Difference between revisions of "Team:UCLA/Notebook/Honeybee Silk"

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<html>
 
  <h2><b>Honeybee Silk Notebook</b></h2>
 
  <details>
 
      <summary>May 10 - May 16</summary>
 
      <details>
 
        <summary>May 11 Sequencing of silk biobricks</summary>
 
        <p>Today we sent in both our constructs to GeneWiz for sequencing. </p>
 
        <ul>
 
            <li> For sequencing primers, I used the igem vf2 primers and vr primers that bind to the psb1c3 backbone, as well as a sequencing primer that we designed to bind to the silk sequence (p13) see https://benchling.com/s/p7bClpzQ/edit  </li>
 
            <li>The sequencing results came back and checked out for sample 1 of construct 1 (silk coding region) and samples 1 and 2 for construc 2 (regulatory elements + silk) <b> i.e. we have our first honeybee biobricks!!! </b>  </li>
 
            <li> For sequencing results of construct 1 see https://benchling.com/s/4XT6VRRU/edit (click on alignments on the right side of screen)  </li>
 
            <li> For sequencing results of construct 2 see https://benchling.com/s/TxD8z7Kq/edit (click on alignments on the right side of screen)  </li>
 
        </ul>
 
      </details>
 
  </details>
 
 
  <details>
 
      <summary>May 3 - May 9 (Cloning Honeybee Biocricks week 2)</summary>
 
    <details>
 
      <summary>May 4</summary>
 
 
 
</details>
 
<details>
 
      <summary>May 5</summary>
 
 
 
</details>
 
<details>
 
      <summary>May 6</summary>
 
 
 
</details>
 
</details>
 
 
  <details>
 
      <summary>April 26 - May 2 (Cloning honeybee biobricks week 1)</summary>
 
      <details>
 
        <summary>4/26 PCR off honey bee gene block</summary>
 
        <ul>
 
            <li>See Benchling for the g block sequence and primer reference code https://benchling.com/s/p7bClpzQ/edit</li>
 
            <li>The goal of this is to clone 2  different constructs and prepare them as biobricks, one that is just the silk gene, and the other that as the promoter, rbs, etc.. required for protein expression.</li>
 
            <li>Using primers p3, p7, and p8. (See Benchling link)</li>
 
            <li>
 
            <p>PCR Reaction 1 (q5 polymerase kit): primers honeybee p#7 and honeybee p#8 with gBlock, and honeybee p#3 and honeybee p#8 amplify just the honeybee coding region and the prom + coding region respectively</p>
 
              <table style="width: 100%">
 
                  <tr>
 
                    <td><b>Component</b></td>
 
                    <td><b>Volume</b></td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>5X Q5 Reaction Buffer</b></td>
 
                    <td>5</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>10 mM dNTPs</b></td>
 
                    <td>0.5</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        10 uM Forward (primer 3/7)
 
                        </b>
 
                    </td>
 
                    <td>1.25</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        10 uM Reverse (primer 8)
 
                        </b>
 
                    </td>
 
                    <td>1.25</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>Template (diluted to 1ng/uL)</b></td>
 
                    <td>0.5</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        Q5 High Fidelity DNA Polymerase
 
                        </b>
 
                    </td>
 
                    <td>0.25</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>Nuclease Free Water</b></td>
 
                    <td>16.25</td>
 
                  </tr>
 
              </table>
 
            </li>
 
            <li>
 
              PCR program (using benchling annealing temperatures)
 
              <table style="width: 100%">
 
                  <tr>
 
                    <td><b>Temperature (C)</b></td>
 
                    <td><b>Time (Min:sec)</b></td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>98</b></td>
 
                    <td>0:30</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>98 x 30 cycles</b></td>
 
                    <td>0:10</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        grad.
 
                        55.4/58.4/61.4 C x 30
 
                        </b>
 
                    </td>
 
                    <td>0:20</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        72 x 30
 
                        </b>
 
                    </td>
 
                    <td>0:30</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>72(diluted to 1ng/uL)</b></td>
 
                    <td>2:00</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>12</b></td>
 
                    <td>hold</td>
 
                  </tr>
 
              </table>
 
            </li>
 
            </li>
 
            <p>The program and pcr reaction recipe were based on the manufacturer protocol. These annealing temperatures were determined using benchling's algorithm. In the future we will use the NEB annealing temperature calculator, because it is more accurate and takes the type of polymerase into account <b>See tomorrow's entry for the gel image of the pcr reaction</b></p>
 
        </ul>
 
      </details>
 
      <details>
 
        <summary>4/27 visualization and purification of honeybee gblock PCR</summary>
 
        <p>We visualized the gradient pcr from 4/26 on a 1% agarose gel. See image below:</p>
 
        <img width="300px" src="https://static.igem.org/mediawiki/2015/6/6e/4.27_honeybee_gel_image_UCLA.PNG"/>
 
        <ul>
 
            <li> The PCR for both constructs (silk coding region) and (regulatory elements + silk coding region) worked relatively well across all gradient temperatures.  </li>
 
            <li> We next scaled up the PCR reaction to 50 ul for each construct and gel extracted the product </li>
 
            <li>
 
From left to right, just silk w/ bb prefix and suffix w/ increasing annealing temp. , ladder, followed by silk+ promoter at increasing temp (see 4/26 for pcr conditions)
 
</li>
 
       
 
</ul>
 
      </details>
 
      <details>
 
        <summary> 4/28 PCR to prepare 2 honeybee constructs for cloning</summary>
 
We are repeating the same PCR from 4/26, but only at one temperature instead of a gradient, and we are scaling the reaction up to 50 ul.
 
<li> PCR recipe
 
  <table style="width: 100%">
 
                  <tr>
 
                    <td><b>Component</b></td>
 
                    <td><b>Volume</b></td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>5X Q5 Reaction Buffer</b></td>
 
                    <td>10</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>10 mM dNTPs</b></td>
 
                    <td>1.0</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        10 uM Forward (primer 3/7)
 
                        </b>
 
                    </td>
 
                    <td>2.5</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        10 uM Reverse (primer 8)
 
                        </b>
 
                    </td>
 
                    <td>2.5</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>Template (diluted to 1ng/uL)</b></td>
 
                    <td>1.0</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        Q5 High Fidelity DNA Polymerase
 
                        </b>
 
                    </td>
 
                    <td>0.50</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>Nuclease Free Water</b></td>
 
                    <td>32.5</td>
 
                  </tr>
 
              </table>
 
            </li>
 
            <li>
 
              PCR program (using benchling annealing temperatures)
 
              <table style="width: 100%">
 
                  <tr>
 
                    <td><b>Temperature (C)</b></td>
 
                    <td><b>Time (Min:sec)</b></td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>98</b></td>
 
                    <td>0:30</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>98 x 30 cycles</b></td>
 
                    <td>0:10</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        grad.
 
                        62 (silk)  58.4 (silk+prom) x 30
 
                        </b>
 
                    </td>
 
                    <td>0:20</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        72 x 30
 
                        </b>
 
                    </td>
 
                    <td>0:30</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>72(diluted to 1ng/uL)</b></td>
 
                    <td>2:00</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>10</b></td>
 
                    <td>hold</td>
 
                  </tr>
 
              </table>
 
            </li>
 
<li>
 
PCR products run on 1% agarose gel.</li>
 
<li>There were some non specific bands, but the appropriate bands were present. However, the bands were kind of off.  I think this is due to the samples running weird on the gel.  B/c the first two lanes were the exact same sample, just aliquoted, and they ran fairly differently. 
 
</li>
 
<li>
 
Performed a gel extraction and purified using Qiagen min elute gel extraction kit
 
</li>
 
<li>
 
Results : 115 ng/ ul for silk tube #1, 86 ng/ul tube #2  (8.8 ul each tube)
 
136 ng/ul promoter + silk
 
 
</li>
 
</ul>
 
 
      </details>
 
<details>
 
        <summary> 4/30 Double digest of Honeybee PCR product and ligation into PSB1C3 plasmid backbone</summary>
 
Vinson and Olivia already prepared ecor1 and pst1 digested psb1c3, (35 ng/ul in honeybee box) which I will be using for ligation. It is column purified as well.
 
I am double digesting both the plain silk construct, as well as the promoter+silk construct with ecoR1 and pst1.
 
<li>
 
Digestion Rxn (50 ul)
 
One for silk and One for prom+silk
 
<table style="width: 100%">
 
                  <tr>
 
                    <td><b>EcoR1 (C)</b></td>
 
                    <td>1 ul</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>Pst1</b></td>
 
                    <td>1 ul</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
NEB buffer 2.1 (10x)
 
</b></td>
 
                    <td>5 ul</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                 
 
Silk DNA / PRom+Silk DNA
 
</b>
 
                    </td>
 
                    <td>
 
8.7 ul / 7.2 ul
 
</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        H20
 
                        </b>
 
                    </td>
 
                    <td>
 
34.2 / 35.8 ul
 
</td>
 
                  </tr>
 
</table>
 
 
</li>
 
<li>
 
Digestion incubation specifications: 37 degrees for 1 hour, and 80 degree heat inactivation for 15 minutes, hold 10C
 
 
</li>
 
<li>
 
<b>Ligation Reaction into PSB1c3 vector</b>
 
 
</li>
 
<li>
 
20 ul ligation reaction according to protocol using T4 ligase
 
 
</li>
 
<li>
 
2 reactions, (1 for silk, 1 for promoter + silk)
 
Both are at a 3:1 molar insert:vector ratio.
 
<table style="width: 100%">
 
                  <tr>
 
                    <td><b>
 
10X T4 Ligase Buffer
 
</b></td>
 
                    <td>1 2 ul</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
PSB1C3 (50 ng)
 
</b></td>
 
                    <td>1.4 ul</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
 
silk construct / prom + silk construct
 
</b></td>
 
                    <td>
 
1.4 ul / 1.06 ul
 
</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                 
 
 
T4 Ligase
 
</b>
 
                    </td>
 
                    <td>
 
1 ul</td>
 
                  </tr>
 
                  <tr>
 
                    <td><b>
 
                        H20
 
                        </b>
 
                    </td>
 
                    <td>
 
 
14.2 / 14.54
 
</td>
 
                  </tr>
 
</table>
 
 
</li>
 
Reaction conditions: 16 C for 12 hours, 65 C for 15 min, hold at 10 C
 
 
 
</details>
 
 
  </details>
 
 
</html>
 
  
 +
=HONEYBEE SILK PROJECT=
  
  
Line 368: Line 11:
  
 
==<u>Achievements</u>==
 
==<u>Achievements</u>==
As of 5/16 we are well on our way to establishing a strong foundation for this project.  We have cloned some honeybee sequences into biobrick approved format, and are on our way to expressing our first batch of honeybee silk protein. (For detailed achievements see below)
+
As of 9/18 we are well on our way to establishing a strong foundation for this project.  We have cloned five honey bee sequences into biobrick approved format, have expressed and purified honey bee silk protein. (For detailed achievements see below)
 
===Cloning===
 
===Cloning===
We have successfully cloned and sequence verified '''two''' honeybee silk constructs into the igem psb1c3 plasmid backbone, in the correct biobrick format. A comprehensive list of our honeybee parts can be found [https://2015.igem.org/Team:UCLA/Parts here]
+
We have successfully cloned and sequence verified '''five''' honeybee silk constructs into the igem psb1c3 plasmid backbone, in the correct biobrick format. A comprehensive list of our biobrick parts can be found [https://2015.igem.org/Team:UCLA/Parts here]
 
*The first sequence is just the honeybee silk protein #3 in the psb1c3 backbone.  The annotated sequence, along with further details on how we designed and cloned this biobrick can be found here. [http://parts.igem.org/Part:BBa_K1763000 BBa_K1763000]  
 
*The first sequence is just the honeybee silk protein #3 in the psb1c3 backbone.  The annotated sequence, along with further details on how we designed and cloned this biobrick can be found here. [http://parts.igem.org/Part:BBa_K1763000 BBa_K1763000]  
*The second sequence is the same silk coding sequence, but with regulatory elements upstream for protein expression, including [http://parts.igem.org/Part:BBa_R0010 BBa_R0010 Promoter] and [http://parts.igem.org/Part:BBa_B0034 RBS BBa_B0034 ].  The sequence can be found here. [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001  ] along with Further details on the design and cloning of this biobrick.
+
*The second sequence is the same silk coding sequence, but with regulatory elements upstream for protein expression, including [http://parts.igem.org/Part:BBa_R0010 BBa_R0010 Promoter] and [http://parts.igem.org/Part:BBa_B0034 RBS BBa_B0034 ].  The sequence can be found here. [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001  ] along with further details on the design and cloning of this biobrick.
 +
*The third sequence is a fusion protein between our honeybee silk and the Spycatcher, protein which has specific affinity for SpyCatcher. It also contains regulatory elements like a promoter and rbs for expression. You can view the sequence [http://parts.igem.org/Part:BBa_K1763008 BBa_K1763008 here .] 
 +
*The fourth sequence [http://parts.igem.org/Part:BBa_K1763007 BBa_K1763007 ] is our honeybee silk sequence with a T7 promoter upstream for effective protein expression in bacterial strains containing t7 polymerase.
 +
*The fifth sequence, [http://parts.igem.org/Part:BBa_K1763015 BBa_K1763015] is our honeybee silk sequence+sfGFP with a T7 promoter and RBS upstream that can be used in bacterial strains that contain T7 polymerase.
  
 
===Protein Expression===
 
===Protein Expression===
We have just finished our first round of honey bee silk expression from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001]
+
*We have just finished our first round of honey bee silk expression from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001]
 +
*[https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/26_May_2015 Here] is our first attempt at protein expression along with the gel.
 +
[[File:UCLAHoneybee.jpg|none|thumb|300px|'''Fig. 1''' Expected size of product is 40.0 kDA]]
 +
 
 +
*Unfortunately the purified protein that we see on the gel (should be in next to last lane on the right) doesn't seem to be the right size.
 +
**We will try again using a T7 promoter and a slightly modified protein expression and purification protocol.
 +
*Here is our most recent attempt at expressing honeybee silk protein and the SDS PAGE results. We see a band at the expected size of 40 kDA!
 +
[[File:UCLA honeybee Growth optimization 37C.jpg|none|thumb|300px|]]
  
 
==<u>What we are working on now</u>==
 
==<u>What we are working on now</u>==
We are currently at the starting phases of honeybee silk protein expression. We have expressed [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001  ].  Next up, we will run our purified protein on an SDS PAGE gel to ascertain the purity of our purified silk protein. For our progress so far, see our lab notebook entries from [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/18_May_2015 5/18],[https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/19_May_2015 5/19], and [https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/26_May_2015 5/26]
+
*We are currently optimizing our honeybee silk expression protocol. We are getting a band at the right size (40 kDa) on our SDS PAGE gels, but there is a fair amount of contamination. See example below (The purified product is in lane 7).
 +
[[File:Honeybee BL21 honeybee SDS PAGE 8.7.15.JPG|none|thumb|300px|'''Fig. 1''' Expected size of product is 40 kDa (lane 6 and 7)). Lane 8 is BSA protein positive control, and lane 1 is Biorad dual color ladder. From lane 1-8 S1,F2,F3,S2,Final product 8/7, Final Product 7/29,BSA positive control, (see table)]]
 +
*We are optimizing out cell lysis and purification protocol in the hopes of getting higher yield and a more pure protein product.
 +
*In terms of producing fibers, we have "purified" honeybee product dissolved in SDS ready to go. We are waiting on the protein concentrators to arrive so that we can have a concentrated enough dope for spinning into fibers.
 +
** We hope that the protein concentration will also help to get rid of some of the contaminating proteins that are under the 30 kDa cutoff.
  
 
==<u>Raw lab notebook entries</u>==
 
==<u>Raw lab notebook entries</u>==
 +
*April 28 - May 6 = cloning of [http://parts.igem.org/Part:BBa_K1763000 BBa_K1763000] and [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001 ]
 +
*May 16-May 19 = Expressing Silk Protein in E. Coli.
 +
*May 26-27 = Purifying Silk Protein and 1st SDS PAGE gel
 +
*July 10-16 = Cloning Silk into Pet vector and preparing BL21 (DE3) competent cells
 +
*July 17-23= Cloning Silk into BL21 (de3) cells.
 +
*July 28-August 3rd = 1st expression in Bl21 (de3) and SDS PAGE results.
 +
*August 7 = 2nd purfication and SDS of Honeybee silk in Bl21 cells along with SDS PAGE results.
 +
*August 11 = BCA protein concentration assay on product from 8/7
 +
*August 12 = Optimization of growth conditions for Honeybee expression and SDS PAGE results.
 +
*Sept 4,7 = SDS PAGE gels for different lysis protocols
 
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Latest revision as of 18:32, 18 September 2015


iGEM UCLA





HONEYBEE SILK PROJECT

Goals

The goal of this project is to recombinantly express honeybee silk proteins, with the intention of creating synthetic honeybee silk fibers. We hope to develop protocols to efficiently produce the raw protein, process the protein into various materials, and characterize these materials. Furthermore, by conjugating the honeybee protein with other proteins, we aim to create honeybee silk materials with a wide array of properties and functionalities.

Achievements

As of 9/18 we are well on our way to establishing a strong foundation for this project. We have cloned five honey bee sequences into biobrick approved format, have expressed and purified honey bee silk protein. (For detailed achievements see below)

Cloning

We have successfully cloned and sequence verified five honeybee silk constructs into the igem psb1c3 plasmid backbone, in the correct biobrick format. A comprehensive list of our biobrick parts can be found here

  • The first sequence is just the honeybee silk protein #3 in the psb1c3 backbone. The annotated sequence, along with further details on how we designed and cloned this biobrick can be found here. [http://parts.igem.org/Part:BBa_K1763000 BBa_K1763000]
  • The second sequence is the same silk coding sequence, but with regulatory elements upstream for protein expression, including [http://parts.igem.org/Part:BBa_R0010 BBa_R0010 Promoter] and [http://parts.igem.org/Part:BBa_B0034 RBS BBa_B0034 ]. The sequence can be found here. [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001 ] along with further details on the design and cloning of this biobrick.
  • The third sequence is a fusion protein between our honeybee silk and the Spycatcher, protein which has specific affinity for SpyCatcher. It also contains regulatory elements like a promoter and rbs for expression. You can view the sequence [http://parts.igem.org/Part:BBa_K1763008 BBa_K1763008 here .]
  • The fourth sequence [http://parts.igem.org/Part:BBa_K1763007 BBa_K1763007 ] is our honeybee silk sequence with a T7 promoter upstream for effective protein expression in bacterial strains containing t7 polymerase.
  • The fifth sequence, [http://parts.igem.org/Part:BBa_K1763015 BBa_K1763015] is our honeybee silk sequence+sfGFP with a T7 promoter and RBS upstream that can be used in bacterial strains that contain T7 polymerase.

Protein Expression

  • We have just finished our first round of honey bee silk expression from [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001]
  • Here is our first attempt at protein expression along with the gel.
Fig. 1 Expected size of product is 40.0 kDA
  • Unfortunately the purified protein that we see on the gel (should be in next to last lane on the right) doesn't seem to be the right size.
    • We will try again using a T7 promoter and a slightly modified protein expression and purification protocol.
  • Here is our most recent attempt at expressing honeybee silk protein and the SDS PAGE results. We see a band at the expected size of 40 kDA!
UCLA honeybee Growth optimization 37C.jpg

What we are working on now

  • We are currently optimizing our honeybee silk expression protocol. We are getting a band at the right size (40 kDa) on our SDS PAGE gels, but there is a fair amount of contamination. See example below (The purified product is in lane 7).
Fig. 1 Expected size of product is 40 kDa (lane 6 and 7)). Lane 8 is BSA protein positive control, and lane 1 is Biorad dual color ladder. From lane 1-8 S1,F2,F3,S2,Final product 8/7, Final Product 7/29,BSA positive control, (see table)
  • We are optimizing out cell lysis and purification protocol in the hopes of getting higher yield and a more pure protein product.
  • In terms of producing fibers, we have "purified" honeybee product dissolved in SDS ready to go. We are waiting on the protein concentrators to arrive so that we can have a concentrated enough dope for spinning into fibers.
    • We hope that the protein concentration will also help to get rid of some of the contaminating proteins that are under the 30 kDa cutoff.

Raw lab notebook entries

  • April 28 - May 6 = cloning of [http://parts.igem.org/Part:BBa_K1763000 BBa_K1763000] and [http://parts.igem.org/Part:BBa_K1763001 BBa_K1763001 ]
  • May 16-May 19 = Expressing Silk Protein in E. Coli.
  • May 26-27 = Purifying Silk Protein and 1st SDS PAGE gel
  • July 10-16 = Cloning Silk into Pet vector and preparing BL21 (DE3) competent cells
  • July 17-23= Cloning Silk into BL21 (de3) cells.
  • July 28-August 3rd = 1st expression in Bl21 (de3) and SDS PAGE results.
  • August 7 = 2nd purfication and SDS of Honeybee silk in Bl21 cells along with SDS PAGE results.
  • August 11 = BCA protein concentration assay on product from 8/7
  • August 12 = Optimization of growth conditions for Honeybee expression and SDS PAGE results.
  • Sept 4,7 = SDS PAGE gels for different lysis protocols
April
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July
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September
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