Latest revision as of 02:29, 2 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.

**Figure 1** Overview of final vectors. First level: The two vectors, unlinearized. Second level: linearized by opening in USER casette. Third level: Insertion of YFP. 4: Produced YFP protein with and without CPP tag. YFP used as example; the same method is used for expression of BFP with and without CPP tag.

**Figure 2** Our initial vector design was built on pRSET, and would have had possibility for 3A assembly and immediate biobrick formation. We worked a lot on this initial vector, but did not succeed in creating it.

Expression of fluorescent proteins

We inserted BFP and YFP with USER cloning, and transformed them into our expression strain BL21 using Mix&Go. BFP and YFP was expressed with the CPP tag from the CPP vector, and without from the vector without CPP. Together with CPP alone, this gave us a total of 5 protein constructs expressed.

CPP-USER-his
CPP-YFP-his
CPP-BFP-his
YFP-his
BFP-his

The expression was verified using western blotting, and fluorescence of YFP and BFP both with and without CPP was verified with fluorescence microscopy.

Expression of fluorescent proteins

In order to evaluate the import of the CPP tagged and non-tagged proteins, we first purified the fluorescent proteins, then incubated E.coli cells with the purified proteins. Afterwards we removed the non-endocytosed fluorescent proteins by washing with water. (Fig 2)

Fluorescence microscopy was used to see if the fluorescent proteins had been imported into the cells. If the import system works with CPP tags, the fluorescent proteins should now be present in the cells treated with CPP-tagged fluorescent proteins, but not in the non-CPP tagged.

**Figure 3** Protein import with YFP A: with and B: without CPP tag - asme is done with BFP. 1) Proteins are expressed in *E.coli*, then 2) purified, before 3) import in other cells is investigated; both in *E.coli* and in eukaryotic cells. Non-endocytosed YFP/BFP is removed before looking at the fluorescence of the treated cells.

Design process/future

Summary: We have made vectors to express YFP and BFP, and checked that they are expressed and fluorescing both with and without the CPP tag. We investigated protein import into cells with fluorescence microscopy.

Vector Design: Our initial vector design was Biobrick compatible, with XXX and XXX as prefixes and suffixes. We designed a long primer to span entire sections and generate the whole vector, but this never succeeded. Instead, we decided to scale down on the design and use a simpler vector based on pET102, where we needed to make fewer modifications.

Import system: Initially, we looked into utilizing existing protein import systems found in endosymbionts: import systems from mitochondria, chloroplasts or even peroxisomes. Quickly, though, we realised that these systems are too complex to be achieved within an iGEM project. The mitochondrial translocation system contains at least 5 proteins in a complex just in the outer membrane (Truscott et al 2003), which is already very different from bacterial membranes. The same problem arises with regards to the chloroplast translocation system (Shi and Theg 2013), which also requires a large number of proteins to function.

After this initial set-back in the design proccess, we found an article on CPP’s: Cell Penetrating Peptides (Chang et al 2014), and decided to attempt to utilize these.

Exit from vesicles: If the protein is endocytosed, but not released from the vesicle it can not be utilized in the cell. If we were able to confirm uptake in vesicles, the next step would be to break the vesicles and release the proteins. For this reason, we looked into a way of breaking the vesicles by adding a small peptide from the influenza hemagglutinin HA2, consisting of the 23 N-terminal amino acids (Erazo-Oliveras, 2012). To attempt to break the vesicles, we would attach HA2 to the N-terminal end of the CPP tagged protein.

References

Chang, M., Chou, J., & Lee, H. (2005). Cellular Internalization of Fluorescent Proteins via Arginine-rich Intracellular Delivery Peptide in Plant Cells. Plant And Cell Physiology, 46(3), 482-488. http://dx.doi.org/10.1093/pcp/pci046

Chang, M., Huang, Y., Aronstam, R., & Lee, H. (2014). Cellular Delivery of Noncovalently-Associated Macromolecules by Cell- Penetrating Peptides. Current Pharmaceutical Biotechnology, 15(3), 267-275. http://dx.doi.org/10.2174/1389201015666140617095415

Erazo-Oliveras, A., Muthukrishnan, N., Baker, R., Wang, T., & Pellois, J. (2012). Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges. Pharmaceuticals, 5(12), 1177-1209. http://dx.doi.org/10.3390/ph5111177

Eudes, F., & Chugh, A. (2008). Cell-penetrating peptides. Plant Signaling & Behavior, 3(8), 549-550. http://dx.doi.org/10.4161/psb.3.8.5696 Schmidt, O., Pfanner, N., & Meisinger, C. (2010). Mitochondrial protein import: from proteomics to functional mechanisms. Nature Reviews Molecular Cell Biology, 11(9), 655-667. http://dx.doi.org/10.1038/nrm2959

Shi, L., & Theg, S. (2013). The chloroplast protein import system: From algae to trees. Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research, 1833(2), 314-331. http://dx.doi.org/10.1016/j.bbamcr.2012.10.002

Truscott, K., Brandner, K., & Pfanner, N. (2003). Mechanisms of Protein Import into Mitochondria. Current Biology, 13(8), R326-R337. http://dx.doi.org/10.1016/s0960-9822(03)00239-2

@@ Line 14: / Line 14: @@
                      <div class="intro-message2">
 <h3></h3>
-                         <h1>P R O T E I N  I M P O R T</h1>
+                         <h1>P R O T E I N &ensp;  I M P O R T</h1>
                      </div>
                  </div>
@@ Line 34: / Line 34: @@
 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.
 <br><br>
-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 <i>et al</i> 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 <i>et al</i> 2005, and Chang<i>et al</i> 2014).
+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 (<u><a href="#P5">Schmidt <i>et al</i> 2010</a></u>). 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) (<u><a href="#P1">Chang <i>et al</i> 2005</a></u>, and <u><a href="#P2">Chang<i>et al</i>, 2014</a></u>).
 </p>
@@ Line 47: / Line 47: @@
                      <h2 class="section-heading">Final Design </h2>
                                          <p class="lead">
-<strong>Background</strong>:  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 <i>et al</i> 2005).
+<strong>Background</strong>:  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 (<u><a href="#P4">Eudes and Chugh, 2008</a></u>). 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 (<u><a href="#P1">Chang <i>et al</i> 2005</a></u>).
 <br><br>
 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.
@@ Line 121: / Line 121: @@
 </figcaption>
 </figure>
-<br><br>
+<br>
-<h4>Vector design</h4>
-<p class="lead">
-A modified version of the <a href="https://www.addgene.org/vector-database/2623/">pET102 vector</a> was used for expression of all genes in this sub-project. See <a href="https://2017.igem.org/Team:UCopenhagen/Protein-Import">protein import</a> for details on design and creation of the vector.<br></p>
-<h4>Codon optimization and synthetic <i>yddG</i></h4>
-<p class="lead">
-Following problems with amplifying <i>yddG</i> from the genomic DNA, we decided to order a synthetically made version, and codon optimized it in the process.<br></p>
-<h4>Point mutations for feedback resistance in <i>trpE</i> and <i>aroG</i></h4>
-<p class="lead">
-We amplified the <i>aroG</i> and <i>trpE</i> genes using genomic DNA from <i>E.coli</i> 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. <br><br>
-The mutations were designed in accordance with previous work by <u><a href="#I2">Gu <i>et al.</i> (2012)</a></u>. In TrpE, position 293 is changed from methionine to threonine, and for AroG position 150 is changed from proline to leucine.
 <figure>
-<br><br>
-                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/5/5d/TrpE_pointmutation.png" alt="" width="250" height="200">
 <br>
-  <figcaption><b>Figure 2 </b>Point mutation in <i>trpE</i>, 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.
+                    <img class="img-responsive" src="https://static.igem.org/mediawiki/parts/d/d3/R9_insert.PNG" alt="" width="250" height="200">
-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.
+<br>
+  <figcaption><b>Figure 2 </b>Our initial vector design was built on pRSET, and would have had possibility for 3A assembly and immediate biobrick formation. We worked a lot on this initial vector, but did not succeed in creating it.
 </figcaption>
 </figure>
-</p>
-<h4>Multi-gene constructs</h4>
+<h4>Expression of fluorescent proteins</h4>
+<p class="lead">We inserted BFP and YFP with USER cloning, and transformed them into our expression strain BL21 using Mix&Go. BFP and YFP was expressed with the CPP tag from the CPP vector, and without from the vector without CPP. Together with CPP alone, this gave us a total of 5 protein constructs expressed. <br><br>
-<p class="lead">
-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.<br><br>
-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.
 <ul style="text-align:left; color:white;">
-<li>YddG-his</li>
+<li>CPP-USER-his</li>
-<li>TrpE-his</li>
+<li>CPP-YFP-his</li>
-<li>AroG-his</li>
+<li>CPP-BFP-his</li>
-<li>YddG-TrpE-his: <a href="http://parts.igem.org/Part:BBa_K2455007">BBa_K2455007</a></li>
+<li>YFP-his</li>
-<li>YddG-AroG-his: <a href="http://parts.igem.org/Part:BBa_K2455008">BBa_K2455008</a></li>
+<li>BFP-his
-<li>YddG-TrpE-AroG-his</li>
+</li>
 </ul>
 </p>
+<p class="lead">The expression was verified using western blotting, and fluorescence of YFP and BFP both with and without CPP was verified with fluorescence microscopy.</p>
-<p class="lead">
+<h4>Expression of fluorescent proteins</h4>
-<figure>
-<br><br>
-                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/f/ff/Construct_insertions2.png" alt="" width="250" height="200">
-<br>
- <figcaption><b>Figure 3 </b>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.
-</figcaption>
-</figure>
-</p>
-<h4>Serial growth experiment of <i>E.coli</i> and yeast</h4>
 <p class="lead">
-<i>E. coli</i> strains MG1655 and BL21 were grown in several media, until we settled on the minimal yeast media YNB with the pH adjusted to 7 (<u><a href="#I1">van Summeren-Wesenhagen and Marienhagen, 2014</a></u>). <br><br>
+In order to evaluate the import of the CPP tagged and non-tagged proteins, we first <a href="https://static.igem.org/mediawiki/2017/8/88/Protein_extractions_and_purification.pdf">purified</a> the fluorescent proteins, then incubated <i>E.coli</i> cells with the purified proteins. Afterwards we removed the non-endocytosed fluorescent proteins by washing with water. (Fig 2) <br><br>
+Fluorescence microscopy was used to see if the fluorescent proteins had been imported into the cells. If the import system works with CPP tags, the fluorescent proteins should now be present in the cells treated with CPP-tagged fluorescent proteins, but not in the non-CPP tagged. <br>
-To ensure that <i>E.coli</i> 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 <i>E.coli</i> 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>
-<br><br>
-                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/4/42/Serialgrowth1.png" alt="" width="250" height="200">
 <br>
-  <figcaption><b>Figure 4 </b>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.
+                    <img class="img-responsive" src="https://static.igem.org/mediawiki/2017/b/bf/CPP_import.png " alt="" width="250" height="200">
+<br>
+  <figcaption><b>Figure 3 </b>Protein import with YFP A: with and B: without CPP tag - asme is done with BFP. 1) Proteins are expressed in <i>E.coli</i>, then 2) purified, before 3) import in other cells is investigated; both in <i>E.coli</i> and in eukaryotic cells. Non-endocytosed YFP/BFP is removed before looking at the fluorescence of the treated cells.
 </figcaption>
 </figure>
-</p>
-<h4>Serial growth experiment with transformed <i>E.coli</i></h4>
-<p class="lead">
-To evaluate, if the complementation of the yeast amino acid auxotrophy by <i>E.coli</i> tryptophan production is successful, we made serial growth experiments with transformed <i>E. coli</i>. <br><br>
-We first growed the E.coli, transformed with the single, double and triple construct vectors described above in <a href="https://static.igem.org/mediawiki/2017/f/f6/Protocol18_UCopenhagen.pdf">YNB media</a> without a tryptophane source. Subsequently, we cleared the media and inoculated it with auxotroph yeast, then evaluated the growth using OD<sub>600</sub> measurements. <br><br>
-We also checked the growth of auxotrophic yeast in the media with and without added tryptophan, to ensure that any growth after <i>E.coli</i> growth was due to tryptophan being produced by <i>E.coli</i> and not due to the yeast overcoming the auxotrophy.
-All serial growth experiments used variations of the same <a href="https://static.igem.org/mediawiki/2017/0/0e/Protocol19_UCopenhagen.pdf">protocol</a>.
-</p>
-            <div class="col-lg-5 col-sm-6">
-                <hr class="section-heading-spacer">
-                <div class="clearfix"></div>
-<h4>HPLC measurements of tryptophan production</h4>
-<p class="lead">
-In addition to the qualitative assessment of tryptophan production by yeast growth, we made a quantitative evaluation of tryptophan production by our transformed <i>E.coli</i>. <br><br>
-We used <a href="https://static.igem.org/mediawiki/2017/b/b8/Protocol20_UCopenhagen.pdf">HPLC-MS </a> 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. <br><br>
-We measure production from the following transformations of E. coli.
-<ul style="text-align:left; color:white;">
-<li>empty vector</li>
-<li>YddG-his</li>
-<li>TrpE-his </li>
-<li>AroG-his</li>
-<li>YddG-TrpE-his</li>
-<li>YddG-AroG-his</li>
-<li>YddG-TrpE-AroG-his</li>
-<li>YddG-TrpE-AroG</li>
-</ul>
-</p>
-<p class="lead">Growth of these transformants are simultaneously monitored with OD<sub>600</sub> measurements. </p>
-</div>
-   <div class="col-lg-5 col-lg-offset-2 col-sm-6">
-<br><br>
-<br><br>
-<figure>
-                    <img class="img-responsive3" src="https://static.igem.org/mediawiki/2017/9/97/HPLC-MS-foto.jpg" alt="" width="250" height="200">
-<br>
- <figcaption><b>Figure 5 </b>Photo of the HPLC-MS set-up.
-</figcaption>
-</figure>
-</div>
 </div>
      </div>
@@ Line 248: / Line 167: @@
                      <h2 class="section-heading">Design process/future</h2>
 <p class="lead">
-<strong>Summary</strong>: We cloned <i>E.coli</i> 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 <i>E.coli</i> in the same media, and have attempted to grow it on the tryptophan produced by <i>E. coli</i> as the single tryptophan source.
+<strong>Summary</strong>: We have made vectors to express YFP and BFP, and checked that they are expressed and fluorescing both with and without the CPP tag. We investigated protein import into cells with fluorescence microscopy. <br><br>
-<br><br>
-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.
+<strong>Vector Design</strong>: Our initial vector design was Biobrick compatible, with XXX and XXX as prefixes and suffixes. We designed a long primer to span entire sections and generate the whole vector, but this never succeeded. Instead, we decided to scale down on the design and use a simpler vector based on pET102, where we needed to make fewer modifications. <br><br>
-<br><br>
-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. <br><br>
+<strong>Import system</strong>: Initially, we looked into utilizing existing protein import systems found in endosymbionts: import systems from mitochondria, chloroplasts or even peroxisomes. Quickly, though, we realised that these systems are too complex to be achieved within an iGEM project. The mitochondrial translocation system contains at least 5 proteins in a complex just in the outer membrane (<u><a href="#P7">Truscott <i>et al</i> 2003</a></u>), which is already very different from bacterial membranes. The same problem arises with regards to the chloroplast translocation system (<u><a href="#P6">Shi and Theg 2013</a></u>), which also requires a large number of proteins to function. <br><br>
-To perform our tryptophan overproduction, we chose genes that, when over-expressed would have the greatest impact, based on the article by <u><a href="#I2">Gu <i>et al.</i> (2012)</a></u>. Initially, we considered simply overexpressing the tryptophan operon, but quickly realised this would be highly downregulated due to negative feedback regulation.<br><br>
-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 <i>E.coli</i>.
+After this initial set-back in the design proccess, we found an article on CPP’s: Cell Penetrating Peptides (Chang <i>et al</i> 2014), and decided to attempt to utilize these.<br><br>
-</p>
+<strong>Exit from vesicles</strong>: If the protein is endocytosed, but not released from the vesicle it can not be utilized in the cell. If we were able to confirm uptake in vesicles, the next step would be to break the vesicles and release the proteins. For this reason, we looked into a way of breaking the vesicles by adding a small peptide from the influenza hemagglutinin HA2, consisting of the 23 N-terminal amino acids (<u><a href="#P3">Erazo-Oliveras, 2012</a></u>). To attempt to break the vesicles, we would attach HA2 to the N-terminal end of the CPP tagged protein.
                    </div>
                  </div>
@@ Line 268: / Line 189: @@
                      <h2 class="section-heading">References</h2>
                      <p class="lead">
-<a name="I1">Summeren-Wesenhagen, P. V., & Marienhagen, J. (2014). Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin. <i>Applied and Environmental Microbiology, 81</i>(3), 840-849. doi:10.1128/aem.02966-14</a>
-<br><br>
+<a name="P1">Chang, M., Chou, J., & Lee, H. (2005). Cellular Internalization of Fluorescent Proteins via Arginine-rich Intracellular Delivery Peptide in Plant Cells. <i>Plant And Cell Physiology, 46</i>(3), 482-488. http://dx.doi.org/10.1093/pcp/pci046</a> <br><br>
-<a name="I2">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. <i>Microbial Cell Factories, 11</i>(1), 30. doi:10.1186/1475-2859-11-30</a>
-<br><br>
+<a name="P2">Chang, M., Huang, Y., Aronstam, R., & Lee, H. (2014). Cellular Delivery of Noncovalently-Associated Macromolecules by Cell- Penetrating Peptides. Current Pharmaceutical Biotechnology, 15(3), 267-275. http://dx.doi.org/10.2174/1389201015666140617095415</a><br><br>
-<a name="I3">Wang, J., Cheng, L., Wang, J., Liu, Q., Shen, T., & Chen, N. (2013). Genetic engineering of Escherichia coli to enhance production of l-tryptophan. <i>Applied Microbiology and Biotechnology, 97</i>(17), 7587-7596. doi:10.1007/s00253-013-5026-3</a>
-<br><br>
+<a name="P3">Erazo-Oliveras, A., Muthukrishnan, N., Baker, R., Wang, T., & Pellois, J. (2012). Improving the Endosomal Escape of Cell-Penetrating Peptides and Their Cargos: Strategies and Challenges. Pharmaceuticals, 5(12), 1177-1209. http://dx.doi.org/10.3390/ph5111177</a><br><br>
-<a name="I4">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</a>
-<br><br>
+<a name="P4">Eudes, F., & Chugh, A. (2008). Cell-penetrating peptides. Plant Signaling & Behavior, 3(8), 549-550. http://dx.doi.org/10.4161/psb.3.8.5696
-<a name="I5">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. <i>Applied and Environmental Microbiology, 66</i>(10), 4503-4509. doi:10.1128/aem.66.10.4503-4509.2000</a>
+Schmidt, O., Pfanner, N., & Meisinger, C. (2010). Mitochondrial protein import: from proteomics to functional mechanisms. Nature Reviews Molecular Cell Biology, 11(9), 655-667. http://dx.doi.org/10.1038/nrm2959</a><br><br>
-<br><br>
-<strong>Reference for USER cloning</strong>:
+<a name="P5">Shi, L., & Theg, S. (2013). The chloroplast protein import system: From algae to trees. Biochimica Et Biophysica Acta (BBA) - Molecular Cell Research, 1833(2), 314-331. http://dx.doi.org/10.1016/j.bbamcr.2012.10.002</a><br><br>
-<br>
-<a name="I6">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. <i>Nucleic Acids Research, 34</i>(18). doi:10.1093/nar/gkl635</a>
+<a name="P6">Truscott, K., Brandner, K., & Pfanner, N. (2003). Mechanisms of Protein Import into Mitochondria. Current Biology, 13(8), R326-R337. http://dx.doi.org/10.1016/s0960-9822(03)00239-2</a><br><br>
-<br><br>
-<strong>Reference for USER fusion</strong>:
-<br>
-<a name="I7">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. <i>Nucleic Acids Research, 35</i>(7). doi:10.1093/nar/gkm106</a>
 </p>

Difference between revisions of "Team:UCopenhagen/Protein-Import"

Latest revision as of 02:29, 2 November 2017

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

Introduction

Final Design

Experiments

Overview

Verifications and biobrick creation

Vector creation

Expression of fluorescent proteins

Expression of fluorescent proteins

Design process/future

References

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