iGEM 2024 — Team hopkins

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## PCR Method

### Overall

1. Day 1: DNA extraction
   1. Using [this](https://www.zymoresearch.com/products/quick-dna-tissue-insect-microprep-kit) Zymo Quick-DNA Tissue/Insect Microprep Kit
   2. [Protocol](https://files.zymoresearch.com/protocols/_d6015_quick-dna_tissue-insect_microprep_kit.pdf)
2. Day 2: Run and optimize PCR
   1. Ingredients:
      1. IDT primers (try both sets)
      2. PCR Reagents
3. Day 3: Run gel and image
   1. Ingredients:
      1. 1 kb ladder
      2. Dye
      3. gel dye
      4. PCR product

Tissue processing- keep it frozen according to Danyon "DNA tends to be stable enough and there aren’t a lot of proteins trying to degrade it, so it can be thawed without majorly compromising its integrity. However, keeping things frozen will prevent proteins from interacting with the DNA and potentially breaking it down. This issue is more important for RNA, but the principle still holds true for DNA. Since the protocol you have works with frozen or fresh tissues, it tends to be good practice to keep it frozen because the tissue is easier to break (if you were going to grind it) and the DNA won’t degrade as easily."

7/18/2024: DNA extraction of caddisfly silk samples to begin PCR

People present: Sophia

Materials:

* Caddisfly samples (silk gland AF-23-SG)
* 2-Mercaptoethanol
* Zymo kit
* Vortexer
* Centrifuge

[Protocol](https://files.zymoresearch.com/protocols/_d6015_quick-dna_tissue-insect_microprep_kit.pdf) → Deviation from protocol: no bashing beads machine instead used vortex for 1 minute

Results:

FAILURE :(- tubes labeled with 23SG-SX-7/18/24 For 119.2 ng/uL there is some kind of DNA/RNA at 260/280 so there is something there. It might be usable for PCR. We need to remeasure the second one due to negative concentration.

![dna1](https://static.igem.wiki/teams/5275/wet-lab/dnaextractionattempt1-1.png)
![dna2](https://static.igem.wiki/teams/5275/wet-lab/dnaextractionattempt1-2.png)

Possible problems:

1. Use of vortexer for 30 minutes instead of the prescribed bead basher does not fully shred the silk glands open (see below image)
   ![tube](https://static.igem.wiki/teams/5275/wet-lab/dnaextractionattempt1-tubeimage.png)
   1. Solution: asked Josh to use his lab's bead basher, use that (scheduled for 3PM on 7/19/24)
2. Nanodrop shows there are clearly contaminants
   1. Solution: multiple centrifugations to remove buffer

Tasks for next time:

* use beadbasher instead of vortexer
* contaminants need to be removed more often (second 3 minute centrifugation until no liquid collects into collection tube for the final step before elution)
* remeasure the negative concentration sample and get an actual measurement

7/19/24: Attempt 2 of DNA extraction of caddisfly silk samples to begin PCR

People present: Sophia

Materials:

* Caddisfly samples (silk gland AF-24-SG)
* 2-Mercaptoethanol
* Zymo kit
* Bead basher (courtesy of josh)
* Centrifuge

[Protocol](https://files.zymoresearch.com/protocols/_d6015_quick-dna_tissue-insect_microprep_kit.pdf)

no deviations from the protocol

Results:

![extraction1](https://static.igem.wiki/teams/5275/wet-lab/dnaextractionattempt2-1.png)
![extraction2](https://static.igem.wiki/teams/5275/wet-lab/dnaextractionattempt2-2.png)

: remeasured negative concentration from 7/18/24

7/28/24: PCR Attempt

PCR Attempt: Gel

People present: Alex

Results:

![gelPCR](https://static.igem.wiki/teams/5275/wet-lab/gelpcrmethod.png)

Conclusion: Fail :(

Notes: Timp + Lucy suggested we continue with this have 2 pots on the stove, wet lab ultimately decided against due to lack of resources and funding.

### Synthesis Method

9/28/24: Construction of pBS+motif1 Strain

People present: Siyona, Sophia, Katie (who is this DIVA 💜)

Goal(s):

* Restriction digest motif with BamHI and XhoI
* T4 Ligase + motif + incubation 22C
* Transformation into DH10B cells -> plate on carb and incubate for 24 hours

Reconstituted motif1

* 4ug DNA -> +20 uL for 200 ng/uL concentration

[Restriction Digest](https://www.neb.com/en-us/protocols/2012/12/07/optimizing-restriction-endonuclease-reactions)

* 5 uL of 200 ng/uL DNA (total 1 ug of DNA)
* 1 uL BamHI
* 1 uL XhoI
* 5 uL rCutsmart Buffer
* 38 uL H2O

labeled digest motif 1

* Run for 1 hour at 37C then 20 minutes 65C

Ligation

* 2 uL T4 ligase buffer
* 10 uL motif1
* 1 ug pBS
* 5 uL
* 1 uL T4 ligase
* (to 20 uL H2O)

labeled ligase motif 1

* Room temp for 1 hour + 10 min 65C inactivation

Transformation

* DH10B cells
* Plated on LB+Carb plates

9/29/24: Liquid Culture Setup

People present: Sophia, Kalen

Liquid Culture set up at 6PM

* Used P1000 to pick 3 colonies and incoluate LB+Carb liquid media

![expculture](https://static.igem.wiki/teams/5275/wet-lab/expculture-motif1.jpg)

9/30/24: Miniprep of pBS+motif1

Miniprep of liquid culture

People present: Siyona, Sophia

Started at 10AM (14 hour mark)

Following protocol: <https://www.neb.com/en-us/protocols/2015/11/20/monarch-plasmid-dna-miniprep-kit-protocol-t1010>

Nano Dropped results below:

* Colony 1: 141.8 ng/uL, 1.93 A260/280
* Colony 2: 266.6 ng/uL, 1.89 A260/280
* Colony 3: 553.9 ng/uL, 1.89 A260/280
* Curves look good noticeable peak at 260 wavelength

![nano1](https://static.igem.wiki/teams/5275/wet-lab/nanodrop-colony1.jpg)
![nano3](https://static.igem.wiki/teams/5275/wet-lab/nanodrop-colony3.jpg)

Submitted to plasmidsaurus order HXFNPR

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## Design

Typically used by caddisflies to collect and attach aquatic debris to themselves for metamorphosis, caddisfly silk is a good prospective material for its high tensile strength, waterproof properties, and non-cytotoxicity (4). The caddisfly silk protein is a heterodimer of heavy and light fibroins (abbreviated H-fibroin and L-fibroin, respectively). Caddisfly silk gets its adhesive properties from the H-fibroin, which has repeated β-sheet motifs with highly conserved proline-glycine turns (4). The main difficulties in this project lie in the H-fibroin, which has a DNA sequence that is not only highly repetitive but also incredibly long at 19kb (Fig. 1). These qualities make the H-fibroin difficult to synthesize and isolate.

Caddisflies can be categorized based on their use of silk: 1. Cocoon makers use the silk to pupate into cocoons, 2. Tube makers use the silk to build cases with surrounding aquatic debris, and 3. Retreat makers use silk to create stationary shelters and catch prey (4). Intuitively, retreat makers have the strongest silk and cocoon makers have the weakest silk strength because their silk must be strong enough to resist water currents and remain stationary. Initial experimentation relied on Arctopysche grandis (a retreat maker) to synthesize the strongest possible silk to maximize successful recovery with a caddisfly and silkworm-based mesh. However, this year’s dry lab subteam modeled H-fibroin subunits from multiple caddisfly species, ultimately finding that not every subunit could synthesize functional protein. Thus, our focus shifted from silk strength to being able to produce a functional protein.

![species](https://static.igem.wiki/teams/5275/wet-lab/species.jpg)

###### Figure 1. “Schematic visualization of the primary structure of the h-fibroin gene of one representative species per clade” from Heckenhauer et al. (DOI: <https://doi.org/10.1016/j.isci.2023.107253>). We used Arctopsyche grandis initially, but switched to Atopsyche Davidsoni in later methods of synthesis of the H-fibroin protein.

Last year, the Hopkins iGEM team sought to accomplish this goal by synthesizing an altered H-fibroin protein with only a few motifs. Unfortunately, the dry lab subteam discovered this year that models of this altered H-fibroin yielded nonfunctional protein. Thus, the goal of the 2024 iGEM team was redirected to synthesizing the full H-fibroin protein.

To begin, we decided to switch from an Escherichia coli expression vector to Saccharomyces Cerevisiae instead because a eukaryote is more likely to express eukaryotic caddisfly silk protein. Additionally, S. Cerevisiae can express larger proteins such as the H-fibroin while still retaining the same low-cost, fast-growing, robust properties with the genetic tools of a [model organism](https://pubmed.ncbi.nlm.nih.gov/19500547/)(2).

Our initial design primarily relies on PCR to isolate the H-fibroin gene. Based on preliminary research from Frandsen Lab, the H-fib subunit gene of the Arctopysche grandis species contains a smaller, less repetitive region in the 10-12kb section of the coding sequence (Fig. 2). Therefore, we can theoretically design two sets of primers that each amplify about half of the H-fib gene. One set would contain a forward primer that binds to the N-terminus sequence and a reverse primer that binds to the low repeat region. The second set would contain a forward primer binding adjacent to the reverse primer of the first set and a reverse primer that binds to the C-terminus. Adding about 10 bases of synthetic complementary sequence on the 5’ of the reverse primers of set 1 and 5’ of forward primers of set 2 would enable us to perform Gibson assembly using the two fragments that we PCR amplify. Unfortunately, we found that even this small low repeat region has some sequence homology scattered throughout the gene. Nevertheless, we expected to be able to purify the correct fragments using gel extraction based on the length of each PCR fragment. Using our design, we should be able to yield one fragment that is approximately 6kb and another that is about 12kb.

## Build

We designed two sets of primers to yield two fragments of the H-fibroin gene, with each set having 2 forward and 2 reverse primers. Overhangs were added (in orange, see Fig. 2) to reverse 1 and forward 2 so the two H-fibroin fragments could be annealed in Gibson Assembly later. In anticipation for future golden gate steps, BsaI cut sites were added to forward primer 1 and reverse primer 2. Theoretically, we would be able to produce a scarless H-fib gene following this method.

![frequency](https://static.igem.wiki/teams/5275/wet-lab/frequency-cds.png)

###### Figure 2. Example Set of Primers for Isolating and Amplifying H-Fibroin with PCR. Two sets of primers were created based on a less repetitive region at the 10-12kb mark of the coding sequence. Each set of primers consist of a forward 1, reverse 1, forward 2, and reverse 2 primer, which some overlap between reverse 1 and forward 2 primers to allow for subsequent Gibson Assembly.

## Test

Using Arctopsyche grandis silk gland samples acquired from Frandsen lab, we extracted caddisfly DNA using the Zymo Tissue/Insect microprep kit and ran this as the template for PCR. A range of annealing temperatures were tested and the reaction was run for an additional 30 seconds per cycle to guarantee that polymerase would be able to replicate sufficient bases to cover 6 or 12 kb. However, we saw that all annealing temperatures instead had bands that are mostly about 1kb, which is far from the expected 12kb and 6kb H-fibroin fragments (Fig. 4). After meeting members from Frandsen Lab, we found that it is likely due to the nonspecific binding of primers to other parts of the bug genome.

![gel-pcr](https://static.igem.wiki/teams/5275/wet-lab/gelpcrmethod.png)

###### Figure 4. Attempted PCR of H-Fibroin Using Two Different Sets of Primers. Lanes labeled as the following: 1. 1kb ladder 4. First half primer set 1 (forward 1, reverse 1) 5. Second half primer set 1 (forward 2, reverse 2) 6. First half primer set 2 (forward 1, reverse 1) 7. Second half primer set 2 (forward 2, reverse 2) 9. Only template 10. Only primers 11. Enzyme mixture 12. Reagents only

## Learn

It is clear that a PCR-based approach is not appropriate for our purposes as it would be difficult to avoid nonspecific primer binding to other parts of the caddisfly genome. Thus, we turned to conceptualization and literature review to find another method for synthesizing the H-fibroin.

Spider silk is similar to caddisfly silk in that the fibroin gene for spider silk is also highly modular and has been researched for biomaterial use. There have been multiple successful [iterations](https://www.sciencedirect.com/science/article/pii/S104659282100022X) of synthetic spider silk expression in E. coli (1, 3, 5, 6, 7), and thus we sought to replicate one such protocol for caddisfly silk. A paper titled A Protocol for the Production of Recombinant Spider Silk-like Proteins for Artificial Fiber Spinning (7) uses a restriction digest, gel extract, ligation, transformation and miniprep rinse-and-repeat protocol to stitch together motifs and form long, highly repetitive sequences.

Opting instead to synthesize the Atopsyche davidsoni instead of the Arctopsyche grandis H-fibroin for its more unimodular structure (Fig. 1), this synthesis-based method adds appropriate restriction digest sites on the 5’ and 3’ ends of special motifs of the Caddisfly gene. On the 5’ end, XhoI, NdeI, and XmaI restriction sites are added adjacent to each other in that sequence. On the 3’ end, BspEI and BamHI sites are adjacently added in that sequence. We were able to add these overhangs on the N-terminus sequence, repeating motif, and C-terminus sequence of Atopsyche davidsoni and codon optimized these sequences to be compatible with IDT synthesis parameters. By digesting these fragments with XhoI and BamHI, we can integrate each fragment into a pBlueScript II SK(+) backbone containing a ScaI cut site. Then, by digesting one fragment with XmaI and ScaI and another fragment with BspEI and ScaI, we can connect the fragment treated with BspEI in front of the other fragment treated with XmaI (Fig. 5). Deviating from the paper, we also added BsaI to flank the N-terminus, x number of motifs, and C-terminus coding sequence in preparation for later golden gate assembly when building the yeast plasmid. Based on the Florence Teulé et al. protocol, we should be able to continuously and sequentially combine fragments until we reach 81 motifs (Fig. 1) to produce a full H-fibroin protein coding sequence with a single serine scar. We are currently attempting this synthesis-based method:

![plasmid](https://static.igem.wiki/teams/5275/wet-lab/plasmid.png)

###### Figure 5. “Strategy to build large synthetic spider silk-like tandem repeat sequences from small double-stranded monomer DNAs flanked by compatible but non regenerable restriction sites” from Florence Teulé et al. (DOI: <https://doi.org/10.1038/nprot.2008.250>) Their description: (a) The engineered silk-like module with appropriate flanking restriction sites is cloned in the plasmid vector. (b) The recombinant plasmid is subjected to two separate restriction digestions and, in both cases, fragments containing the insert are isolated and ligated to each other. (c) The resulting plasmid contains an insert that was doubled in size and has a nonfunctional internal XmaI/BspEI hybrid site. The black stars (★) indicate the restriction digestion of DNA and N× means that the strategy can be repeated as many times as needed.

## Before Jamboree

Before Jamboree we hope to complete an initial proof-of-concept pilot run of the synthesis method with the final product including a N terminus, 3 motifs, and a C terminus. The N and C terminus each contain one motif to yield a total of three motifs after two rounds of ligation. Theoretically, after the addition of enough motifs, the addition of N and C-termini would be added last.

So far, we have started the first phase of this proof-of-concept run; we have already digested our IDT-synthesized motif and pBluescript II SK(+) with XhoI and BamHI, and ligated the two with T4 Ligase. We then transformed the ligated plasmid into DH10B which was then plated on LB plates with Carbenicillin alongside a negative control (Fig. 6). As of 9/30/24, surviving colonies have been inoculated in liquid culture, miniprepped, and sent for sequencing.

![negative-control](https://static.igem.wiki/teams/5275/wet-lab/controlculture-motif1.jpg)
![expculture](https://static.igem.wiki/teams/5275/wet-lab/expculture-motif1.jpg)

###### Figure 6. Transformation Results for Proof of Concept Synthesis Experiment. Left hand side: negative control, air bubbles under the agar due to lack of pre-warming step, ultimately observed no opaque colonies on top of agar; Right hand side: experimental plate, air bubbles under agar due to lack of pre-warming step, observed 10 opaque colonies (in particular, an opaque colony near the middle of the plate).

## Future Direction

The diagram below would be an example of the final product of our synthesis approach. The virtual assembly displays a 3x repeat plasmid with both the N and C terminus added as well as BsaI cut sites flanking both ends of the gene in preparation for future Golden Gate assembly to build the final yeast expression plasmid. Further, given more time and resources, we would be able to go exponentially from 3 repeats to n repeats doubling the number of repeat for every round of synthesis we perform.

![assembled-pbl](https://static.igem.wiki/teams/5275/wet-lab/pbs-updated.png)

Ultimately, we hope to introduce plasmids containing H-fibroin and L-fibroin genes into S. Cerevisiae (lab strain BY4741) for expression of caddisfly silk (or at least the components) synthetically (Fig. 7). Because the main obstacles in synthesizing the caddisfly silk protein involve the H-fibroin, there was no attempt to construct the L-fibroin plasmid. Construction of these plasmids would involve Golden Gate Assembly with parts from the OYC (Open Yeast Collection) provided by the iGEM Distribution Kits (Table 1). This includes parts provided in previous years as well:

![h-fib](https://static.igem.wiki/teams/5275/wet-lab/hfibplasmidupdated.png)
![l-fib](https://static.igem.wiki/teams/5275/wet-lab/lfibplasmidupdated.png)

###### Figure 7. H-Fib and L-Fib Final Plasmids to be Transformed into Yeast after Golden Gate Construction.

#### Table 1a. H-Fibroin Yeast Plasmid Construction with Distribution Kit Parts

| Sequence | Description | Linked Sequence | Part ID |
| --- | --- | --- | --- |
| AAAA | plasmid backbone (OYC-eforRed-dropout) | GCAA | BBa\_J435281 |
| GCAA | Linker | AAAA | IDT |
| AAAA | 5’ homology arm (ScHR5'-HO) | AAGG | BBa\_J435241 |
| AAGG | URA3 selection marker | ATGA | BBa\_J435253 |
| ATGA | Left connector (uses BbsI sites for lvl2) | GGAG | BBa\_J435232 |
| GGAG | Adh1 promoter | AATG | BBa\_J435200 |
| AATG | CDS (L-fibroin or H-fibroin) | CGTT | synthesized |
| CGTT | Terminator (TDH1- can mix and match promoters and terminators) | CGCT | BBa\_J435230 |
| CGCT | Right connector (uses BbsI sites for lvl2) | AGAC | BBa\_J435270 |
| AGAC | 3’ homology arm (ScHR3'-HO) | CGAA | BBa\_J435242 |
| CGAA | Origin of transfer | GCAA | BBa\_J435286 |
| GCAA | E. coli- Amp resistance | ACTA | BBa\_J435256 |
| ACTA | E. coli- origin | AAAA | BBa\_J435289 |

#### Table 1b. L-Fibroin Yeast Plasmid Construction with Distribution Kit Parts

| Sequence | Description | Linked Sequence | Part ID |
| --- | --- | --- | --- |
| AAAA | plasmid backbone (OYC-eforRed-dropout) | GCAA | BBa\_J435281 |
| GCAA | Linker | AAAA | IDT |
| AAAA | 5’ homology arm (ScHR5'-HO) | AAGG | BBa\_J435241 |
| AAGG | URA3 selection marker | ATGA | BBa\_J435253 |
| ATGA | Left connector (uses BbsI sites for lvl2) | GGAG | BBa\_J435232 |
| GGAG | Adh1 promoter | AATG | BBa\_J435200 |
| AATG | CDS (L-fibroin or H-fibroin) w/His-tag on C-terminus | CGTT | synthesized |
| CGTT | Terminator (TDH1- can mix and match promoters and terminators) | CGCT | BBa\_J435230 |
| CGCT | Right connector (uses BbsI sites for lvl2) | AGAC | BBa\_J435270 |
| AGAC | 3’ homology arm (ScHR3'-HO) | CGAA | BBa\_J435242 |
| CGAA | Origin of transfer | GCAA | BBa\_J435286 |
| GCAA | E. coli- Amp resistance | ACTA | BBa\_J435256 |
| ACTA | E. coli- origin | AAAA | BBa\_J435289 |

Two items of note are the linker sequence and the polyhis tag. The linker sequence provided in the OYC collection did not match our needs. In particular, we needed a sequence that has a 5’ GCAA and 3’ AAAA overhang sequence to enable smooth golden gate assembly. Therefore we manually added the above overhangs via IDT synthesis. Further, we added His-tags on the N-terminus of L-fib. This is to be able to perform the purification of L-fib as the L-fib subunit is much smaller (~30 kD) than the H-fib subunit and will elute with other native cellular proteins.

Should we successfully transform the plasmid into BY4741 (confirmed through miniprep and sequencing), we would then proceed to protein purification through HPLC-SEC and His-Tag purification. Later research and literature review would be conducted to determine next steps for constructing the heterodimer protein and post-translational modifications and/or environmental conditions required for functional protein.

1. Bhattacharyya, G., Oliveira, P., Krishnaji, S. T., Chen, D., Hinman, M., Bell, B., Harris, T. I., Ghazitabatabaei, A., Lewis, R. V., & Jones, J. A. (2021). Large scale production of synthetic spider silk proteins in Escherichia coli. Protein Expression and Purification, 183, 105839. <https://doi.org/10.1016/j.pep.2021.105839>
2. Demain, A. L., & Vaishnav, P. (2009). Production of recombinant proteins by microbes and higher organisms. Biotechnology Advances, 27(3), 297–306. <https://doi.org/10.1016/j.biotechadv.2009.01.008>
3. Fukushima, Y. (1998). Genetically engineered syntheses of tandem repetitive polypeptides consisting of glycine-rich sequence of spider dragline silk. Biopolymers, 45(4), 269–279. [https://doi.org/10.1002/(sici)1097-0282(19980405)45:4](https://doi.org/10.1002/%28sici%291097-0282%2819980405%2945%3A4)
4. Heckenhauer, J., Stewart, R. J., Ríos-Touma, B., Powell, A., Dorji, T., Frandsen, P. B., & Pauls, S. U. (2023). Characterization of the primary structure of the major silk gene, h-fibroin, across caddisfly (Trichoptera) suborders. iScience, 26(8), 107253. <https://doi.org/10.1016/j.isci.2023.107253>
5. Lewis, R. V., Hinman, M., Kothakota, S., & Fournier, M. J. (1996). Expression and purification of a spider silk protein: a new strategy for producing repetitive proteins. Protein Expression and Purification, 7(4), 400–406. <https://doi.org/10.1006/prep.1996.0060>
6. Prince, J. T., McGrath, K. P., DiGirolamo, C. M., & Kaplan, D. L. (1995). Construction, cloning, and expression of synthetic genes encoding Spider Dragline Silk. Biochemistry, 34(34), 10879–10885. <https://doi.org/10.1021/bi00034a022>
7. Teulé, F., Cooper, A. R., Furin, W. A., Bittencourt, D., Rech, E. L., Brooks, A., & Lewis, R. V. (2009). A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning. Nature Protocols, 4(3), 341–355. <https://doi.org/10.1038/nprot.2008.250>

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## PCR Method

### Overview

1. DNA Extraction
2. PCR

DNA Extraction

Used the Zymo Quick-DNA Tissue/Insect Microprep Kit D6015 to extract DNA from Caddisfly samples.
Protocol for DNA extraction (from Zymo):
For optimal performance, add beta-mercaptoethanol (user supplied) to the Genomic Lysis Buffer to a final dilution of 0.5%(v/v) i.e., 500 µl per 100 ml.

1. Add specimen(s) to a ZR BashingBead™ Lysis Tube (2.0 mm). Add 750 µl BashingBead™ Buffer to the tube and cap tightly. Note: Generally, no more than 10 mg tissue should be sampled, since larger samples will exceed the DNA binding capacity of the spin column (See Specifications on page 1). Up to 200 µl of whole blood or up to 1.7 x106 cells suspended in 200 µl PBS can also be sampled.
2. Secure in a bead beater fitted with a 2 ml tube holder assembly (e.g., Disruptor Genie™) and process at maximum speed for 10 minutes. Note: Processing time will vary based on sample input and bead beater. Times may be as little as 5 minutes when using high-speed cell disrupters (FastPrep® -24).
3. Centrifuge the ZR BashingBead™ Lysis Tube (2.0 mm) in a microcentrifuge at ≥10,000 x g for 1 minute.
4. Transfer up to 400 µl supernatant to the Zymo-Spin™ III-F Filter in a Collection Tube and centrifuge at 8,000 x g for 1 minute. Discard the Zymo-Spin™ III-F Filter.
5. Add 1,200 µl of Genomic Lysis Buffer to the filtrate in the Collection Tube from Step 4. Mix well.
6. Transfer 800 µl of the mixture from Step 5 to a Zymo-Spin™ IC Column1 in a Collection Tube and centrifuge at 10,000 x g for 1 minute.
7. Discard the flow through from the Collection Tube and repeat Step 6.
8. Add 200 µl DNA Pre-Wash Buffer to the Zymo-Spin™ IC Column in a new Collection Tube and centrifuge at 10,000 x g for 1 minute.
9. Add 500 µl g-DNA Wash Buffer to the Zymo-Spin™ IC Column and centrifuge at 10,000 x g for 1 minute.
10. Transfer the Zymo-Spin™ IC Column to a clean 1.5 ml microcentrifuge tube and add ≥ 20 µl (10 µl minimum) DNA Elution Buffer directly to the column matrix. Centrifuge at 10,000 x g for 30 seconds to elute the DNA.

PCR

Use repliQa HiFi ToughMix

PCR Reaction Mix

* 1.25 uL Forward Primer
* 1.25 uL Reverse Primer
* 12.5 uL repliQa HiFi ToughMix (2X)
* 10 uL dH2O
* Single colony (DNA)

PCR Cycle

* 98°C 3 min
* 32 Cycles:
  + Denaturation: 98°C 30s
  + Gradient Annealing for 30s
    - First half of H-Fibroin gene: 55-65.3°C
    - Second half of H-Fibroin gene: 58-65.4°C
  + Extension: 68°C 130s
* 72°C 5 min
* 4°C ∞

## Synthesis Method

Proof of concept

1. Restriction digest of pBS and motif1 (from IDT) with BamHI and Xho
2. Ligation of motif 1 and pBS
3. Transformation into DH10B
4. Liquid Inoculation
5. Miniprep
6. Begin from step 1 from paper: Restriction digest of pBS+motif1 with ScaI and XmaI (aliquot 1)
7. Restriction digest of pBS+motif1 with ScaI and BspEI (aliquot 2)
8. Gel extraction of both aliquots
9. Ligation of aliquot 1+N terminus and aliquot 2+C terminus
10. Transformation into DH10B
11. Liquid inoculation
12. Miniprep

If we had more time...

1. Starting from step 5, we would repeat steps 6-12 but step 9 would only include ligation of aliquot 1 and 2 and we would continue to add motifs until the desired number, then finally digest and ligate in the N/C termini
2. Prepping level 0 assembly
3. Level 1 Golden Gate Assembly
4. Level 2 Golden Gate Assembly → Assembled yeast plasmid!
5. Yeast transformation
6. Protein Purification

Restriction Digest

Adapted Optimizing Restriction Endonuclease Reactions from NEB

Prepared Restriction Digest Using the Following:

* 5 uL of 200 ng/uL DNA (total 1 ug of DNA)
* 1 uL BamHI
* 1 uL XhoI
* 5 uL rCutsmart Buffer
* 38 uL H2O

Digest for 1 hour at 37°C then deactivate for 15 minutes 65C

Ligation

Prepared Ligation Using the Following:

* 2 uL T4 ligase buffer
* 10 uL Motif1 diges
* 5 uL pBlueScript
* 1 uL T4 ligase
* 2 uL H2O

Room temp for 1 hour + 10 min 65C inactivation

Bacterial (DH10B) Transformation

Using High Efficiency Transformation Protocol using NEB® 10-beta Competent E. coli (High Efficiency) (C3019H/C3019I)

1. Thaw a tube of DH10B Competent E. coli cells on ice for 10 minutes.
2. Add 1 µl of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.
3. Place the mixture on ice for 30 minutes. Do not mix.
4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
6. Pipette 950 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture.
7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C. (did not do so had bubbles)
9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in NEB 10-beta/Stable Outgrowth Medium.
10. Spread 50-100 µl of each dilution onto LB/carb plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

Bacterial Yeast Inoculation

1. Using a sterile pipette tip select a single colony from your plate.
2. “Dig” the pipette tip into the liquid + selection marker
3. Incubate the culture at 37°C (E. Coli) or 30°C (Yeast) for 12-18 hr in a shaking incubator.
4. After incubation, check for growth, which is characterized by a cloudy haze in the media.
5. For long term storage, you can suspend strain in a glycerol stock.

Miniprep

Adapted from Monarch® Plasmid DNA Miniprep Kit Protocol (NEB #T1010)

1. Pellet 1–5 ml bacterial culture (not to exceed 15 OD units) by centrifugation for 30 seconds. Discard supernatant.
2. Resuspend pellet in 200 μl Plasmid Resuspension Buffer (B1) (pink). Vortex or pipet to ensure cells are completely resuspended. There should be no visible clumps.
3. Incubate the culture at 37°C (E. Coli) or 30°C (Yeast) for 12-18 hr in a shaking incubator.
4. Lyse cells by adding 200 μl Plasmid Lysis Buffer (B2) (blue/green). Invert tube immediately and gently 5–6 times until color changes to dark pink and the solution is clear and viscous. Do not vortex! Incubate for one minute.
5. Neutralize the lysate by adding 400 μl of Plasmid Neutralization Buffer (B3) (yellow). Gently invert tube until color is uniformly yellow and a precipitate forms. Do not vortex! Incubate for 2 minutes.
6. Clarify the lysate by spinning for 2–5 minutes at 16,000 x g.
7. Carefully transfer supernatant to the spin column and centrifuge for 1 minute. Discard flow-through.
8. Re-insert column in the collection tube and add 200 μl of Plasmid Wash Buffer 1. Plasmid Wash Buffer 1 removes RNA, protein and endotoxin. (Add a 5 minute incubation step before centrifugation if the DNA will be used in transfection.) Centrifuge for 1 minute. Discarding the flow-through is optional.
9. Add 400 μl of Plasmid Wash Buffer 2 and centrifuge for 1 minute.
10. Transfer column to a clean 1.5 ml microfuge tube. Use care to ensure that the tip of the column has not come into contact with the flow-through. If there is any doubt, re-spin the column for 1 minute before inserting it into the clean microfuge tube.
11. Add 12 μl nuclease-free water to the center of the matrix. Wait for 1 minute, then spin for 1 minute to elute DNA.
12. Nanodrop and record results.

Yeast Transformation

Day -2

1. Thaw strain to be transformed on YPG plate.
2. Prepare DNA for transformation (using appropriate PCR protocol located in dropbox).

Day 1

1. Inoculate each strain to be transformed from the YPG plate into either 5 ml YPD (if step 1) or SC-Ura (if step 2) and grow overnight at 30C.

Day 2

1. For each transformation, start one subculture by diluting 0.2 ml of the overnight culture into either 5 ml of YPD (if step 1) or SC-Ura (if step 2). Grow 4 hrs at 30C (to 5 x 10^6 cells/ml).
2. Turn on 42C water bath and make sure all materials are ready (boiled salmon sperm DNA, DMSO, PEG, water, LiAc at 1M and 0.1M).
3. Harvest cells by centrifuging at 4000 rpm for 5 min. Discard supernatant by pouring, resuspend in 1 ml water and transfer to clean microcentrifuge tube.
4. Centrifuge at 10,000 rpm for 1 min.
5. Aspirate off supernatant and resuspend in 1ml 0.1M LiAc.
6. Centrifuge at 10,000 rpm for 1 min.
7. Aspirate off supernatant and resuspend in 1ml 0.1M LiAc.
8. Centrifuge at 10,000 rpm for 1 min.
9. Aspirate off supernatant and add following solutions in order (directly on top of cell pellet):
   * 0.240 ml 50% PEG
   * 0.035 ml 1 M LiAc
   * 0.025 ml 2 mg/ml ssDNA (boiled 20 min at 99C)
   * 0.036 ml 100% DMSO
   * 0.034 ml DNA
10. Vortex 15 sec to resuspend cells.
11. Incubate for 30 min in 30C incubator.
12. Incubate for 30 min in 42C water bath.
13. Centrifuge at 10,000 rpm for 1 min
14. Discard supernatant and resuspend cells in 0.1 ml water.
15. Plate 0.1 ml on YPD plate. Grow plates at 30C for 14 hours.

Day 3

1. Replica plate from YPD to either SC-URA (if step 1) or 5-FOA (if step 2) plates using velvets.

Protein Purification

1. Collect supernatant from step H centrifuging step and add to bead column (noting to add samples evenly along the wall as to keep the bead level for slow and thorough his-tag purification); Collect flow through; Repeat until samples completely passes (By now, all desired proteins should adhere onto beads)
2. Wash beads using salt solution with gradient change. Then wash with iminazole wash solution ( 300µM of Iminazole + sec buffer contents) 3 times; wait 5 mins for every addition and collect flow through (ensure slow flow for thorough wash);
3. For samples that require a protease cut, seal the bottom of the bead column with parafilm and add sec buffer until beads get submerged. Then add on Eppendorf tube worth of enzyme (ULP1 cut enzyme; enzyme also has his-tags); Seal the top with parafilm and then incubate for overnight on rotating plate
4. Condense flow through volume using filtration tubes with right protein kD size such that all proteins ≥certain threshold gets filtered; add flow through and centrifuge until all liquid either gets condensed or flows through; Take bottle out of centrifuge and flip gently periodically to prevent over condensation which could lead to precipitation and loss of protein products; Wash condensing tube using UP water for future reuse.
5. Wash gel filtration column with sec buffer and calibrate/set up the protein purification machine
6. Load samples to column and calculate predicted harvest volume based on kD value of H-L fibroin complex
7. Start harvesting +2ml before predicted harvest volume and note number of AKTA peaks to determine purity of sample empirically; (ideally, there should be a single peak at the harvest volume) if multiple peaks exist near predicted harvest volume, run western blots to determine the correct tube that contains the target protein
8. Measure concentration mg/ml using nanodrop and cover to concentration.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project and what you learned from them. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}