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

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MaSp2 AB:              BBa_K1763002
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Apis mellifera (honeybee) silk fibroin 3:              BBa_K1763000
 
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MaSp2 BC: BBa_K1763003
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Apis mellifera (honeybee) silk fibroin 3 + regulatory elements: BBa_K1763001
 
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MaSp2 CA: BBa_K1763004
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Honeybee silk protein + T7 promoter: BBa_K1763007
 
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MaSp2 SeqAB: BBa_K1763009
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Lac Promoter + Apis mellifera (honeybee) silk fibroin 3 + SpyCatcher: BBa_K1763008
 
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MaSp1 AB: BBa_K1763010
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T7 Promoter + Apis mellifera (honeybee) silk fibroin 3 + sfGFP: BBa_K1763015
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MaSp1 BC: BBa_K1763011
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MaSp1 CA: BBa_K1763012
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MaSp1 SeqAB2:         BBa_K1763423
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M2-3(1C3): BBa_K1763424
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M2-3(T7): BBa_K1763425
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M2-6(1C3): BBa_K1763426
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M2-6(T7): BBa_K1763427
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M2-9(1C3): BBa_K1763428
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M2-9(T7): BBa_K1763429
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M2-12(1C3): BBa_K1763430
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M2-12(T7): BBa_K1763431
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M2-15(1C3): BBa_K1763432
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M2-15(T7): BBa_K1763433
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M1-9(1C3): BBa_K1763434
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M1-9(T7): BBa_K1763435
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M1-12(1C3): BBa_K1763436
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M1-12(T7): BBa_K1763437
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M1/2[2:1]-12(1C3): BBa_K1763438
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M1/2[2:1]-12(T7): BBa_K1763439
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M1/2[1:1]-12(1C3): BBa_K1763440
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M1/2[1:1]-12(T7): BBa_K1763441
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M1/2[1:2]-12(1C3): BBa_K1763442
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M1/2[1:2]-12(T7): BBa_K1763443
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Revision as of 02:39, 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. Submitted and sequenced 5 honey bee silk biobricks Expressed honey bee silk protein and verified presence of SDS PAGE gel 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
  • Honeybee silk protein + 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

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.