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

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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 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).
 
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.  
 
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.  
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Existing techniques seek to remove reliance of cloning on primer annealing (Tokareva et al, 2013). Generally, these techniques break the silk genetic into monomeric sequences, then assemble the monomers into the final construct. Head-to-tail cloning assembles gene constructs by ligating two plasmid halves together. Each half carries one of the silk monomers, and the resulting complete plasmid has been doubled. This technique can be used recursively to assemble increasingly large silk genes in a specified manner. Directional recursive ligation uses a similar tactic, where individual monomers are ligated one at a time into a receiving plasmid. Concatemerization is another technique where a pool of monomers are ligated in a single reaction, then cloned into plasmids. This particular technique is useful for creating a library of sizes and compositions.
 
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These existing techniques are not ideal for engineering recombinant silks, because they require repeated and extensive cloning for large constructs, as in the case of head-to-tail assembly and directional recursive ligation, or do not offer any control over the length or genetic composition, as is the case for concatemerization. As it currently stands, there is no one technique that offers rapid and controllable assembly of recombinant spider silk genes.
 
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Iterative Capped Assembly (ICA) is a cloning method that is used to sequentially assemble long,
 
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repetitive DNA sequences. This technique was developed by Briggs et al. in 2012 as a method
 
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to assemble Transcription Activator-Like Effector Nucleases (TALENs) which are sequence
 
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specific DNA binding proteins that consist of multiple repetitive monomers. Each repeat
 
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monomer is responsible for binding to a specific nucleotide in the target sequence. Due to the
 
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repetitive nature of TALE genes, conventional PCR is unable to reliably amplify these sequences
 
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due to non-specific primer binding.
 
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Although ICA was developed using TALE construction as a model problem, this technique can
 
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be used to construct long, repetitive DNA constructs in a directly controllable fashion. ICA assembles repetitive sequences one monomer unit at a time, while preventing the elongation of
 
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incomplete nucleotide chains. The full length sequence is flanked by unique primer annealing
 
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sites, which allows the PCR amplification of the final product. This entire process is performed
 
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using a solid substrate, which greatly facilitates the construction of long sequences.
 
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This summer, we adapt the use of Iterative Capped Assembly (ICA) as a technique for cloning recombinant spider silks in a time-efficient and specific manner that is unparalleled by existing methods. Our experience shows that ICA can be used as a method for the quick assembly of spider silk genes.
 
 
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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.  
  
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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) 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.
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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.
 
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Revision as of 02:21, 19 September 2015

iGEM UCLA




BACKGROUND

METHODOLOGY

RESULTS

BIOBRICKS























Honey Bee Silk

Background

Abstract

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.

Introduction

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). 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.

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(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 (Parts) and the respective biobrick pages for more detailed design information and characterization of each biobrick.
  2. 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.

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. 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) 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. 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 honey bee silk materials for functionality

Achievements

  1. Successfully improved last year’s biobrick BBa_K1384000
    1. Redesigned MaSp1 and MaSp2 monomers with modified sticky ends, and cloned these into biobricks.
  2. Created a collection of parts to be used for Iterative Capped Assembly
  3. Demonstrated that ICA is adaptable to silk using our designed sticky ends.
    1. Optimized ICA for use with spider silk genes to enable fast, efficient assembly of repetitive constructs.
  4. Used ICA to assemble a variety of silk genetic constructs of different length and composition to examine their properties.
    1. Used ICA to create 10 different silk constructs ranging from 3-mers to 15-mers, and constructs with MaSp1 and MaSp2 in ratios of [1:2], [1:1] and [2:1].

List of Biobricks

  • MaSp2 AB: BBa_K1763002
  • MaSp2 BC: BBa_K1763003
  • MaSp2 CA: BBa_K1763004
  • MaSp2 SeqAB: BBa_K1763009
  • MaSp1 AB: BBa_K1763010
  • MaSp1 BC: BBa_K1763011
  • MaSp1 CA: BBa_K1763012
  • MaSp1 SeqAB2: BBa_K1763423
  • M2-3(1C3): BBa_K1763424
  • M2-3(T7): BBa_K1763425
  • M2-6(1C3): BBa_K1763426
  • M2-6(T7): BBa_K1763427
  • M2-9(1C3): BBa_K1763428
  • M2-9(T7): BBa_K1763429
  • M2-12(1C3): BBa_K1763430
  • M2-12(T7): BBa_K1763431
  • M2-15(1C3): BBa_K1763432
  • M2-15(T7): BBa_K1763433
  • M1-9(1C3): BBa_K1763434
  • M1-9(T7): BBa_K1763435
  • M1-12(1C3): BBa_K1763436
  • M1-12(T7): BBa_K1763437
  • M1/2[2:1]-12(1C3): BBa_K1763438
  • M1/2[2:1]-12(T7): BBa_K1763439
  • M1/2[1:1]-12(1C3): BBa_K1763440
  • M1/2[1:1]-12(T7): BBa_K1763441
  • M1/2[1:2]-12(1C3): BBa_K1763442
  • M1/2[1:2]-12(T7): BBa_K1763443

References

Briggs A., Rios X., Chari R., Luhan Y., Zhang F., Mali P., and Church G. Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers. Nucleic Acids Research. 2012;40(15): e117

Hinman, M.B., Lewis, R. Isolation of a clone encoding a second dragline silk fibroin. Nephila clavipes dragline silk is a two-protein fiber. J. Biol. Chem. 1992;267: 19320–19324.

Tokareva O., Michalczechen-Lacerda V., Rech E., and Kaplan D. Recombinant DNA production of spider silk proteins. Microbial Biotechnology. 2013;6(6): 651-663

Xu, M., Lewis, R.V. Structure of a protein superfiber: spider dragline silk. PNAS;1990;87, 7120.

FIGURES: I DON'T KNOW WHERE TO PUT THEM

Fig 1:In ICA, the initiator of the growing DNA chains are immobilized on magnetic streptavidin beads to facilitate removal. The first monomer is ligated to the initiator. (Step A) Subsequent ligation steps incorporate capping oligos, which prevent the extension of incorrect chains: those that failed to ligate in the previous round. (Steps B, C) Ligation continues until the construct reaches the final desired length. The final step incorporates a terminator oligo. (Final Step) Only DNA constructs of the correct size can ligate to the terminator, as all incorrect constructs will have been capped.
Fig 2:Downstream Cloning after Iterative Capped Assembly. After elution from the beads, the ICA constructs are amplified using PCR primers that anneal to the initiator and the terminator. These primer binding sites are unique in the construct, and can be found nowhere else in the sequence. The only constructs that are amplified are those that have the initiator and terminator. All other constructs, while present, are excluded from amplification. After amplification, the construct can be cloned into a vector using traditional techniques.
Fig 4:Schematic of examples of initiator (a), terminator (b), and capping oligos (c), used in our ICA project. The cap shown has the B-type sticky end.
Fig 5:Schematic of the three types of sticky ends we designed for ICA. Sticky end A is 5’-AGTT-3’. Sticky end B is 5’-TGTC-3’. Sticky end C is 5’-CGTG-3’. An assembled 3-mer construct AB+BC+CA is shown as an example of how these biobricks would be used.
Fig 6:Gel image of constructs we created this summer using ICA. pSB1C3 Plasmids containing the sequence verified construct were digested using XbaI and PstI. Results were run on 1% TAE gel. The expected band size for the pSB1C3 is ~2070. Expected sizes for inserts fragments are indicated on the right hand side.