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Revision as of 18:25, 1 November 2017

P R O T E I N I M P O R T

Introduction

The third mechanism that we have investigated in our project is protein import into bacterial cells, as a stand in for a symbiont. In the two best known endosymbiotic organelles, mitochondria and chloroplasts, a majority of gene expression has moved from the symbiont to the host. Because this relationship seems to be an evolutionary foundation of the known endosymbiotic relationships, we will attempt to imitate this concept.

Besides our interest in the evolutionary aspect of protein import, we believe it is valuable when approaching modular endosymbiosis. The principle of protein import would enable additional modularity in an endosymbiotic system, as one host can be manipulated to produce proteins to be utilized by a range of symbionts.

The majority of the proteins, destined for mitochondria, are expressed in the cytosol and subsequently imported across the membranes via transport complexes taking up unfolded peptides with an N-terminal signal sequence targeting them to the mitochondria (Schmidt et al 2010). Similar transport complexes are found in chloroplast. We want a similar targeted uptake, but in a simpler system. Thus, we utilise a small peptide shown to penetrate many types of cells; a Cell Penetrating Peptide (CPP) (Chang et al 2005, and Changet al 2014).

Final Design

Background: CPPs are small peptides, typically rich in arginines, which are able to facilitate transport of a wide variety of cargos across plasma membranes. Their origin in nature comes from viral domains such as the viral HIV tat domain (Eudes and Chugh, 2008). In recent years, research on creating synthetics CPPs has been conducted and especially peptides constructed solely from arginine residues have been of interest. The arginine rich sequence has been shown to trigger endocytosis in a wide range of cell types, including onion and potato cells. These experiments have shown that GFP connected to a CPP has entered the cells contained in vesicles (Chang et al 2005).

If CPP can be used as a protein tag for import into an endosymbiotic symbiont, the host proteins targeted to the symbiont would simply need the CPP added.

Goal: Evaluate the efficiency of protein uptake by our Escherichia coli chassis in presence and absence of the cell penetrating peptide (CPP).

Circuits and Biobricks: The parts in our circuit are fluorescent proteins and CPP.

We have chosen the yellow and blue fluorescent proteins (YFP and BFP) from the Biobrick repository, and improved them by adding the CPP sequence to the C-terminal end.

YFP: BBa_K2455002

BFP: BBa_K2455005

Additionally, we created a biobrick of CPP with a USER casette, ready for insertion of any protein to be imported: BBa_K2455003.

YFP and BFP were chosen to avoid overlapping colours with FM4-64, a red staining used for membranes, in case we wanted to look into the localization and potential vesicle breaking using confocal microscopy. As it turned out, we did not get far enough in the lab to do this.

Experiments

Overview

General verification


Vector creation

  • Biobrick compatible (failed)
  • Vector design
  • CPP tag insertion


Evaluating protein import:

  • Expression of fluorescent proteins
  • Purification and import of the expressed proteins

Verifications and biobrick creation

In all three sub projects, we have used gel electrophoresis and sequencing to verify our stepwise experiments. Read more about our general verifications and biobrick creation under interdependency.

Vector creation

Our vector is a modified version of the pET102 vector, which contains a USER cassette, and a his tag after the USER cassette. The USER cassette is used for cloning genes into. We build the vector design on prSET102; an existing vector in our lab, containing the USER cassette and some restriction sites.

We have made two versions of the vector: one for expression of untagged YFP and BFP, that was also used in the interdependency subproject. The other has a CPP tag before the USER cassette, which will add CPP to the proteins.

Vector design

A modified version of the pET102 vector was used for expression of all genes in this sub-project. See protein import for details on design and creation of the vector.

Codon optimization and synthetic yddG

Following problems with amplifying yddG from the genomic DNA, we decided to order a synthetically made version, and codon optimized it in the process.

Point mutations for feedback resistance in trpE and aroG

We amplified the aroG and trpE genes using genomic DNA from E.coli strain MG1655. The amplification was done using two sets of primers per gene, with overhangs allowing for insertion of a point mutation in each gene and for cloning with USER assembly.

The mutations were designed in accordance with previous work by Gu et al. (2012). In TrpE, position 293 is changed from methionine to threonine, and for AroG position 150 is changed from proline to leucine.




Figure 2 Point mutation in trpE, and insertion in vector with USER assembly. To perform the point mutation, we designed two sets of primers for each gene. The outer primers are the same as used for amplification from the genomic DNA. The central primers have overhangs containing the point mutations. This way, the genes are split in two, and the two parts are combined when inserting in the USER cassette in the expression vector - and now containing a point mutation in the middle.

Multi-gene constructs

To evaluate the effect of the proteins, we made multi-gene constructs. The constructs were made by designing primers with overhangs encoding Ribosome Binding Sites (RBS). RBS makes sure all genes will be transcribed, and the overhangs enable combination of the genes into multi-gene constructs (fig 3) and USER assembly.

We created biobricks of one or two genes in combination. We attempted to make all the following constructs into biobricks, but did not succeed with the 3 gene construct.




Figure 3 Multi-gene constructs were created with a series of primers with overhangs coding for Ribosome Binding Sites (RBS) between the genes. The outer primers coded for USER overhangs for USER ligation.

Serial growth experiment of E.coli and yeast

E. coli strains MG1655 and BL21 were grown in several media, until we settled on the minimal yeast media YNB with the pH adjusted to 7 (van Summeren-Wesenhagen and Marienhagen, 2014).

To ensure that E.coli do not produce substances hindering yeast growth, we performed an initial serial growth experiment, shown in figure 4. The serial growth experiment was performed with ON growth of E.coli in YNB pH 7, clearing the media of E.coli by spinning and filtration through 0.2 µm filters, and inoculating with yeast (AM94).




Figure 4 Serial growth experiment. 1) E.coli from liquid culture in LB media is inoculated in YNB media. 2) After sufficient growth of E.coli in YNB, the media is cleared of E.coli by spinning and filtration. 3) E.coli free media is then inoculated with tryptophan auxotroph yeast.

Serial growth experiment with transformed E.coli

To evaluate, if the complementation of the yeast amino acid auxotrophy by E.coli tryptophan production is successful, we made serial growth experiments with transformed E. coli.

We first growed the E.coli, transformed with the single, double and triple construct vectors described above in YNB media without a tryptophane source. Subsequently, we cleared the media and inoculated it with auxotroph yeast, then evaluated the growth using OD600 measurements.

We also checked the growth of auxotrophic yeast in the media with and without added tryptophan, to ensure that any growth after E.coli growth was due to tryptophan being produced by E.coli and not due to the yeast overcoming the auxotrophy. All serial growth experiments used variations of the same protocol.

HPLC measurements of tryptophan production

In addition to the qualitative assessment of tryptophan production by yeast growth, we made a quantitative evaluation of tryptophan production by our transformed E.coli.

We used HPLC-MS with a program for amino acid detection to measure the concentration of tryptophan in the media after 10-40 hours growth. Internal and external standards were used. Simultaneously, we measured concentrations of tyrosine and phenylalanine, which should be increased when AroG is expressed.

We measure production from the following transformations of E. coli.

  • empty vector
  • YddG-his
  • TrpE-his
  • AroG-his
  • YddG-TrpE-his
  • YddG-AroG-his
  • YddG-TrpE-AroG-his
  • YddG-TrpE-AroG

Growth of these transformants are simultaneously monitored with OD600 measurements.






Figure 5 Photo of the HPLC-MS set-up.

Design process/future

Summary: We cloned E.coli strains to express proteins in the tryptophan synthesis pathway, and have measured the production of these cells. We have ensured that auxotroph yeast can grow after E.coli in the same media, and have attempted to grow it on the tryptophan produced by E. coli as the single tryptophan source.

In our design process, we have considered a wide range of possible gene combinations and interdependency mechanisms. We settled on a simple amino acid interdependency which is easy to test with an amino acid auxotroph yeast.

In a final version of an endosymbiotic relationship, amino acid dependency might not be the optimal dependency as it would not allow the system to be grown on a media with all amino acids that might be required for co-cultures for added modularity. Instead of tryptophan, a different, but vital metabolite could be supplied by the symbiont.

To perform our tryptophan overproduction, we chose genes that, when over-expressed would have the greatest impact, based on the article by Gu et al. (2012). Initially, we considered simply overexpressing the tryptophan operon, but quickly realised this would be highly downregulated due to negative feedback regulation.

To release the feedback regulation, we decided on an amino acid translocator to reduce the intracellular tryptophan concentration. Several deletions of e.g. endogenous trpR has also been considered, but this would make our project overly complicated due to the difficulty of making such deletions in E.coli.

References

Summeren-Wesenhagen, P. V., & Marienhagen, J. (2014). Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin. Applied and Environmental Microbiology, 81(3), 840-849. doi:10.1128/aem.02966-14

Gu, P., Yang, F., Kang, J., Wang, Q., & Qi, Q. (2012). One-step of tryptophan attenuator inactivation and promoter swapping to improve the production of L-tryptophan in Escherichia coli. Microbial Cell Factories, 11(1), 30. doi:10.1186/1475-2859-11-30

Wang, J., Cheng, L., Wang, J., Liu, Q., Shen, T., & Chen, N. (2013). Genetic engineering of Escherichia coli to enhance production of l-tryptophan. Applied Microbiology and Biotechnology, 97(17), 7587-7596. doi:10.1007/s00253-013-5026-3

Sigma-Aldrich. (2017). Yeast Synthetic Drop-out Medium Supplements Y1501. (n.d.). Retrieved October 31, 2017, from http://www.sigmaaldrich.com/catalog/product/sigma/y1501?lang=en&region=DK

Bianciotto, V., Lumini, E., Lanfranco, L., Minerdi, D., Bonfante, P., & Perotto, S. (2000). Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae. Applied and Environmental Microbiology, 66(10), 4503-4509. doi:10.1128/aem.66.10.4503-4509.2000

Reference for USER cloning:
Nour-Eldin, H. H., Hansen, B. G., Nørholm, M. H., Jensen, J. K., & Halkier, B. A. (2006). Advancing uracil-excision based cloning towards an ideal technique for cloning PCR fragments. Nucleic Acids Research, 34(18). doi:10.1093/nar/gkl635

Reference for USER fusion:
Geu-Flores, F., Nour-Eldin, H. H., Nielsen, M. T., & Halkier, B. A. (2007). USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Research, 35(7). doi:10.1093/nar/gkm106

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