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

 
(17 intermediate revisions by 2 users not shown)
Line 43: Line 43:
 
<!--BACKGROUND-->
 
<!--BACKGROUND-->
 
<div class= "content_subsection" id="background">
 
<div class= "content_subsection" id="background">
−
<h1>Honey Bee Silk</h1>
+
<h1>Honeybee Silk</h1>
 
<h2>Background</h2>
 
<h2>Background</h2>
 
<h4>Abstract</h4>
 
<h4>Abstract</h4>
 
<p>
 
<p>
−
In addition to the more well known silks from spiders and silkworms, we decided to also explore silk from the honey bee Apis mellifera. We cloned the honey bee silk gene as well as several variants of it and submitted them as the first honey bee silk biobricks. To investigate its potential as a biomaterial, we expressed the silk protein and confirmed its presence using SDS PAGE.
+
In addition to the more well known silks from spiders and silkworms, we decided to also explore silk from the honeybee <i>Apis mellifera</i>. We cloned the honeybee silk gene as well as several variants of it and submitted them as the first honeybee silk biobricks. To investigate its potential as a biomaterial, we expressed the silk protein and confirmed its presence using SDS PAGE.
  
 
</p>  
 
</p>  
 
<h4>Introduction</h4>
 
<h4>Introduction</h4>
 
<p>
 
<p>
−
Silk from Apis Mellifera represents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honey bee silk has certain advantages over spider and silkworm silk. The size of the honey bee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non repetitive, which allows us to synthesize and make modifications to the gene without the complications that are inherent to repetitive DNA sequences (citation). Honey bee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure (Tara D. Sutherland et al. Mol Biol Evol 2007;24:2424-2432)(http://mbe.oxfordjournals.org/content/24/11/2424/F8.large.jpg). In wild type honey bee silk these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4. However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers (citation).
+
Silk from Apis Mellifera presents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honeybee silk has certain advantages over spider and silkworm silk. The size of the honeybee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non-repetitive, which allows us to synthesize and make modifications to the gene, without the complications that are inherent to repetitive DNA sequences. Honeybee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure.
−
A major goal of our project is to give biological fibers entirely new functionalities. Therefore, in addition to expressing wild type honey bee silk, we have also created constructs in which honey bee silk protein is fused to other proteins. Our first fusion construct is honey bee silk fused to super folder green fluorescent protein (sfGFP), which will act as our proof of principle that silk can be functionalized while still maintaining exceptional physical properties. Our second fusion is silk fused to SpyCatcher protein, which would allow for the capture of any protein modified to contain the short SpyTag peptide.  
+
 
 +
<figure align="middle"><img width="500px" height="400" src= "http://mbe.oxfordjournals.org/content/24/11/2424/F8.large.jpg" />
 +
<figcaption style="width: 100%; margin: auto;" align="middle">
 +
<b>Fig 1:</b>Honeybee Silk Coiled Coil Structure
 +
</figcaption>
 +
</figure>
 +
 
 +
 
 +
In wild type honeybee silk these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4. However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers.
 +
A major goal of our project is to give biological fibers entirely new functionalities. Therefore, in addition to expressing wild type honeybee silk, we have also created constructs in which honeybee silk protein is fused to other proteins. Our first fusion construct is honeybee silk fused to super folder green fluorescent protein (sfGFP), which will act as our proof of principle that silk can be functionalized while still maintaining exceptional physical properties. Our second fusion is silk fused to SpyCatcher protein, which would allow for the capture of any protein modified to contain the short SpyTag peptide.  
 
</p>
 
</p>
 
</div>
 
</div>
Line 66: Line 75:
 
Although previous literature has shown that wild type honeybee silk is composed of four highly similar proteins, we decided to only produce one. This decision was based on the result of Tara Sutherland's research, which claimed to be able to produce honeybee fibers with comparable physical properties to wild type fibers using only one of the four proteins. The protein they used was AmelF3, which is the protein we focused on.  
 
Although previous literature has shown that wild type honeybee silk is composed of four highly similar proteins, we decided to only produce one. This decision was based on the result of Tara Sutherland's research, which claimed to be able to produce honeybee fibers with comparable physical properties to wild type fibers using only one of the four proteins. The protein they used was AmelF3, which is the protein we focused on.  
  
−
We obtained the sequence of the AmelF3 gene from genbank.( http://www.ncbi.nlm.nih.gov/gene/100192201), synthesized it using IDT’s synthesis service and used this construct as our baseline sequence from which to construct our biobricks. Because we aimed to express honeybee silk protein, we added regulatory elements such as promoters and ribosome binding sites to several our constructs. Two of our biobricks represent fusion proteins between silk and another functional protein. The first fusion biobrick is silk fused to sfGFP(http://parts.igem.org/Part:BBa_K1763015), which will serve as our proof of principle that honeybee silk can be functionalized while still retaining its mechanical properties. The second fusion biobrick is honey bee silk fused to SpyCatcher protein, which allow for capture of and protein modified to contain a Spytag peptide.(http://parts.igem.org/Part:BBa_K1763008) Additionally, we fused sfGFP to AmelF3. Please see our list of biobricks (<a href="https://2015.igem.org/Team:UCLA/Parts"?>Parts</a>) and the respective biobrick pages for more detailed design information and characterization of each biobrick.
+
We obtained the sequence of the AmelF3 gene from genbank.( http://www.ncbi.nlm.nih.gov/gene/100192201), synthesized it using IDT’s synthesis service and used this construct as our baseline sequence from which to construct our biobricks. Because we aimed to express honeybee silk protein, we added regulatory elements such as promoters and ribosome binding sites to several our constructs. Two of our biobricks represent fusion proteins between silk and another functional protein. The first fusion biobrick is silk fused to sfGFP(<a href="http://parts.igem.org/Part:BBa_K1763015">BBa_K1763015</a>), which will serve as our proof of principle that honeybee silk can be functionalized while still retaining its mechanical properties. The second fusion biobrick is honeybee silk fused to SpyCatcher protein, which allow for capture of and protein modified to contain a Spytag peptide.(<a href="http://parts.igem.org/Part:BBa_K1763008">BBa_K1763008</a>) Additionally, we fused sfGFP to AmelF3. Please see our list of biobricks (<a href="https://2015.igem.org/Team:UCLA/Parts"?>Parts</a>) and the respective biobrick pages for more detailed design information and characterization of each biobrick.
 
</li>
 
</li>
  
 
<li>
 
<li>
−
In addition to creating honey bee genetic constructs, we needed protocols to express and purify honey bee silk proteins. We drew heavily from previous literature by T. Sutherland that had established a method of expressing and purifying honeybee silk proteins.  According to previous literature, honeybee silk proteins aggregate and form insoluble inclusion bodies within the e. coli cell. Therefore, it is reported that these proteins can be purified by lysing the cells and collecting the insoluble fraction. In order to determine protein concentration. Furthermore, because we wanted to use a T7 promoter to drive expression of our honeybee constructs, therefore, we used a strain of bacteria that expressed T7 polymerase, BL21 (DE3).  For more detailed descriptions of our protein expression and purification, please see our lab notebook entries.
+
In addition to creating honeybee genetic constructs, we needed protocols to express and purify honeybee silk proteins. We drew heavily from previous literature by T. Sutherland that had established a method of expressing and purifying honeybee silk proteins.  According to previous literature, honeybee silk proteins aggregate and form insoluble inclusion bodies within the e. coli cell. Therefore, it is reported that these proteins can be purified by lysing the cells and collecting the insoluble fraction. In order to determine protein concentration. Furthermore, because we wanted to use a T7 promoter to drive expression of our honeybee constructs, therefore, we used a strain of bacteria that expressed T7 polymerase, BL21 (DE3).  For more detailed descriptions of our protein expression and purification, please see our lab notebook entries.
−
?
+
 
 
</li>
 
</li>
  
Line 84: Line 93:
 
We used standard restriction digest cloning to clone our five biobrick constructs. We sequence verified all our parts and submitted them to the registry.  
 
We used standard restriction digest cloning to clone our five biobrick constructs. We sequence verified all our parts and submitted them to the registry.  
 
Here is a diagram of all of our sequence verified parts.  
 
Here is a diagram of all of our sequence verified parts.  
 +
<li>
  
 +
<figure align="middle"><img width="800px" height="450px" src= "https://2015.igem.org/File:Screen_Shot_2015-09-18_at_7.15.27_PM.png" />
 +
<figcaption  style="width: 100%; margin: auto;" align="middle">
 +
<b>Fig 2:</b>Schematic of our honeybee silk constructs
 +
</figcaption>
 +
</figure>
  
−
<figure align="middle"><img width="500px" height="200" src= "https://2015.igem.org/File:Screen_Shot_2015-09-18_at_7.15.27_PM.png" />
+
 
 +
From top to bottom they are the honeybee silk coding region, the coding region under control of lac promoter, the silk protein fused to Spycatcher, the coding region under control of a T7 promoter, and silk protein fused to sfGFP.
 +
 
 +
We also successfully made competent BL21 (DE3) cells using the Zymo mix and go chemical competency kit. We performed a competency test and we determined that the competency of our cells was 3e7 colony forming units / microgram of DNA. (<a href="https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/12_July_2015"?>Parts</a>) 
 +
Finally, we were able to express our honeybee silk proteins and confirm its presence through SDS PAGE. 
 +
 
 +
<figure align="middle"><img width="800px" height="450" src= "https://2015.igem.org/File:UCLA_honeybee_Growth_optimization_37C.jpg?" />
 
<figcaption  style="width: 100%; margin: auto;" align="middle">
 
<figcaption  style="width: 100%; margin: auto;" align="middle">
−
<b>Fig 1:</b>Schematic of our honey bee silk constructs
+
<b>Fig 3:</b>SDS PAGE optimization image
 
</figcaption>
 
</figcaption>
 
</figure>
 
</figure>
  
  
−
From top to bottom they are the honey bee silk coding region, the coding region under control of lac promoter, the silk protein fused to Spycatcher, the coding region under control of a T7 promoter, and silk protein fused to sfGFP.
 
  
−
We also successfully made competent BL21 (DE3) cells using the Zymo mix and go chemical competency kit. We performed a competency test and we determined that the competency of our cells was 3e7 colony forming units / microgram of DNA. (https://2015.igem.org/Team:UCLA/Notebook/Honeybee_Silk/12_July_2015)
+
We also obtained some rough estimate of protein yield by doing a BCA protein assay. This estimate may not be entirely reliable because we observed some contaminating bands on our SDS PAGE gels, indicating that there are proteins other than honeybee silk in the solution. From our BCA assay, we determined that we got around 12 mg of protein from a 300ml overnight cell culture.  
  
−
Finally, we were able to express our honeybee silk proteins and confirm its presence through SDS PAGE (https://2015.igem.org/File:UCLA_honeybee_Growth_optimization_37C.jpg?).  We also obtained some rough estimate of protein yield by doing a BCA protein assay. This estimate may not be entirely reliable because we observed some contaminating bands on our SDS PAGE gels, indicating that there are proteins other than honeybee silk in the solution. From our BCA assay, we determined that we got around 12 mg of protein from a 300ml overnight cell culture.
 
  
−
<figure style= "margin: 10px; float: left;"><img width="600px" src= "https://2015.igem.org/File:Honeybee_silk_SDS_PAGE.jpg" />
+
<figure align="middle"><img width="200px" height="500" src= "https://2015.igem.org/File:Honeybee_silk_SDS_PAGE.jpg" />
−
<figcaption  style="margin: auto; width: 600px;">
+
<figcaption  style="width: 100%; margin: auto;" align="middle">
 +
<b>Fig 4:</b>SDS PAGE image of honeybee silk
 +
</figcaption>
 +
</figure>
  
 
Future Directions
 
Future Directions
Line 107: Line 129:
 
Assess the mechanical properties of these fibers
 
Assess the mechanical properties of these fibers
 
Express and process functionalized silk proteins
 
Express and process functionalized silk proteins
−
Assay functionalized honey bee silk materials for functionality   
+
Assay functionalized honeybee silk materials for functionality   
  
 
</p>
 
</p>
Line 113: Line 135:
 
<ol>
 
<ol>
 
<li>
 
<li>
−
Submitted and sequenced 5 honey bee silk biobricks
+
Submitted and sequenced 5 honeybee silk biobricks
−
Expressed honey bee silk protein and verified presence of SDS PAGE gel
+
<li>
 +
Expressed honeybee silk protein and verified presence of SDS PAGE gel
 +
<li>
 
Had a great summer!   
 
Had a great summer!   
 
</li>
 
</li>
Line 127: Line 151:
 
<ul>
 
<ul>
 
<li>
 
<li>
−
MaSp2 AB:               BBa_K1763002
+
Apis mellifera (honeybee) silk fibroin 3: <a href="http://parts.igem.org/Part:BBa_K1763000">BBa_K1763000</a>                                     
 
</li>
 
</li>
 
<li>
 
<li>
−
MaSp2 BC: BBa_K1763003
+
Apis mellifera (honeybee) silk fibroin 3 + regulatory elements: <a href="http://parts.igem.org/Part:BBa_K1763001">BBa_K1763001></a>        
 
</li>
 
</li>
 
<li>
 
<li>
−
MaSp2 CA: BBa_K1763004
+
Apis mellifera (honeybee) silk fibroin 3 + T7 promoter: <a href="http://parts.igem.org/Part:BBa_K1763007">BBa_K1763007</a>
 
</li>
 
</li>
 
<li>
 
<li>
−
MaSp2 SeqAB: BBa_K1763009
+
Lac Promoter + Apis mellifera (honeybee) silk fibroin 3 + SpyCatcher: <a href="http://parts.igem.org/Part:BBa_K1763008">BBa_K1763008</a>
 
</li>
 
</li>
 
<li>
 
<li>
−
MaSp1 AB: BBa_K1763010
+
T7 Promoter + Apis mellifera (honeybee) silk fibroin 3 + sfGFP: <a href="http://parts.igem.org/Part:BBa_K1763015">BBa_K1763015</a>
−
</li>
+
−
<li>
+
−
MaSp1 BC: BBa_K1763011
+
−
</li>
+
−
<li>
+
−
MaSp1 CA: BBa_K1763012
+
−
</li>
+
−
<li>
+
−
MaSp1 SeqAB2:         BBa_K1763423
+
−
</li>
+
−
<li>
+
−
M2-3(1C3): BBa_K1763424
+
−
</li>
+
−
<li>
+
−
M2-3(T7): BBa_K1763425
+
−
</li>
+
−
<li>
+
−
M2-6(1C3): BBa_K1763426
+
−
</li>
+
−
<li>
+
−
M2-6(T7): BBa_K1763427
+
−
</li>
+
−
<li>
+
−
M2-9(1C3): BBa_K1763428
+
−
</li>
+
−
<li>
+
−
M2-9(T7): BBa_K1763429
+
−
</li>
+
−
<li>
+
−
M2-12(1C3): BBa_K1763430
+
−
</li>
+
−
<li>
+
−
M2-12(T7): BBa_K1763431
+
−
</li>
+
−
<li>
+
−
M2-15(1C3): BBa_K1763432
+
−
</li>
+
−
<li>
+
−
M2-15(T7): BBa_K1763433
+
−
</li>
+
−
<li>
+
−
M1-9(1C3): BBa_K1763434
+
−
</li>
+
−
<li>
+
−
M1-9(T7): BBa_K1763435
+
−
</li>
+
−
<li>
+
−
M1-12(1C3): BBa_K1763436
+
−
</li>
+
−
<li>
+
−
M1-12(T7): BBa_K1763437
+
−
</li>
+
−
<li>
+
−
M1/2[2:1]-12(1C3): BBa_K1763438
+
−
</li>
+
−
<li>
+
−
M1/2[2:1]-12(T7): BBa_K1763439
+
−
</li>
+
−
<li>
+
−
M1/2[1:1]-12(1C3): BBa_K1763440
+
−
</li>
+
−
<li>
+
−
M1/2[1:1]-12(T7): BBa_K1763441
+
−
</li>
+
−
<li>
+
−
M1/2[1:2]-12(1C3): BBa_K1763442
+
−
</li>
+
−
<li>
+
−
M1/2[1:2]-12(T7): BBa_K1763443
+
 
</li>
 
</li>
 
</ul>
 
</ul>
  
 
</div>
 
</div>
 +
  
  
Line 225: Line 181:
 
Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. <i>PLoS ONE</i> 2011. e16489
 
Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. <i>PLoS ONE</i> 2011. e16489
 
</p>
 
</p>
 +
 +
<p>Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks.<i> Mol Biol Evol</i> 2007;24:2424-2432
  
 
</div>
 
</div>

Latest revision as of 03:59, 19 September 2015

iGEM UCLA




BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS























Honeybee Silk

Background

Abstract

In addition to the more well known silks from spiders and silkworms, we decided to also explore silk from the honeybee Apis mellifera. We cloned the honeybee silk gene as well as several variants of it and submitted them as the first honeybee silk biobricks. To investigate its potential as a biomaterial, we expressed the silk protein and confirmed its presence using SDS PAGE.

Introduction

Silk from Apis Mellifera presents an intriguing alternative to silks from spiders or silkworms. Although it is not quite as strong as these other types of silks, working with honeybee silk has certain advantages over spider and silkworm silk. The size of the honeybee silk protein gene is considerably smaller than the silk genes of spiders or silkworms. More importantly, the gene sequence is non-repetitive, which allows us to synthesize and make modifications to the gene, without the complications that are inherent to repetitive DNA sequences. Honeybee silk also has a very different secondary and tertiary structure than spider and silkworm silks. It forms primary alpha helices, and four silk proteins come together to form a coiled coil structure.

Fig 1:Honeybee Silk Coiled Coil Structure
In wild type honeybee silk these coiled coils are formed from four similar, yet unique proteins, Amelf 1-4. However, a study has shown that using one of these proteins, (Amelf3) is sufficient to reproduce the physical properties of the wild type fibers. A major goal of our project is to give biological fibers entirely new functionalities. Therefore, in addition to expressing wild type honeybee silk, we have also created constructs in which honeybee silk protein is fused to other proteins. Our first fusion construct is honeybee silk fused to super folder green fluorescent protein (sfGFP), which will act as our proof of principle that silk can be functionalized while still maintaining exceptional physical properties. Our second fusion is silk fused to SpyCatcher protein, which would allow for the capture of any protein modified to contain the short SpyTag peptide.

Methodology

  1. Although previous literature has shown that wild type honeybee silk is composed of four highly similar proteins, we decided to only produce one. This decision was based on the result of Tara Sutherland's research, which claimed to be able to produce honeybee fibers with comparable physical properties to wild type fibers using only one of the four proteins. The protein they used was AmelF3, which is the protein we focused on. We obtained the sequence of the AmelF3 gene from genbank.( http://www.ncbi.nlm.nih.gov/gene/100192201), synthesized it using IDT’s synthesis service and used this construct as our baseline sequence from which to construct our biobricks. Because we aimed to express honeybee silk protein, we added regulatory elements such as promoters and ribosome binding sites to several our constructs. Two of our biobricks represent fusion proteins between silk and another functional protein. The first fusion biobrick is silk fused to sfGFP(BBa_K1763015), which will serve as our proof of principle that honeybee silk can be functionalized while still retaining its mechanical properties. The second fusion biobrick is honeybee silk fused to SpyCatcher protein, which allow for capture of and protein modified to contain a Spytag peptide.(BBa_K1763008) Additionally, we fused sfGFP to AmelF3. Please see our list of biobricks (Parts) and the respective biobrick pages for more detailed design information and characterization of each biobrick.
  2. In addition to creating honeybee genetic constructs, we needed protocols to express and purify honeybee silk proteins. We drew heavily from previous literature by T. Sutherland that had established a method of expressing and purifying honeybee silk proteins. According to previous literature, honeybee silk proteins aggregate and form insoluble inclusion bodies within the e. coli cell. Therefore, it is reported that these proteins can be purified by lysing the cells and collecting the insoluble fraction. In order to determine protein concentration. Furthermore, because we wanted to use a T7 promoter to drive expression of our honeybee constructs, therefore, we used a strain of bacteria that expressed T7 polymerase, BL21 (DE3). For more detailed descriptions of our protein expression and purification, please see our lab notebook entries.

Results

We used standard restriction digest cloning to clone our five biobrick constructs. We sequence verified all our parts and submitted them to the registry. Here is a diagram of all of our sequence verified parts.

  • Fig 2:Schematic of our honeybee silk constructs
    From top to bottom they are the honeybee silk coding region, the coding region under control of lac promoter, the silk protein fused to Spycatcher, the coding region under control of a T7 promoter, and silk protein fused to sfGFP. We also successfully made competent BL21 (DE3) cells using the Zymo mix and go chemical competency kit. We performed a competency test and we determined that the competency of our cells was 3e7 colony forming units / microgram of DNA. (Parts) Finally, we were able to express our honeybee silk proteins and confirm its presence through SDS PAGE.
    Fig 3:SDS PAGE optimization image
    We also obtained some rough estimate of protein yield by doing a BCA protein assay. This estimate may not be entirely reliable because we observed some contaminating bands on our SDS PAGE gels, indicating that there are proteins other than honeybee silk in the solution. From our BCA assay, we determined that we got around 12 mg of protein from a 300ml overnight cell culture.
    Fig 4:SDS PAGE image of honeybee silk
    Future Directions Optimize protein expression and purification protocols for higher yields and greater purity Process purified silk proteins into materials such as films or fibers Assess the mechanical properties of these fibers Express and process functionalized silk proteins Assay functionalized honeybee silk materials for functionality

    Achievements

    1. Submitted and sequenced 5 honeybee silk biobricks
    2. Expressed honeybee silk protein and verified presence of SDS PAGE gel
    3. Had a great summer!

    • List of Biobricks

    • Apis mellifera (honeybee) silk fibroin 3: BBa_K1763000
    • Apis mellifera (honeybee) silk fibroin 3 + regulatory elements: BBa_K1763001>
    • Apis mellifera (honeybee) silk fibroin 3 + T7 promoter: BBa_K1763007
    • Lac Promoter + Apis mellifera (honeybee) silk fibroin 3 + SpyCatcher: BBa_K1763008
    • T7 Promoter + Apis mellifera (honeybee) silk fibroin 3 + sfGFP: BBa_K1763015

    References

    Weisman, S., Haritos, V., Church, J., Huson, M., Mudie, S., Rodgers, A., Dumsday, G., Sutherland, T. Honeybee silk: Recombinant protin production, assembly, and fiber spinning. Elsevier Ltd. 2009.

    Sutherland, T., Church, J., Hu, X., Huson, M., Kaplan, D., Weisman, S. Single Honeybee Silk Protein Mimics Properties fo Multi-Protein Silk. PLoS ONE 2011. e16489

    Sutherland, T. Conservation of Essential Design Features in Coiled Coil Silks. Mol Biol Evol 2007;24:2424-2432