used in this report were autoclaved and treated following Linköping University’s guidelines. The experiments were done in small-scale in order to lower the usage of antibiotics. No genetic modification other than addition of genes in plasmid form was done.

Data Availability

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Contents

Financial Disclosure 2 Ethics statement 2 Data Availability 2 Abbreviations 4 1. Introduction 5 1.1 Protein production 5

1.2 The GroE chaperone system 6

1.3 GroES possible individual function 8

1.4 Neurodegenerative diseases 9

1.4.1 Amyloid-beta 9

1.4.2 α-synuclein 9

1.4.3 Tau 9

1.5 Fluorescent Proteins 10

1.5.1 Enhanced Green Fluorescent Protein 10

1.5.2 mNeonGreen 10

Aim 11

2. Experimental Design 11

2.1 Inducible GroES-gene 11

2.2 Plasmid design for co-expression 12

2.3 Experimental design 12

3. Methods 13

3.1 Assembly of the TetR-pTet-GroES to the vector pSB4A5 13

4. Results/Data 15

4.1 Creating a plasmid with an inducible GroES-gene 15

4.2 Kinetic fluorescence spectroscopy results 16

5. Discussion 19

5.1 Assembly and characterization 19

5.2 mNG-Aß1-42 19

5.3 EGFP-Aß1-42 19

5.4 α-synuclein-EGFP 19

5.5 Tau0N4R-EGFP 20

5.6 The dubious role of GroES 20

6.1 Conclusions about the role of GroES 20

6.2 Further characterization 21

6.3 Data acquisition 21

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Abbreviations

SP Substrate Protein

GroES Growth Essential Small

GroEL Growth Essential Large

GroE Growth Essential (GroEL and GroES)

Hsp Heat shock protein

Amyloid-beta

APP Amyloid Precursor Protein

AD Alzheimer’s Disease

PD Parkinson’s Disease

EGFP Enhanced Green Fluorescent Protein

mNG mNeonGreen

LanYFP Yellow Fluorescent Protein

IPTG Isopropyl β-D-1-thiogalactopyranoside

OD Optical Density

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1. Introduction

1.1 Protein production

When bacteria are used in large scale industrial production or for expression of large recombinant proteins in research, certain challenges arise. These challenges occur because the machinery of the organism is not adjusted to some parts of the protein’s components. One such problem occurs in the folding process when proteins become too complex, or larger than 60 kDa. This may lead to misfolding followed by the aggregation of the protein in question [1].

Some proteins can fold to their native state on their own due to the fact that proteins prefer to be in a state of low free energy. However, local minimums in free energy can occur due to energy barriers. In order to overcome these barriers, the need of additional energy is required. Organisms have solved this problem by the creation of chaperones (Figure 1) which are able to add the needed energy [1,2]. As can be seen in Figure 1. It is challenging for a misfolded protein to refold to its correct form on its own [1,2].

Figure 1. ​A refined version of Gibbs free energy concept related to the protein folding energetics. The energy state of misfolded proteins (red) is close to the native state (green). Chaperones are able to let a protein refold itself by being able to add enough energy for the protein to refold.

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1.2 The GroE chaperone system

Chaperones are proteins that can interact with substrate proteins (SP), and assist in their folding-process. By folding proteins to their native form they also have an important role in maintaining proteostasis; homeostasis of proteins. There are several types of chaperones and they all have varying affinities for SP:s and SP states [3].

Chaperones can work in different subsystems. One of the essential subsystems in ​E. coli is the Growth essential (GroE)-system consisting of two parts: Growth essential small (GroES) and Growth essential large (GroEL) (Figure 2). Chaperones are generally a very conserved group of proteins, indicating their importance. The eukaryotic version of the GroE-system is Hsp60 (GroEL) and Hsp10 (GroES).The GroE-system is not protein specific and is mainly limited by protein size [3].

A B

Figure 2. ​A. GroEL (green) and GroES (red). ​B. A top view of GroEL (green) illustrating how the subunits create a ring. The helices H and I in the apical domain subunits are directed inwards, which can be shown as the small strands pointing inwards towards the GroEL center. Exposing the helices allows SP:s to be bound to the GroEL and later on to be encapsulated. From PDB entry 1AON.

GroEL consists of two heptameric rings of ~57 kDa subunits which creates a hollow barrel-like shape [4]. The subunits contain an ATP-binding domain and an apical domain which binds to GroES, and creates the entry-point of the cavity (Figure 2, B). Bound to the apical domains are helices (known as helix H and I) which exposes hydrophobic sites towards the center of the hollow for the purpose of binding SP:s (Figure 2) [4].

GroES can be compared to a lid to the GroEL-barrel, encapsulating SP:s for the folding process (Figure 2). It consists of a heptameric ring containing ~10 kDa subunits which exposes flexible loops that interacts with helices H and I. However, this interaction is ATP-dependent. When ATP binds to GroEL’s subunit sites there will be a conformational change that allows GroES to interact with it [4].

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Figure 3 ​. A simplification of the E. coli folding pathway. The first step is synthesis of a protein by mRNA (black strand) being read by a ribosome (maroon) which transcribes it to an amino acid chain (short multi-colored chain beside the ribosome) which becomes an unfolded protein (long multi-colored chain). The protein will then either spontaneously fold to its correct/native form (green origami crane), misfold (green cloud) or get folded by GroE (orange) which then fold the protein to its correct form. The gradient of black to pale gray arrows illustrates the most likely to least likely pathway, respectively.

GroE binds to an unfolded or misfolded protein (Figure 3) by hydrophobic interaction via GroEL’s apical domain. Encapsulation is energetically unfavourable and is therefore driven by a conformational change by the binding of one ATP per GroEL subunit [4,5].

During the addition of ATP, GroEL will internalize the bound protein. The ATP-interaction will act as a timer and the hydrolyzation of ATP will be the executing alarm which will lead to the release of the natively folded SP (Figure 3). During the internalization the protein will have been rearranged due to the GroE-system adding energy and letting the protein reach a new state of low free energy (Figure 1) [5,6].

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1.3 GroES possible individual function

Today the GroE system is thought to consist of the main functional part GroEL and its co-chaperone GroES. GroES’s function is thought to be a mere “shielding” or lid-like function which protects the SP from the outside environment while GroEL encapsulate the SP and refolds it [4,7].

In the article “Transient conformational remodeling of folding proteins by GroES-individually and in concert with GroEL” by Moparthi et al. it was reported that GroES ​can mediate in protein folding dynamic remodeling individually ​in vitro​. GroES might do this both by mediating and by interacting in protein unfolding and compression induced by GroEL binding [7]. This mechanism is illustrated in Figure 4 by a question mark.

Figure 4. ​Schematic representation of the GroE mechanism When the unfolded/misfolded protein binds to GroEL, GroES can bind to GroEL-Substrate-complex. By addition of ATP to the GroEL-Substrate-GroES-complex, the conformational structure of the complex is altered and folding/refolding can transpire. The question mark points to the possible lone function of GroES as an alternative first interactive step for unfolded/misfolded proteins.

The data from Moparthi SB et al. is corroborated by previous findings such as Neidhart FC et al. who studied GroEL and GroES concentrations before and after heat shock. In 37 ℃ the ratio of GroES to GroEL is 1,9 and after induction it is 4,7. Also, the concentration of free GroES after heat induction is almost the same as before even though exposed to ATP, this information points to a lone function of GroES [7-9].

Another supporting fact is that GroES might assist in the folding of GroEL. Lissin NM et al. in “(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL” has indications of this, supported by data from Montero Llopis P et al. article “Spatial organization of the flow of genetic information in bacteria.”. Data from Montero Llopis P et al. shows that GroEL and GroES’s mRNA are spatially close to each of other during transcription, also GroES is transcribed before GroEL. This might indicate that GroES assists in the folding of GroEL after being produced [7,10,11].

The eukaryotic equivalents of GroE, as previously mentioned, are Hsp10 (GroES) and Hsp60 (GroEL). Dubaquié Y et al. in “Identification of in vivo substrates of the yeast mitochondrial

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chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10” has published data confirming that Hsp10 mediate the import of Hsp60 in to the mitochondria [7,12]. By chaperones being evolutionary conserved [3]; this is yet another indication that GroES might have a individual function.

The high ratio of GroES should have a functional explanation; it might be as mentioned by Moparthi SB et al. They described that the function of GroES might be more than a co-chaperone of GroEL, and that its functioning would be one of mediating dynamic remodeling, interacting in protein unfolding and compression induced by GroEL binding, and also in the folding process of GroEL transcription [7-12].

1.4 Neurodegenerative diseases

To investigate chaperone function one can use hard-to-fold and aggregation-prone proteins. Proteins known for these abilities can for example be linked to neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s disease (PD) [13].

1.4.1 Amyloid-beta

Amyloid-beta (Aβ) is a peptide derived from amyloid precursor protein (APP). It is linked to AD, and can in high concentrations misfold and become a toxic oligomer with a strong intermolecular interaction ability. This is due to a secondary structure conversion, thought to arrive from an interactive tail domain, protruding from the misfolded Aβ-peptide. This leads the oligomer to have more β-sheets, which interacts with free monomer Aβ and includes them into the oligomer. Aβ-oligomers are thought to be the cause of AD. The misfolded Aβ and Aβ-oligomers can be refolded, but the oligomers can turn into fibrils that in turn can become aggregates. These two forms can not be refolded [14,15]. The most toxic form of Aβ is known as Aβ 1-42. Aβ 1-42 is a commonly used for research in hard-to-fold, aggregation prone proteins [16]. The protein structure of Aβ can be observed in Figure 5, A and B, the red part.

1.4.2 α-synuclein

Misfolding and aggregation of α-synuclein is correlated to PD and partly to AD [17]. α-synuclein-oligomers and fibrils in PD is thought to cause harmful Lewy bodies which are aggregates of protein within neurons [17]. The middle of the α-synuclein protein (61-95) has also been found in Aβ-plaques in abnormal amounts and contributes to the pathology of AD [18]. Chaperones are able to refold α-synuclein-oligomers to less harmful correctly folded α-synuclein [17]. The protein structure of α-synuclein can be observed in Figure 5, C.

1.4.3 Tau

Hyperphosphorylated tau can be found in both AD and PD. Tau’s activity is regulated by its phosphorylation but toxic during hyperphosphorylation. The reason tau is harmful in a hyperphosphorylated form is because it’s unable to be regulated. This is due to neutralization of inhibitory domains leading to an uncontrollable tau-tau interaction. This can further lead to neurofibrillary tangles (NFT). Aβ and α-synuclein are able to interact with, and bind to tau, increasing phosphorylation [19,20].

Tau consists of six isoforms in the human brain due to different forms of splicing. There are three to four repeats in the microtubule-binding (3R and 4R) and none, one or two insertions in the N-terminal projection domain (0N-2N). The R-domains have a microtubule-binding and assembly-promoting

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activity. The 4R isoform of tau can be seen in a higher concentration in tau-linked diseases with a genetic background due to the higher ability of hyperphosphorylation. The 4R isoform such as 0N4R is a preferred form of tau used in research [19,20]. The protein structure of tau can be observed in Figure 5, D.

1.5 Fluorescent Proteins

Fluorescent proteins are able to emit light when they are shone on by light of the correct wavelength. One way to use fluorescent proteins is by linking them to SP:s. The fluorescent protein will work as a tag to the SP and the higher the fluorescence; the more SP is expressed.

1.5.1 Enhanced Green Fluorescent Protein

Enhanced green fluorescent protein (EGFP) is a modified version of green fluorescent protein (GFP). The excitation peak of EGFP is at 488 nm with an emission peak at 507 nm [21,22]. The protein structure can be seen in Figure 5, B-D where EGFP (green) is linked to proteins.

1.5.2 mNeonGreen

mNeonGreen (mNG) is a mutated form of yellow fluorescent protein (LanYFP) from ​Branchiostoma

Lanceolatum. ​The mutation resulted in a green color instead of LanYFP’s yellow and became a monomer instead of the previous LanYFP tetramer. The change of color resulted in an excitation peak at 506 nm with an emission peak at 517 nm. It also increased the brightness 1,5 -3 times compared to GFPs. The protein mNG is also more easily folded in comparison to EGFP [23].

A. mNG-Aβ1-42 ​ B. ​EGFP-Aβ1-42

C. ​α-synuclein-EGFP​ D. ​Tau0N4R-EGFP

Figure 5. A. mNG, illustrated with an EGFP protein (lime green) linked to Aβ 1-42 (red). B. EGFP (green) linked to Aβ 1-42 (red). C. EGFP (green) linked to α-synuclein (yellow). D. EGFP (green) linked to tau0N4R (blue) [24][25][26].

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Aim

The aim of the research is to investigate and characterize the individual function of GroES in vivo. Further investigation can be conducted if results indicate that GroES might be more than a co-chaperone to GroEL.

The characterization of GroES is intended to be executed by expressing hard to fold, aggregation prone proteins linked to a fluorescent protein. It will be done by overexpressing GroES in a separate plasmid, studying the potential change in protein expression afterwards. The fluorescent proteins enables measurement of protein production. EGFP linked proteins will also be more difficult to fold and will therefore add to the characterization of GroES. The GroES-plasmid will be compared to a Takara ® -plasmid with the GroE-system. The proteins expressed were mNG-AB, EGFP-AB, EGFP-Tau and EGFP-a-synuclein.

The research was heavily inspired by “Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL” written by Moparthi SB, Sjölander D, Villebeck L, Jonsson B-H, Hammarström P and Carlsson U.

2. Experimental Design

2.1 Inducible GroES-gene

The design of the inducible GroES gene (from E.coli K12 strain) can be seen in Figure 6. No termination sequence had to be added to the end of this part, because of the termination found downstream in the vector, pSB4A5. The pTet-system with the necessary repressor protein for control [27], was chosen because it was not present in any other of the plasmids we intended to co-transform. Why the vector pSB4A5 was chosen is explained further below. The sequence data can be found in the appendix.

Figure 6. A cartoon representation of the system used to express GroES. Cons. P is a constitutive promoter with a medium strength. RBS is the Shine-Delgarno sequence most commonly found in E.coli ribosomes. ORF1 = Open reading frame for the repressor protein of the tetracycline promoter. ORF2 = Gene encoding for GroES (E.coli K12 strain)

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2.2 Plasmid design for co-expression

A full list of plasmid names and general information can bee seen in table 1. The Takara ®-plasmid pGro7 is used to express both GroEL and GroES. pGro7’s expression of the GroE-system is used as comparison for the investigation of GroES and the role of GroES overexpression. It is important to note that there is always a baseline of GroEL expression in the bacterial genome.

As seen in Figure 7, GroES had to be ligated into pSB4A5 to avoid having the same ORI and the same antibiotic resistance as the other plasmids. The tetracycline promoter was chosen to enable fine-tuning of the GroES expression.

Figure 7. An illustration of the plasmids used in the experiments. pSub*, is either mNG-Aß1-42, EGFP-Aß1-42, α-synuclein-EGFP or Tau0N4R-EGFP. They were used as a substrate proteins for co-expressions.

Table 1 ​. Showing the different plasmids used in this report. The name indicates which protein is at the

N-terminal, respectively the C-terminal.

2.3 Experimental design

A simple overview of the experimental design is shown in Figure 8. Four combinations were heat shocked (HS) into ​E.coli (BL21(DE3) Gold). The plasmids are named after which ORI they have. See table 1 and yellow segments of figure 7.

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The combinations were: Substrate (figure 7A) , substrate and GroES (Figure 7 A+C), substrate, GroES and GroE (Figure 7 A+B+C), substrate and GroE (Figure 7 A+C). Figure 7A can be either mNG-Aß1-42, EGFP-Aß1-42, α-synuclein-EGFP or Tau0N4R-EGFP. Figure 7B is always pGro7 and figure 7C is always pSB4A5-GroES.

The plasmids were co-transformed in the cases where multiple plasmids needed to be transformed. Bacteria containing plasmids were grown in LB-media containing antibiotic for respective plasmid until desired OD was achieved. The chaperones were induced with 0.25 mg/ml L-arabinose (from Takara®) and 200 ng/ml tetracycline (this concentration was tested in this report).

For the induction of substrate plasmids 0.5 mM IPTG (this concentration was tested in this report) was used, and the substrate was induced 30 mins after the chaperone plasmids (Takara® recommendation). The bacterial samples were placed in a 96-well plate that ran for 16 hours at 37 °C in a Galaxy FLUOstar microplate reader. Measurements were conducted every 15 minutes.

Figure 8. ​The experimental procedure for the co-expression. The plasmids used are: Substrate plasmid (A), pGro7 from Takara ® (B) and GroES plasmid (C). The substrate plasmids contain one of the following substrates: mNG-Aß1-42, EGFP-Aß1-42, α-synuclein-EGFP or Tau0N4R-EGFP.

3. Methods

3.1 Assembly of the TetR-pTet-GroES to the vector pSB4A5

Gibson assembly master mix (New England Biolabs) was used to ligate the insert to the vector. A molar ratio of insert 2:1 vector was used and followed the protocol for 2-3 parts assembly. The overhangs used were the restriction enzyme sites, found both on the insert and on the vector.

Colony screening and agarose gel: ​Traditional colony screening was used. Colonies was picked

with pipette tips and first moved to a new LB-agar plate with a grid. The rest of the bacteria left on the tip was shaken down into a PCR tube with Q5® Hot Start High-Fidelity 2X Master Mix (12.5 uL) from New England Biolabs. The PCR products ran on an agarose gel (1.3 %) for 90 minutes at 80 V. After completion the gel were stained with ethidium bromide. A gel camera (Azure biosystems) was used to illuminate the bands.

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SDS-PAGE gel: ​Bacteria containing the GroES plasmid were grown (10 mL) and induced, a negative control was also done. Cells (500 uL) was taken and placed in an eppendorf tube. For the GroES purification an additional step was included, incubation at 80 ℃ for 40 minutes. After the incubation, cells from both the control and the GroES induced bacteria were centrifuged (12000 g, 10 minutes). For cells not incubated at 80 ℃ the supernatant was removed and milliQ-water (50 uL) was added. The control cells (10 uL) were added to a new tube, and SDS sample buffer (10 uL) was added to both samples. For the incubated GroES cells were taken from the supernatant (10 uL). All samples were incubated (95 ℃, 5 minutes). The samples (15 uL) were added onto a SDS-gel. The gel started at 150 V, after all the samples had wandered into the gel, the voltage was increased to 200 V. The gel ran until the loading dye had reached the bottom of the gel. The gel was washed with dH 2​O on a shaking table (5 minutes, repeat 3 times). The gel was then stained with a dye, used in this report is: Comassie G250, R250. The gel was destained with a destaining solution (50 % MeOH, 40% dH2O, 10 % acetic acid) until a satisfying color was obtained.

Purifying plasmids with GenElute™ Plasmid Miniprep Kit: To start isolating plasmids with the kit

“GenElute™ Plasmid Miniprep Kit” from Sigma Aldrich, 5 ml of overnight grown competent cells was centrifuged for 1 min at 1200 g. The supernatant was discarded and cells resuspended. With 200 µl of the Resuspension Solution and vortex, the bacterial pellet was completely resuspended. The resuspended cells were lysed by adding 200 µl of the Lysis Solution. The contents were immediately mixed by gentle inversion (6–8 times) until the mixture became clear and viscous. The lysis reaction was not to exceed 5 minutes. By adding 350 µl of the Neutralization/Binding Solution neutralization of the Lysis Solution was successful. The tube was inverted 4–6 times. To pellet the cell debris, a centrifugation at 12,000 g for 10 min was done. A GenElute Miniprep Binding Column was inserted into a provided microcentrifuge tube. 500 µl of the Column Preparation Solution was added to the miniprep column and centrifuged at 12,000 g for 1 min. Flow-through was discarded.

The cleared lysate was transferred to the column and centrifuged at 12,000 g for 1 min. All liquid that flowed through was discarded. The column was washed with 700 µl of the diluted Wash Solution by adding it to the column. Centrifugation at 12,000 g for 1 min was done after the Wash step. Flow-through was discarded and centrifuged again at maximum speed for 1 to 2 min without any additional Wash Solution. Additional ethanol that flowed through was discarded. Transfer the column to a new collection tube. To separate the DNA from the column, 50 µl molecular biology reagent water was added to the column and then centrifuged at 12,000 g for 1 min. The DNA was now in the eluate and was ready for immediate use or storage at –20 °C.

Purifying PCR products with MinElute PCR Purification Kit: By using the MinElute PCR

Purification Kit from Qiagen, purification of PCR products was done. 5 volumes of Buffer PB to 1 volume was added to the PCR reaction. The mixture was added to a spin column with a collecting tube. The column was centrifuged for 1 min and flow-through was discarded. 750 µl Buffer PE was added to the column and centrifuged for 1 min. Flow-through was discarded after which another centrifuge for 1 min was needed in order to remove residual ethanol from Buffer PE. The column was then placed in a clean 1,5 ml microcentrifuge tube. To elute DNA, 10 µl Buffer PB or water was added to the center of the MinElute membrane. The column stood for 1 min, and was then centrifuged for 1 min.

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4. Results/Data

4.1 Creating a plasmid with an inducible GroES-gene

The sequencing data shows the ligation of TetR-pTet-GroES into the pSB4A5 vector (see appendix). Colony screening was used to find successful ligations. An agarose gel (1.3 %) of the PCR results can be seen below (Figure 9). A band at 1500 bp could be found in one of the clones screened. A SDS-PAGE (see Figure 10) analysis was also done to confirm if the promoter and gene were working as intended.

Figure 9. Agarose gel results for colony screening. One band at 1500 bp (arrow) could be seen after screening 40 clones (picture has been cropped for readability), which confirms a successful ligation of TetR-pTet-GroES. The ladder is a 2-log ladder from New England Biolabs (left and right asterisks).

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Figure 10. SDS-PAGE results for an induced pTet-GroES in pSB4A5. A= Induction of GroES with tetracycline 200 ng/ml, B= Uninduced E.coli (BL21 (DE3)), C= leakage from adjacent wells, D= BLUeye Prestained Protein ladder from Sigma Aldrich. The arrow indicates the 10 kDa region where GroES is found.

4.2 Kinetic fluorescence spectroscopy results

Results from the experimental measurements are shown in Figure 11. The y-axis represents the fluorescence intensity at 520 nm [28] divided by the start OD 600​. The excitation was carried out at 485 nm [28]. The excitation and emission wavelengths were chosen to fit both mNG and EGFP. The pGro7-plasmid has a more predictable SP-production than pSB4A5-GroES and both plasmids together.

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Figure 11. Showing the kinetic data for all the substrates and combinations of chaperones. The y-axis in graph C,D is zoomed to better show the difference between the data. The mean is only showed in the figure to make it easier to read the results, n = 4. For a indication of the error, Figure 12 shows this partly. The asterisk indicates the part made for this project.

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Figure 12. Bar graphs for all substrates. The asterisks written behind -ES (GroES) indicates that the part was made for this specific project. Control means that the substrate was induced without containing any chaperone plasmid. The y-axis represent the max exchange, which is the highest fluorescence point of the measurements. The error bars represent CI 95 % and n = 4. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

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5. Discussion

5.1 Assembly and characterization

The band at 1500bp in figure 9 confirms that the ligation of TetR-pTet-GroES into pSB4A5 was successful. Furthermore, the results from the SDS-PAGE gel (seen in figure 10) indicates that expression of GroES through the Tet-inducible system was accomplished as well.

As can be seen in the SDS-PAGE gel and the sequencing results, the gene is fully inducible and produces a protein of the same size as GroES. It would’ve been good to confirm the amino acid sequence through MALDI/TOF-TOF MS [29], because of the GroES gene being hard to synthesize. This was circumvented by using the Codon Optimization Tool (Integrated DNA Technologies), and checked for any error in the amino acid sequence. However the project faced a shortage of time and thus no amino acid sequencing could be done. Do note that GroE is always expressed in the E.coli by default.

5.2 mNG-Aß1-42

The folding rate slows down when pGro7 is present compared to when only pSB4A5-GroES is expressed. The combination with additional pSB4A5-GroES and pGro7 seems to prevent the production of the substrate. The best fit for the SP is only pSB4A5-GroES, or a high concentration of GroE. A combination of the two chaperone plasmids seems to be too much for the bacterium, and the substrate folding rate is hindered. In Figure 12, C, a significant difference between the control and GroES alone could be seen, as well as for the GroEL/ES and the control.

5.3 EGFP-Aß1-42

As seen in Figure 11, D, when inducing pSB4A5-GroES and pGro7 together with the substrate containing bacteria, the concentration of substrate-yield increases. The line representing the results for pSB4A5-GroES and pSB4A5-GroES+pGro7, has not even reached the maximum concentration of the substrate. It would be interesting to increase the experimental time to see when the system reaches its maximum point. The graphs presenting the folding rate of each expressed system shows little difference. In Figure 12, D, a significant difference between the control and GroES could be seen, as well as for the GroEL/ES and the control.

5.4 α-synuclein-EGFP

When expressing α-synuclein-EGFP with pSB4A5-GroES there is little difference in concentration compared to expressing α-synuclein-EGFP without a chaperone plasmid. With pGro7 the

concentration of the substrate greatly increases compared to all other expression systems. The kinetics of the folding rate for α-synuclein-EGFP expressed with pGro7 and pSB4A5-GroES is noticeably decreased. It seems that when pGro7 is expressed with an increased concentration of pSB4A5-GroES, it decreases the folding rate of α-synuclein-EGFP. If pGro7 is not expressed, there is no difference in the folding rate. In Figure 12, B, a significant difference could only be seen between the control and GroEL/ES.

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5.5 Tau0N4R-EGFP

There is no decrease in intensity for Tau0N4R-EGFP combined with chaperones. A study with longer experiment time can be useful to measure the maximum concentration. The folding rate of

Tau0N4R-EGFP slows down. However, there is a difference in how much it is slowed down between the chaperones. In presence of pSB4A5-GroES, the folding rate is higher than with pSB4A5-GroES and pGro7 co-expressed. With pGro7, the folding rate is in between the others. In Figure 12, A, a significant difference could be seen between the control and GroES. GroES also showed a significant difference compared to the whole system, GroEL/ES.

5.6 The dubious role of GroES

Due to the fact that this was an incomplete characterization of GroES; it is difficult to detail the exact mechanism to why the increased production of proteins occurred. Three possible options are discussed in the following paragraph:

If the increased protein production is the result of a total increase in activity of the GroE-system it might be due to reasons stated in the previously mentioned articles: “(Mg-ATP)-dependent self-assembly of molecular chaperone GroEL” by Lissin NM et al. and “Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL” written by Moparthi SB et al. The articles indicate that GroES may assist in the assembly of GroEL [7,11]. This might be the cause of the increased protein production. However, this means that GroES has two possible functions: either GroES can only interact with GroEL or GroES can interact with proteins and assist in assembly to some point. The latter alternative means that the increase in production is due to both an increase of the GroE-system, and a lone function of GroES. The most likely lone function of GroES is that it has a holdase function. However, this must be investigated further.

If either of the previous statements are correct so does the induction of GroES in pSB4A5-GroES lead to a higher protein production. However, the reason why expression of GroES together with some SP:s are more or less effective is difficult to explain.

6. Conclusions

6.1 Conclusions about the role of GroES

The results display a clear effect of GroES overexpression on a variety of SP:s. A much higher yield for some of the different SP:s can be seen, indicating that the GroES concentration is important, and perhaps even more important than the GroEL concentration in some cases. However, it is hard to tell if this increase is because of a faster working GroE-system because of the added GroES, or if GroES has a total individual chaperone function.

The result also illustrated that pSB4A5-GroES may be an alternative to the Takara ®-plasmid. However, this won’t be an alternative until it can be explained why some SP:s were helped by the addition of GroES while some weren’t.

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It is also not possible to tell if GroES binds to the substrate first and uses a holdase effect to prevent misfolding and aggregation, further studies of this are needed. In some of the results it is shown that the folding rate is sometimes slowed down when GroES is overexpressed. This occurrence is mostly seen in the triple plasmid system which indicates that this could be happening because of the metabolic strain three plasmids put on the bacterium. These results does, however, support the importance of further characterization of GroES.

6.2 Further characterization

It would be interesting to test GroES ability to bind SP:s while not in concert with GroEL and to crystallize this complex. This would show if GroES has the ability to work as a holdase outside of the GroE complex. Other methods testing affinity or showing a bound substrate protein to GroES could also be used to characterise this chaperone further.

A knock-out/down of the GroE operon could also be interesting to show how this affects the survival of the bacterium; this would show the GroES function better. Because of the constant presence of GroEL in all E.coli strains, GroES function in vivo is hard to confirm. By adding GroES and GroEL in plasmid form after knock-out/down of the GroE operon, one can investigate the function of GroES in vivo.

Many would state that the result of this kind of knock-out/down would either cause bacterial death or severe side effects on the bacteria due to the large amount of proteins GroE folds. However one can not be completely confident of this until a knock-out/down is actually performed.

Investigating homologs of GroES in eukaryotic organisms (Hsp10) and in other bacteria could also be interesting. Especially the more extremophilic bacterium which has already been shown to have some interesting chaperones present in them [30]. Investigation of GroES in other organisms would also show how well conserved the function of this chaperone is between species and through time.

6.3 Data acquisition

Two other experiments of the same kind were done, and deemed inaccurate because of uneven inducement due to large differences in volume of the medium. A constant volume after the OD600

compensation was taken and that volume was induced to create an even inducement. The last experiment is shown in this report, it should have been done multiple times to ensure a strong data acquisition. Due to the lack of time this could not be done.

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7. Acknowledgements

The group would like to thank our supervisors, ​Professor Per Hammarström,​Associate Professor

Lars-Göran Mårtensson, for providing feedback in multiple aspect of the project. In providing lab equipment and in providing knowledge and solutions to the problems that occured during this project, but also moral support and inspiration for our research.

Thank you; ​Hammarström Lab who inspired us to research on this particular field and which lent us equipment and experience.

We would also like to thank the ​Department of Physics, Chemistry and Biology at Linköping University​ for providing a laboratory during the summer and equipment.

We would also like to thank ​Linköping University for providing some economical support which allowed us to research and compete in the iGEM competition.

Great appreciation and thanks to ​Associate Professor, Patrik Lundström, Principal Research

Engineer, Cecilia Andrésen,​Principal Research Engineer Sofie Nyström, ​for support, equipment and laboratory troubleshooting. ​BSc, Max Lindström for helping us with equipment and questions related to methods.

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8. References

[1] Rosano GL, Ceccarelli EA. Recombinant protein expression in Escherichia coli: advances and challenges. Front Microbiol. 2014;5:172.

[2] Kumar V, Sami N, Kashav T, Islam A, Ahmad F, Hassan MI. 2016.Protein aggregation and neurodegenerative diseases: from theory to therapy. Eur. J. Med. Chem. 124, 1105–1120.

[3] Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Ulrich Hartl F. Molecular Chaperone Functions in Protein Folding and Proteostasis. Annu Rev Biochem. 2013 Jun 2;82(1):323–55.

[4] Hayer-Hartl M, Bracher A, Hartl FU. The GroEL–GroES Chaperonin Machine: A Nano-Cage for Protein Folding. Trends Biochem Sci. 2016;41(1):62–76.

[5] Hartl FU. Molecular chaperones in cellular protein folding. Nature (1996) 381:571–9. doi: 10.1038/381571a0.

[6] Fukui N, Araki K, Hongo K, Mizobata T, Kawata Y. Modulating the Effects of the Bacterial Chaperonin GroEL on Fibrillogenic Polypeptides through Modification of Domain Hinge Architecture. J Biol Chem. 2016;291(48):25217-25226.

[7] Moparthi SB, Sjölander D, Villebeck L, Jonsson B-H, Hammarström P, Carlsson U. Transient conformational remodeling of folding proteins by GroES—individually and in concert with GroEL. J Chem Biol. 2014;7(1):1–15.

[8] Neidhardt FC, VanBogelen RA. Heat shock response. In: Neidhardt FC, Ingraham JL, Low KB, Magasanik B, Schaechter M, Umbarger HE, editors. Escherichia coli and Salmonella typhimurium: cellular and molecular biology. Washington, DC: American Society for Microbiology; 1987. pp. 1334–1345.

[9] Buchner et al. published data that GroES is in a 11,2-fold molar excess compared to GroEL.( Rudolph B, Gebendorfer KM, Buchner J, Winter J (2010) Evolution of Escherichia coli for growth at high temperatures. J Biol Chem 285:19029–19034

[10] Montero Llopis P, Jackson AF, Sliusarenko O, Surovtsev I, Heinritz J, Emonet T, Jacobs-Wagner C (2010) Spatial organization of the flow of genetic information in bacteria. Nature 466:77–82

[11] Lissin NM, Venyaminov SY, Girshovich AS (1990) (Mg-ATP)-dependent self-assembly of molecular chaperone GroEL. Nature 348:339–342

[12] Dubaquié Y, Looser R, Fünfschilling U, Jenö P, Rospert S (1998) Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for Hsp60 and Hsp10. EMBO J 17:5868–5876

[13] Ross, C. A. and Poirier, M. A. (2004) ‘Protein aggregation and neurodegenerative disease’, Nature Medicine, 10, pp. S10–S17. doi: 10.1038/nm1066.

[14] Kumar A. Singh A. Ekavali. A review on Alzheimer’s disease pathophysiology and its management: an update. 2015; vol 67: Issue 2 (195-203)

[15] Chaturvedi Sumit Kumar, Siddiqi Mohammad Khursheed, Alam Parvez, Khan Rizwan Hasan.Protein misfolding and aggregation: Mechanism, factors and detection.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2016.05.015.

[16] Marsh J, Alifragis P. Synaptic dysfunction in Alzheimer’s disease: the effects of amyloid beta on synaptic vesicle dynamics as a novel target for therapeutic intervention. Neural Regen Res. 2018 Apr;13(4):616–23.

[17] L. Breydo, J.W. Wu, V.N. Uverskyα-Synuclein misfolding and Parkinson's disease. Biochim. Biophys. Acta, 1822 (2) (2012), pp. 261-285

[18] Bendor JT, Logan TP, Edwards RH. The function of α-synuclein. Neuron. 2013;79(6):1044-66. [19] P. Lei, S. Ayton, D.I. Finkelstein, P.A. Adlard, C.L. Masters, A.I. Bush. Tau protein: relevance to Parkinson's disease. Int. J. Biochem. Cell Biol., 42 (2010), pp. 1775-1778)(Kumar A. Singh A. Ekavali.

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A review on Alzheimer’s disease pathophysiology and its management: an update. 2015; vol 67: Issue 2 (195-203).

[20] Iqbal, K., Liu, F., Gong, C.-X., Alonso, A. del C., & Grundke-Iqbal, I. (2009). Mechanisms of tau-induced neurodegeneration. Acta Neuropathologica, 118(1), 53–69.

[21] Thorn K. Genetically encoded fluorescent tags. Mol Biol Cell. 2017 Apr 1;28(7):848–57.

[22]Saeed IA, Ashraf SS. Denaturation studies reveal significant differences between GFP and blue fluorescent protein. Int J Biol Macromol. 2009;45(3):236–41.

[23] Shaner NC, Lambert GG, Chammas A, Ni Y, Cranfill PJ, Baird MA, et al. A bright monomeric green fluorescent protein derived from Branchiostoma lanceolatum. Nat Methods. 2013 Mar 24;10:407.

[24] Y Zhang. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics, 9: 40 (2008). [25] A Roy, A Kucukural, Y Zhang. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols, 5: 725-738 (2010).

[26] J Yang, R Yan, A Roy, D Xu, J Poisson, Y Zhang. The I-TASSER Suite: Protein structure and function prediction. Nature Methods, 12: 7-8 (2015).

[27] Structural basis of gene regulation by the tetracycline inducible Tet repressor–operator system Peter Orth, Dirk Schnappinger, Wolfgang Hillen, Wolfram Saenger, Winfried Hinrichs. Nature Structural Biology 7, 215–219 (2000)

[28] Mol Biol Cell. (2016) Nov 7; 27(22): 3385–3394.Comparative assessment of fluorescent proteins for in vivo imaging in an animal model system. Jennifer K. Heppert, Daniel J. Dickinson, Ariel M. Pani, Christopher D. Higgins, Annette Steward, Julie Ahringer, Jeffrey R. Kuhn and Bob Goldsteina

[29] Journal of the American Society for Mass Spectrometry

July (2002), Volume 13, Issue 7, pp 784–791. De novo sequencing of peptides using MALDI/TOF-TOF. Alfred L. Yergey, Jens R. Coorssen, Peter S. Backlund, Paul S. Blank, Glen A. Humphrey, Joshua Zimmerberg, Jennifer M. Campbell, Marvin L. Vestal

[30] Alexander Bepperling, Ferdinand Alte, Thomas Kriehuber, Nathalie Braun, Sevil Weinkauf, Michael Groll, Martin Haslbeck, and Johannes Buchner. Proc Natl Acad Sci USA. (2012) Dec 11; 109(50): 20407–20412.Alternative bacterial two-component small heat shock protein systems

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9. Appendix

Takara ® plasmid pGro7

pNIC vector, used for TAU, Syn, AB-EGFP

Primers

Vector reverse (VR):​ attaccgcctttgagtgagc (sequencing and colony screening)

Vector forward 2 (VF2):​ tgccacctgacgtctaagaa (sequencing and colony screening)

Backbone SNP reverse (BSNPR):​ ctctagaagcggccgcgaat (used to amplify pSB4A5)

Backbone ENX forward (BENXF):​ tactagtagcggccgctg (used to amplify pSB4A5)

DNA sequences used in this report

psf4RTAU

CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGG CTGAGCCCCGCCAGGAGTTCGAAGTGATGGAAGATCACGCTGGGACGTACGGGTTGGGGGACAGGAAAGA TCAGGGGGGCTACACCATGCACCAAGACCAAGAGGGTGACACGGACGCTGGCCTGAAAGCTGAAGAAGCA GGCATTGGAGACACCCCCAGCCTGGAAGACGAAGCTGCTGGTCACGTGACCCAAGCTCGCATGGTCAGTA AAAGCAAAGACGGGACTGGAAGCGATGACAAAAAAGCCAAGGGGGCTGATGGTAAAACGAAGATCGCCAC ACCGCGGGGAGCAGCCCCTCCAGGCCAGAAGGGCCAGGCCAACGCCACCAGGATTCCAGCAAAAACCCC GCCCGCTCCAAAGACACCACCCAGCTCTGGTGAACCTCCAAAATCAGGGGATCGCAGCGGCTACAGCAGC CCCGGCTCCCCAGGCACTCCCGGCAGCCGCTCCCGCACCCCGTCCCTTCCAACCCCACCCACCCGGGAGC CCAAGAAGGTGGCAGTGGTCCGTACTCCACCCAAGTCGCCGTCTTCCGCCAAGAGCCGCCTGCAGACAGC CCCCGTGCCCATGCCAGACCTGAAGAATGTCAAGTCCAAGATCGGCTCCACTGAGAACCTGAAGCACCAGC CGGGAGGCGGGAAGGTGCAGATAATTAATAAGAAGCTGGATCTTAGCAACGTCCAGTCCAAGTGTGGCTCA AAGGATAATATCAAACACGTCCCGGGAGGCGGCAGTGTGCAAATAGTCTACAAACCAGTTGACCTGAGCAA GGTGACCTCCAAGTGTGGCTCATTAGGCAACATCCATCATAAACCAGGAGGTGGCCAGGTGGAAGTAAAAT CTGAGAAGCTTGACTTCAAGGACAGAGTCCAGTCGAAGATTGGGTCCCTGGACAATATCACCCACGTCCCT

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GGCGGAGGAAATAAAAAGATTGAAACCCACAAGCTGACCTTCCGCGAGAACGCCAAAGCCAAGACAGACCA CGGGGCGGAGATCGTGTACAAGTCGCCAGTGGTGTCTGGGGACACGTCTCCACGGCATCTCAGCAATGTC TCCTCCACCGGCAGCATCGACATGGTAGACTCGCCCCAGCTCGCCACGCTAGCTGACGAGGTGTCTGCCTC CCTGGCCAAGCAGGGTTTGAGCAGTAAAGGTGGATACGGCCTGAATGATACTAGTATGGTGAGCAAGGGCG AGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAG CGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGC AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACC CCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATC TTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACC GCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTA CAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCC ACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCC CGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGC GATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGT GATAAGGATCCGAA

psfASYN

CATATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGG ACGTGTTCATGAAGGGTCTGAGCAAAGCGAAAGAGGGCGTGGTTGCGGCGGCGGAAAAGACCAAACAGGG TGTTGCGGAGGCGGCGGGCAAGACCAAAGAAGGCGTGCTGTACGTTGGTAGCAAGACCAAAGAGGGTGTG GTTCACGGCGTGGCGACCGTTGCGGAGAAGACCAAAGAACAGGTGACCAACGTTGGTGGCGCGGTGGTTA CCGGTGTGACCGCGGTTGCGCAAAAGACCGTGGAGGGTGCGGGCAGCATTGCGGCGGCGACCGGTTTTG TTAAGAAAGACCAGCTGGGCAAAAACGAGGAAGGTGCGCCGCAAGAGGGCATTCTGGAAGATATGCCGGT GGACCCGGATAATGAAGCGTATGAGATGCCGAGCGAAGAAGGCTACCAGGACTATGAACCGGAGGCGAGC AGTAAAGGTGGATACGGCCTGAATGATACTAGTATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGG TGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGG GCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC CACCCTCGTGACCACCCTGACCTGGGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCAC GACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAA CTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATC GACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACATCAGCCACAACGTCTATAT CACCGCCGACAAGCAGAAGAACGGCATCAAGGCCAACTTCAAGATCCGCCACAACATCGAGGACGGCAGC GTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC ACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGA GTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGATAAGGATCCGAA

psfFABETA

ATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTTCCAATCCATGGTGA GCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCC ACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTG CACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTC AGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGA GCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACC CTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGG AGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCA AGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGG CGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAAC GAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGC TGTACAAGGGAGATATACATATGGACGCTGAATTCCGTCACGACTCTGGTTACGAAGTTCACCACCAGAAGC TGGTGTTCTTCGCTGAAGACGTGGGTTCTAACAAGGGTGCTATCATCGGTCTGATGGTTGGTGGCGTTGTGA TCGCTTAATAGCAGTAAAGGTGGATACGGATCCGAA

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TetR-pTet-GroES

TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCCTAGAGAAAGAGGAGAAATACTAGATGTCCAGATTAG ATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTAATGAGGTCGGAATCGAAGGTTTAACAACCCGTA AACTCGCCCAGAAGCTAGGTGTAGAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCG ACGCCTTAGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAAGCTGGCAAG ATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTCATCGCGATGGAGCAAAAGTACATTTA GGTACACGGCCTACAGAAAAACAGTATGAAACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTT CACTAGAGAATGCATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAG AGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATTATTACGACAAGCTA TCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATTCGGCCTTGAATTGATCATATGCGGATTAGA AAAACAACTTAAATGTGAAAGTGGGTCCGCTGCAAACGACGAAAACTACGCTTTAGTAGCTTAATAACACTGA TAGTGCTAGTGTAGATCACCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTT ATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTT TATATCCCTATCATTGATAGAGATTGACATCCCTATCAGTGATAGAGATACTGAGCACCTAGAGAAAGAGGA GAAATACTAGATGAACATCCGTCCGCTGCATGATCGCGTGATTGTCAAGCGCAAGGAGGTTGAGACAAAATC AGCCGGTGGAATTGTTTTGACGGGGAGTGCTGCTGCAAAAAGCACCCGTGGAGAAGTTTTGGCGGTTGGTA ATGGGCGTATTCTGGAGAACGGCGAAGTGAAGCCTCTTGATGTGAAGGTCGGCGATATCGTGATCTTTAAT GATGGGTATGGAGTCAAGTCTGAAAAGATCGATAACGAGGAAGTGCTTATTATGAGTGAATCGGACATTTTA GCCATTGTAGAGGCTTAA

AraC-pBAD-mNG-AB

TTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCC GGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGAT AGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGC TAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTG GCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATC GGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAG CAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCT GGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAG TAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGAT GAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCA CCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGA GATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGG GGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCA TCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAG CATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAA AGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGAT CCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGCTAGCCTAGAGAAAG AGGAGAAATACTAGATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTT CCAATCCGTGAGCAAGGGCGAGGAGGATAACATGGCCTCTCTCCCAGCGACACATGAGTTACACATCTTTG GCTCCATCAACGGTGTGGACTTTGACATGGTGGGTCAGGGCACCGGCAATCCAAATGATGGTTATGAGGAG TTAAACCTGAAGTCCACCAAGGGTGACCTCCAGTTCTCCCCCTGGATTCTGGTCCCTCATATCGGGTATGGC TTCCATCAGTACCTGCCCTACCCTGACGGGATGTCGCCTTTCCAGGCCGCCATGGTAGATGGCTCCGGATA CCAAGTCCATCGCACAATGCAGTTTGAAGATGGTGCCTCCCTTACTGTTAACTACCGCTACACCTACGAGGG AAGCCACATCAAAGGAGAGGCCCAGGTGAAGGGGACTGGTTTCCCTGCTGACGGTCCTGTGATGACCAACT CGCTGACCGCTGCGGACTGGTGCAGGTCGAAGAAGACTTACCCCAACGACAAAACCATCATCAGTACCTTT AAGTGGAGTTACACCACTGGAAATGGCAAGCGCTACCGGAGCACTGCGCGGACCACCTACACCTTTGCCAA GCCAATGGCGGCTAACTATCTGAAGAACCAGCCGATGTACGTGTTCCGTAAGACGGAGCTCAAGCACTCCA AGACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATGTGATGGGCATGGACGAGCTGTACAAG GGCAGCGGTTCGGGCTCAGGAAGCGGTAGTGGCTCGGACGCTGAGTTCCGTCACGACTCTGGTTACGAAG TTCACCACCAGAAGCTGGTGTTCTTCGCTGAAGACGTGGGTTCTAACAAGGGTGCTATCATCGGTCTGATGG TTGGTGGCGTTGTGATCGCTTAATAGCAGTAAAGGTGGATACGGATCCGAA

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pSB1C3 used for AraC-pBAD-mNG-AB

TTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCC GGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGAT AGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGC TAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTG GCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATC GGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAG CAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCT GGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAG TAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGAT GAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCA CCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGA GATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGG GGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCA TCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAG CATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAA AGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGAT CCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGCTAGCCTAGAGAAAG AGGAGAAATACTAGATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTT CCAATCCGTGAGCAAGGGCGAGGAGGATAACATGGCCTCTCTCCCAGCGACACATGAGTTACACATCTTTG GCTCCATCAACGGTGTGGACTTTGACATGGTGGGTCAGGGCACCGGCAATCCAAATGATGGTTATGAGGAG TTAAACCTGAAGTCCACCAAGGGTGACCTCCAGTTCTCCCCCTGGATTCTGGTCCCTCATATCGGGTATGGC TTCCATCAGTACCTGCCCTACCCTGACGGGATGTCGCCTTTCCAGGCCGCCATGGTAGATGGCTCCGGATA CCAAGTCCATCGCACAATGCAGTTTGAAGATGGTGCCTCCCTTACTGTTAACTACCGCTACACCTACGAGGG AAGCCACATCAAAGGAGAGGCCCAGGTGAAGGGGACTGGTTTCCCTGCTGACGGTCCTGTGATGACCAACT CGCTGACCGCTGCGGACTGGTGCAGGTCGAAGAAGACTTACCCCAACGACAAAACCATCATCAGTACCTTT AAGTGGAGTTACACCACTGGAAATGGCAAGCGCTACCGGAGCACTGCGCGGACCACCTACACCTTTGCCAA GCCAATGGCGGCTAACTATCTGAAGAACCAGCCGATGTACGTGTTCCGTAAGACGGAGCTCAAGCACTCCA AGACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATGTGATGGGCATGGACGAGCTGTACAAG GGCAGCGGTTCGGGCTCAGGAAGCGGTAGTGGCTCGGACGCTGAGTTCCGTCACGACTCTGGTTACGAAG TTCACCACCAGAAGCTGGTGTTCTTCGCTGAAGACGTGGGTTCTAACAAGGGTGCTATCATCGGTCTGATGG TTGGTGGCGTTGTGATCGCTTAATAGCAGTAAAGGTGGATACGGATCCGAA

pSB4A5 used for TetR-TetP-GroES

TTATGACAACTTGACGGCTACATCATTCACTTTTTCTTCACAACCGGCACGGAACTCGCTCGGGCTGGCCCC GGTGCATTTTTTAAATACCCGCGAGAAATAGAGTTGATCGTCAAAACCAACATTGCGACCGACGGTGGCGAT AGGCATCCGGGTGGTGCTCAAAAGCAGCTTCGCCTGGCTGATACGTTGGTCCTCGCGCCAGCTTAAGACGC TAATCCCTAACTGCTGGCGGAAAAGATGTGACAGACGCGACGGCGACAAGCAAACATGCTGTGCGACGCTG GCGATATCAAAATTGCTGTCTGCCAGGTGATCGCTGATGTACTGACAAGCCTCGCGTACCCGATTATCCATC GGTGGATGGAGCGACTCGTTAATCGCTTCCATGCGCCGCAGTAACAATTGCTCAAGCAGATTTATCGCCAG CAGCTCCGAATAGCGCCCTTCCCCTTGCCCGGCGTTAATGATTTGCCCAAACAGGTCGCTGAAATGCGGCT GGTGCGCTTCATCCGGGCGAAAGAACCCCGTATTGGCAAATATTGACGGCCAGTTAAGCCATTCATGCCAG TAGGCGCGCGGACGAAAGTAAACCCACTGGTGATACCATTCGCGAGCCTCCGGATGACGACCGTAGTGAT GAATCTCTCCTGGCGGGAACAGCAAAATATCACCCGGTCGGCAAACAAATTCTCGTCCCTGATTTTTCACCA CCCCCTGACCGCGAATGGTGAGATTGAGAATATAACCTTTCATTCCCAGCGGTCGGTCGATAAAAAAATCGA GATAACCGTTGGCCTCAATCGGCGTTAAACCCGCCACCAGATGGGCATTAAACGAGTATCCCGGCAGCAGG GGATCATTTTGCGCTTCAGCCATACTTTTCATACTCCCGCCATTCAGAGAAGAAACCAATTGTCCATATTGCA TCAGACATTGCCGTCACTGCGTCTTTTACTGGCTCTTCTCGCTAACCAAACCGGTAACCCCGCTTATTAAAAG CATTCTGTAACAAAGCGGGACCAAAGCCATGACAAAAACGCGTAACAAAAGTGTCTATAATCACGGCAGAAA AGTCCACATTGATTATTTGCACGGCGTCACACTTTGCTATGCCATAGCATTTTTATCCATAAGATTAGCGGAT

(29)

CCTACCTGACGCTTTTTATCGCAACTCTCTACTGTTTCTCCATACCCGTTTTTTTGGGCTAGCCTAGAGAAAG AGGAGAAATACTAGATGCACCATCATCATCATCATTCTTCTGGTGTAGATCTGGGTACCGAGAACCTGTACTT CCAATCCGTGAGCAAGGGCGAGGAGGATAACATGGCCTCTCTCCCAGCGACACATGAGTTACACATCTTTG GCTCCATCAACGGTGTGGACTTTGACATGGTGGGTCAGGGCACCGGCAATCCAAATGATGGTTATGAGGAG TTAAACCTGAAGTCCACCAAGGGTGACCTCCAGTTCTCCCCCTGGATTCTGGTCCCTCATATCGGGTATGGC TTCCATCAGTACCTGCCCTACCCTGACGGGATGTCGCCTTTCCAGGCCGCCATGGTAGATGGCTCCGGATA CCAAGTCCATCGCACAATGCAGTTTGAAGATGGTGCCTCCCTTACTGTTAACTACCGCTACACCTACGAGGG AAGCCACATCAAAGGAGAGGCCCAGGTGAAGGGGACTGGTTTCCCTGCTGACGGTCCTGTGATGACCAACT CGCTGACCGCTGCGGACTGGTGCAGGTCGAAGAAGACTTACCCCAACGACAAAACCATCATCAGTACCTTT AAGTGGAGTTACACCACTGGAAATGGCAAGCGCTACCGGAGCACTGCGCGGACCACCTACACCTTTGCCAA GCCAATGGCGGCTAACTATCTGAAGAACCAGCCGATGTACGTGTTCCGTAAGACGGAGCTCAAGCACTCCA AGACCGAGCTCAACTTCAAGGAGTGGCAAAAGGCCTTTACCGATGTGATGGGCATGGACGAGCTGTACAAG GGCAGCGGTTCGGGCTCAGGAAGCGGTAGTGGCTCGGACGCTGAGTTCCGTCACGACTCTGGTTACGAAG TTCACCACCAGAAGCTGGTGTTCTTCGCTGAAGACGTGGGTTCTAACAAGGGTGCTATCATCGGTCTGATGG TTGGTGGCGTTGTGATCGCTTAATAGCAGTAAAGGTGGATACGGATCCGAA

Sequencing results

TetR-pTet-GroES

http://parts.igem.org/File:T--Linkoping_Sweden--GroES4A5.zip

References

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