iGEM 2023 — Team hopkins

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{% block title %}Notebook{% endblock %}
{% block lead %}Documentation for our project over time{% endblock %}
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###### Feb-March (Team)

- Training and ideation!

###### 21 March (HP)

- We arranged an hour-long call with Dr. Stewart from the University of Utah to share our proposed project goals discuss caddisfly silk and gene properties and predict major obstacles. He also shared an unpublished article with information on the modular structure of the h-fibroin gene.

###### 9 April (Team)

- After months of ideation and development we selected caddisfly silk as our Hopkins 2023 project!

###### April-May (Hardware)

- We have ideated designed and integrated new components and modifications to our Hopkins iGEM bioreactor which was first assembled in 2021. This came from substantial discussion and mentorship from Sreeni Eadara (Hopkins 2020-2022). So far we have taken the 3D printing software Marlin and modified it to work with a bioreactor allowing the user to modify parameters such as temperature and pH through a simple LCD interface--similar to how you can change extruder and bed temperatures with a 3D printer to modify output quality.

###### April-May (Hardware)

- Beyond the bioreactor our second substantial idea is for the design and assembly of a spinner kit. The goal is to process our raw silk/fibroin solution into a usable adhesive (spinner) that we can administer to a surface with controlled physical properties (applicator). We have proposed the design identified and ordered parts and are evaluating possible test materials.

###### April 16 (Dry Lab)

- We discussed ideas for modeling our protein and planned a promotional video to show to the JHU Student Initiatives Fund to present our teamÆs activity to JHU engineering faculty.

###### April 30 (Dry Lab)

- We submitted our promotional video for the Student Initiatives Fund.

###### 8 May (Wet Lab)

- We received h-fibroin and l-fibroin sequences from Dr. Russell Stewart.

###### 27 May (Wet Lab)

- Russell Stewart sent us a paper which contained a script to visualize the locations of serine-rich repeats in the sequence. However we were unable to get the script to work properly.

###### May-Jun (Wet Lab)

- We discovered that the heavy chain sequence was too long for a feasible plasmid. Research into shortening our sequences by truncating highly repetitive regions.

###### 1-15 Jun (Hardware)

- Conceptualization and Design: We finalized the design for the portable bioreactor focusing on simplicity affordability and integration possibilities for sensors like pH and optical density. We successfully integrated the temperature sensor into the bioreactor and we have added CAD designs and have 3D-printed new parts for the system.

###### Jun-Jul (Hardware)

- We contacted Dr. Yayuan Liu @ the Liu group (at JHU) to help us with all electrospinner inquiries. She allowed us to test with her electrospinner and learn the intricacies of how it works. For the glue gun we are working out the details of including a microfluidic device. We have developed a model to determine the force and shear to justify using microfluidics over conventional pressurizing methods and are looking for ways to improve the parameters/outputs.

###### 3 May - 19 July (HP)

- We contacted Drs. Paul Frandsen and Gabi Jijon (introduced to us by Dr. Stewart) to find out more about available gene sequences for L- and H- fibroin and obtaining silk samples.

###### 27-29 May (Wet Lab)

- We requested caddisfly samples for rtPCR of our target sequences and we researched BACs and other methods to transform larger plasmids into a chassis. From this we formulated our first experimental plan.

###### 28 May (Dry Lab)

- We modified a script provided by Dr. Stewart to visualize repeats in the H-fibroin sequence.

###### 30 May (Dry Lab)

- Submitted a request to access NovaFold AI for more powerful protein visualization.

###### 30 May (HP)

- We shared our new experimental plan for reduction of repeats with Frandsen Stewart and Jijon for discussion and feedback.

###### 31 May (HP)

- Meetings and feedback from Dr. Stewart on the experimental plan. He provided key information on the known locations of repeats in several species-specific h-fibroins and also suggested that we investigate the requirement of disulfide bond formation and phosphorylation in our E. coli.

###### 1-10 Jul (Hardware)

- Material Procurement: We ordered a mason jar 3D printing materials pH sensor optical density sensor and other necessary components for the bioreactor project.

###### 6 Jun (Dry Lab)

- We developed additional scripts and used repeat finding and local alignment tools to identify repeat motifs. We indexed occurrences of these repeat motifs as candidates for removal from the coding sequence.

###### 9 Jun (Dry Lab)

- Met with Carl-Erik Tornqvist from DNASTAR about visualizing our H-fibroin using tools such as NovaFoldAI and I-Tasser. However we decided not to go forward because there was no guarantee the software would work on our model.

###### 10 Jun (Wet Lab)

- Discussion of the post-translation modification problems. We adjusted the wet lab plan switching our species to G. pellucidus and using a synthetic truncated sequence in lieu of rtPCR.

###### 24 Jun (Wet Lab)

- We completed manual annotations on the entire heavy chain of G. pellucidus and we designed a truncated control h-fibroin sequence.

###### 3 July (Dry Lab)

- We tested the script from Calgary toward reducing truncated sequences though this was not our final approach.

###### 3 July (Wet Lab)

- We finalized our protocol for disulfide bonding in T7 Shuffle Express competent E. coli. We also designed two longer truncated h-fibroins informed by gene annotations. With viable truncated sequences we eliminated the requirement for BACs.

###### 12 July (Dry Lab)

- We did a preliminary red and blue visualization of the GC content of our sequences using a MATLAB script we wrote. This further guided our efforts to truncate our h-fibroin sequences since we could see the frequency of the GC content.

###### 16 July (Dry Lab)

- We further modified the MATLAB script to output both frequency and percentages of the GC content over a 125 base pair sliding frame. This helped us understand the complexity of trying to reduce the GC content.

###### 1-15 Aug (Hardware)

- 3D Printing and Sensor Calibration: We 3D printed the housing for electronics in the bioreactor. We calibrated the pH and optical density sensors for accurate readings.

###### 21 Aug (Wet Lab)

- We conducted an inventory of all the supplies in our lab to determine what we needed to order. Our ProMega plasmid miniprep kit had enough reagents to miniprep eight samples. We found that we were short on outgrowth medium and out of LB.

###### 23 Aug (Wet Lab)

- We resuspended and transformed pJUMP29-1A pJUMP29-1B pJUMP29-1C pJUMP29-1D using kanamycin plates for selection.

###### 24 Aug (Wet Lab)

- After the transformed pJUMP bacteria incubated on the plates for 24 hours we took our plates out of the incubator wrapped in parafilm and put in 4C chilled storage.

###### 31 Aug (Wet Lab)

- We inoculated liquid LB with the transformed pJUMP29 colonies to grow liquid culture.

###### 1-10 Sep (Hardware)

- Chassis Assembly and Sensor Integration: Assemble the bioreactor components into the mason jar. Integrate pH and optical density sensors securely into the system.

###### 1 Sep (Wet Lab)

- We miniprepped the four pJUMP29 parts that we inoculated one day before. We stored the purified DNA in the -20C freezer.

###### 27 Sep (Wet Lab)

- The following supplies arrived at the lab: spectinomycin plates miniprep kit SOC outgrowth medium LB and chloramphenicol plates.We transformed the pJUMP49-2A part along with mCherry ribosome binding site and terminator using chloramphenicol or spectinomycin for selection. We then took the colonies and inoculated media to grow liquid culture.

###### 29 Sep (Wet Lab)

- We miniprepped the four parts that we transformed two days previously and measured concentrations (ng/uL) A280/260 and A260/230 ratios (to check sample quality/purity) using the nanodrop machine. We stored the purified DNA in the -20C freezer.

###### 30 Sep (Wet Lab)

- We resuspended the promoter and transformed the bacteria using ampicillin plates for the selection.

###### 1-15 Oct (Hardware)

- Electronics Integration: We integrated electronic components into the 3D printed housing including microcontrollers for sensor readings and control systems (pH buffer OD600 temperature oxygenation readings).

###### 1 Oct (Wet Lab)

- We inoculated LB media with the promoter colonies.

###### 2 Oct (Wet Lab)

- We miniprepped the promoter plasmid

###### 3 Oct (Wet Lab)

- We assembled the level 1 vectors using the golden braid protocol.

###### 4 Oct (Wet Lab)

- We transformed the level 1 vectors into bacteria

###### 5 Oct (Wet Lab)

- We inoculated LB media with colonies from the four level 1 vectors and noticed the colonies were green but if the assemblies worked the cells wouldnÆt express GFP. We decided to do a diagnostic restriction digest to check if our BsaI enzyme works.

###### 6 Oct (Wet Lab)

- We miniprepped the four level 1 vectors that we transformed two days previously and measured concentrations (ng/uL) A280/260 and A260/230 ratios (to check sample quality/purity) using the nanodrop machine. We sent 12uL to Plasmidsaurus for whole plasmid sequencing. We stored the rest of purified DNA in the -20C freezer. We set up a diagnostic restriction digest to check if our BsaI enzyme works.

###### 8 Oct (Wet Lab)

- We ran the products of restriction digest with BsaI on a gel and compared the resulting bands with expected bands if BsaI worked properly. We received the results of the whole plasmid sequencing and confirmed that our samples from the level 1 assembly contained GFP. We decided to order a new vial of BsaI enzyme to repeat the level 1 assemblies.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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## Purpose And Function

The purpose of our design is to allow E. coli bacteria to produce caddisfly silk solution: specifically, silk of the caddisfly species Glyphotaelius pellucidus. The silk made by caddisflies (including G. pellucidus) consists of two proteins: a long “heavy chain” h-fibroin and a “light chain” l-fibroin; these two proteins are fused together via disulfide bond formation to help create the structure of the caddisfly silk. [[1]](#ref1)

## Choosing A Species:

The first design-related decision was choosing which particular species of caddisfly to use for this project. First, for a species to be used for expressing its silk in E. coli, it needs to have the sequence for its silk proteins published; this alone narrows the possible choices considerably. Second, in order to further narrow our choices, we must pick between the three broad types of caddisflies: cocoon-makers, case-makers, and retreat-makers (i.e. net spinners). [[2]](#ref2) The larvae of cocoon-making caddisflies use their silk to form cocoons, the larvae of case-making caddisflies use their silk to create protective cases around their growing bodies, and the larvae of retreat-making caddisflies use their silk to spin nets which catch food particles. [[3]](#ref3)

For this project, we chose a case-making caddisfly for several reasons. First, retreat-making caddisflies exhibit a high proline content, ranging from 9.9 to 12.3%, while case-makers have a much lower proportion of proline, ranging from 4 to 5.6%. [[2]](#ref2) Since proline inhibits secondary structure formation in proteins, a lower proline content will make it easier to ensure the constructed sequence is capable of forming beta sheets. Second, the repeat pattern in the h-fibroin gene of retreat-makers is much more complicated than that of case-makers,3 which would make it more difficult to truncate the h-fibroin gene of a retreat maker. (See next section below, “Overcoming Challenges In Building The Design,” for more details on why the h-fibroin has to be truncated in size.)

Cocoon-making caddisflies have lower proline content, and an even more simple genetic repeat pattern than that of case-makers. [[2]](#ref2) However, we decided against using a cocoon-making caddisfly, due to concerns that cocoon silk would not work well as an adhesive. In contrast to silk sticking to itself in order to form a cocoon, the silk spun by case-making caddisfly larvae sticks to pebbles and other underwater objects, in order to form the larvae’s protective case.

Prof. Russell Stewart of the University of Utah pointed us to a pre-print (now a published study), which sequenced the genes of several different caddisfly species, including different case-making species. While the paper describes in words the genetic repeat patterns for each of the species sequenced, there is only one case-making caddisfly species for which the paper has a visual diagram, showcasing the structure of the h-fibroin gene: Glyphotaelius pellucidus. [[3]](#ref3) Knowing that a visual illustration of the h-fibroin genetic pattern would greatly help us in truncating the gene, we chose G. pellucidus, it being the only case-making caddisfly species for which a visual diagram of the h-fibroin gene exists.

![](https://static.igem.wiki/teams/4676/wiki/engineering/silk-structure-hires.jpg )

The above figure comes from Heckenhauer, J., & Stewart, R. J., et al. (2023, August 18). “Characterization of the primary structure of the major silk gene, h-fibroin, across caddisfly (Trichoptera) suborders.” DOI: 10.1016/j.isci.2023.107253

## Overcoming Challenges In Building The Design:

The first design challenge arises with the length of the heavy chain: the h-fibroin of G. pellucidus, without introns, is 23,595 base pairs long - simply too long to give to bacteria as a regular plasmid. [[2]](#ref2) A second challenge is that the h-fibroin of Caddisflies, including G. pellucidus, has too many repeated motifs in its DNA to be ordered and synthesized from a biotech company - in our case, our sponsor IDT (Integrated DNA Technologies). With regards to G. pellucidus, the h-fibroin contains two DNA motifs, labeled RM1 and RM2, which are repeated throughout most of the protein. [[3]](#ref3)
Our multiple approaches to sequence analysis, which includes scripts for the identification of repeats and visualization of GC content, may be found on our [software GitLab](https://gitlab.igem.org/2023/software-tools/hopkins).

Fortunately, this repetitive nature of the h-fibroin allows for a solution to both the first and second challenges: the length of the h-fibroin can be truncated by reducing the number of repeats. In our design, we truncated the h-fibroin of G. pellucidus to contain only one RM1 and one RM2 region.

One might ask if changing the gene, by shrinking it via a reduction in the number of repeats, will affect the efficacy of the silk proteins produced. As Prof. Stewart informed us, however, research has shown that the number of repeats naturally varies within a population of caddisfly larvae of the same species. [[2]](#ref2),[[5]](#ref5) This suggests that an exact number of genetic repeats is not needed for silk protein efficacy.

The third design challenge comes from the need for disulfide bond formation. As mentioned before, the silk structure of caddisflies involves the h-fibroin protein and the l-fibroin protein being connected together with disulfide bonding.[[1]](#ref1) But E. coli are not normally well adapted to disulfide bond formation in proteins they express.[[6]](#ref6) One solution implemented was use of T7 Shuffle strain E. coli as a chassis, since that strain of bacteria has been engineered with the purpose of boosting disulfide bond formation in proteins expressed. [[7]](#ref7) A second solution was co-expressing the fusion protein PDI-GPx7 - a protein created to further boost disulfide bonding for proteins forming in T7 Shuffle strain E. coli. [[8]](#ref8)

A fourth design challenge, unfortunately, was created by our use of T7 Shuffle bacteria. T7 Shuffle E. coli are resistant to the same antibiotic, Spectinomycin, used to plate the level 2 Golden Gate plasmid backbone (pJUMP49-2A(sfGFP)) used in this project. [[9]](#ref9) This could severely hinder protein purification, as we would not know which colonies to select for purification, since we would have no way of knowing which E. coli colonies took up the plasmid and which did not. (If the T7 Shuffle strain was not naturally resistant to Spectinomycin, we would be able to assume any surviving colony must have been given resistance from the backbone of our assembly.) The best possible solution was adding mCherry to the fourth slot in our Golden Gate plasmid assembly; this way, we will be able to see which colonies take up the plasmid and which don’t, as colonies which do take up the second assembly will glow red.

This brings us to the final challenge: protein purification. In order to conduct protein purification, we added a His-Tag and a Sumo sequence to the c-terminus of the l-fibroin. We specifically chose to add these sequences to the c-terminus of the l-fibroin, for the following reasons. One, the n-terminus of the l-fibroin is the docking site for the disulfide bond with the c-terminus of the h-fibroin [[10]](#ref10); thus neither the n-terminus of the l-fibroin nor the c-terminus of the h-fibroin could work. Two, the h-fibroin is a monomer whereby, in forming the silk structure, it uses its n-terminus to attach to other h-fibroins [[11]](#ref11); thus the n-terminus of the h-fibroin could not work either. Therefore, the c-terminus of the l-fibroin is the only possible location where a His-Tag and Sumo sequence can be added for protein purification.

## Testing The Design, And Learning From The Results:

## A. Testing The Assembly

One key test we implemented on our design is determining if the plasmid, described above, is properly assembled. One way to test assembly is the color of the bacterial colonies transformed. If the colonies are green, that would suggest the GFP sequence from the plasmid backbone was not removed by the enzyme (for our project, this would be Bsa1 enzyme), and thus was not replaced with the four parts (truncated h-fibroin, l-fibroin, fusion protein, and mCherry) listed above.

![](https://static.igem.wiki/teams/4676/wiki/engineering/green-colonies.jpeg)

Green colonies

After our first attempt at assembly resulted in green colonies (shown above), indicating a failed attempt, we did two more tests. First, a restriction digest, to check if our Bsa1 enzyme is actually cutting; and whole plasmid sequencing, to see if the desired plasmid ended up being produced. The results of the restriction digest were visualized using a gel, as shown below. Read from left to right, lanes 1 and 7 are 1kb plus ladders, and lanes 2 and 8 are 100bp ladders. In the middle are the pJUMP1A-h-fibroin, pJUMP1B-l-fibroin, pJUMP1C-fusion protein, and pJUMP1D-mCherry respectively:

![](https://static.igem.wiki/teams/4676/wiki/engineering/restriction-gel.png )

diagnostic restriction digest gel

The gel bands indicate the Bsa1 enzyme used was not cutting. The expected bands that should be in lane 3 of the gel above, if the digest worked, are shown below in the virtual digest. (All five lanes in the image below are for the pJUMP1A-H-fibroin.) Since there is no band around 800 to 900 bp, or any near 100 bp, there were no cuts. The three bands on the gel are from the lengths of the different uncut plasmids and the h-fib gene fragment.

![](https://static.igem.wiki/teams/4676/wiki/engineering/virtual-digest-sequence-a-1.png)

virtual restriction digest for pJUMP1A-h-fibroin

A similar lack of expected results occurred with the other lanes in the gel. The expected results are shown below:

![](https://static.igem.wiki/teams/4676/wiki/engineering/virtual-digest-sequence-b-1.png)

virtual restriction digest for pJUMP1B-l-fibroin

![](https://static.igem.wiki/teams/4676/wiki/engineering/virtual-digest-sequence-c-1.png)

virtual restriction digest for pJUMP1C-fusion protein

![](https://static.igem.wiki/teams/4676/wiki/engineering/virtual-digest-sequence-d-1.png)

virtual restriction digest for pJUMP1D-mCherry

We learned from these results that the Bsa1 we were using was not functional, and that new Bsa1 enzyme needed to be obtained for the next attempt at assembly.

The whole plasmid sequencing further confirmed that the enzyme did not remove GFP from the plasmid backbone, as shown below:

![](https://static.igem.wiki/teams/4676/wiki/engineering/plasmid-a.png)

virtual restriction digest for pJUMP1A-h-fibroin

![](https://static.igem.wiki/teams/4676/wiki/engineering/plasmid-b.png)

annotated plasmid for pJUMP1B-l-fibroin

![](https://static.igem.wiki/teams/4676/wiki/engineering/plasmid-c.png)

annotated plasmid for pJUMP1C-fusion protein

![](https://static.igem.wiki/teams/4676/wiki/engineering/plasmid-d.png)

annotated plasmid for pJUMP1D-mCherry

## B. Testing Protein Expression

Another key test is simply obtaining a product at the end. We designed the plasmid to have a His-Tag on the l-fibroin c-terminus, in order to do protein purification. Therefore, if the plasmid is expressed correctly inside the Shuffle T7 cells, we will be able to purify a protein product.

## C. For Future Projects: Ordering A Bigger H-Fibroin

Russell Stewart advised us that the smallest possible truncated h-fibroin could function as a control, against which longer truncated versions can be compared for silk properties such as tensile strength and adhesion.

In our endeavor to make a bigger sequence: we tested it on the IDT wizard and ran into a few issues with the compatibility score. The GC rich regions would have to be removed in order for the sequence to be compatible with the IDT regions. Additionally, sequences that were too repetitive would have to be axed. We discarded the idea of attempting to create a longer h-fibroin than our control because it seemed unlikely that we would be able to express and then purify enough protein to compare the qualities of the variations.

## D. Our Plan For Before Jamboree

Once we acquire new BsaI, we will repeat the Level 1 assembly with BsaI into pJUMP29-1A thru 1D. After transformation in stable competent cells, we will inoculate the transformed cells in LB and miniprep to get DNA for the four level 1 transcriptional units. Then we will do Level 2 assembly with BsmBI into pJUMP49-2A. Following transformation, inoculation, and miniprep, we will send our final assembled plasmid for whole plasmid sequencing to get
confirmation of successful assembly. Next, we will transform the level 2 assembled plasmid into Shuffle T7 Express cells for protein expression. From the plate, we will pick the red colonies that express mCherry to inoculate in a large liquid culture. Lastly, we will use that culture to perform His-Tag protein purification using column filtration and use a SUMO protease to cleave off the His-Tag.

## References

1. [https://pubs.acs.org/doi/10.1021/bm060663u](https://pubs.acs.org/doi/10.1021/bm060663u%20)
2. [Heckenhauer, J., & Stewart, R. J., et al. (2023, August 18). Characterization of the primary structure of the major silk gene, h-fibroin, across caddisfly (Trichoptera) suborders. iScience. DOI: 10.1016/j.isci.2023.107253](https://pubmed.ncbi.nlm.nih.gov/37529107/)
3. Ibid
4. Ibid
5. <https://royalsocietypublishing.org/doi/10.1098/rstb.2019.0206>
6. [https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896424/](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896424/%20)
7. Ibid
8. [Lenon, M., Ke, N., Szady, C., Sakhtah, H., Ren, G., Manta, B., Causey, B., & Berkmen, M. (2020, September 30). Improved production of Humira antibody in the genetically engineered escherichia coli shuffle, by co-expression of human PDI-GPX7 fusions - Applied Microbiology and Biotechnology. SpringerLink.](https://link.springer.com/article/10.1007/s00253-020-10920-5)
9. <https://www.neb.com/en-us/faqs/0001/01/01/do-streptomycin-and-or-spectinomycin-need-to-be-added-to-shuffle-strains>
10. [Collin, M.A., Mita, K., Sehnal, F. et al. Molecular Evolution of Lepidopteran Silk Proteins: Insights from the Ghost Moth, Hepialus californicus . J Mol Evol 70, 519–529 (2010).](https://doi.org/10.1007/s00239-010-9349-8%20)
11. [Naoyuki Yonemura, František Sehnal, Kazuei Mita, and Toshiki Tamura Biomacromolecules 2006 7 (12), 3370-3378 DOI: 10.1021/bm060663u](https://pubs.acs.org/doi/10.1021/bm060663u)

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Description of our experimental procedure, wetlab protocols, and parts used. {% endblock %}
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## Experimental Overview

Preparing Bacteria and Level 0 Assembly

#### First, we put our parts into Level 0 plasmid assembly vectors.

1. Make 2L LB media and 2L LB + agar for plates **(plate pouring protocol below)**
2. Resuspend the following dry plasmid DNA samples:

* pJUMP29-1A(sfGFP)
* pJUMP29-1B(sfGFP)
* pJUMP29-1C(sfGFP)
* pJUMP29-1D(sfGFP)
* pJUMP49-2A(sfGFP)
* Ribosome Binding Site: BBa\_B0030
* Terminator: BBa\_B0015
* mCherry
* Promoter: AB\_T7

3. Thaw and transform NEB StableCompetent E.coli cells with the resuspended plasmids **(see transformation protocol below)**. Each part should be in its own bacterial culture.
4. After 24 hours of incubation, pick colonies from the selection plate and inoculate LB media to grow liquid culture.**(links to protocols coming soon)**
5. Miniprep the colonies using a kit of your choice. We used the ProMega PureYield Miniprep kit (see protocol below).
6. Find the DNA concentration of each component using a nanodrop machine.
7. Store the miniprepped plasmids in -20˚C.

Levels 1 and 2 Plasmid Assembly

#### For our assembly, we used the Golden Braid protocol.

1. Resuspend powdered IDT products in to concentration specified on the bottle.

* H-fibroin
* L-fibroin SUMO
* PDI-GPx7 Fusion Protein

2. Make 2L LB + Kan + agar solution for plates; Pour plates
3. The four coding sequences are the three IDT products and mCherry. Assemble each one into a Level 1 plasmid vector (protocol below).

* The reaction volume for the assembly will be 20µL.
* Calculate volumes so that the reaction tube contains 135.97ng of coding sequence, and 75ng each of the promoter, RBS, terminator, and respective pJUMP backbone.

+ pJUMP29-1A goes with H-fibroin
+ pJUMP29-1B goes with L-fibroin SUMO
+ pJUMP29-1C goes with the Fusion Protein
+ pJUMP29-1D goes with mCherry

* Top up to 20µL by adding an appropriate amount of DI PCR-grade water.
* To each reaction tube, add the following

+ 0.5 ul of Hi-T4 DNA ligase
+ Add 2 ul of 10x T4 ligase buffer
+ 1 ul of BsaI restriction enzyme

* Put the reaction tubes in a thermocycler using appropriate run settings.
* When the run is over, put the assembled Level 1 plasmids into storage at -20˚C.

4. Transform the level 1 plasmids into their own bacterial cultures, using the same protocol. Use about 2-5 µL of DNA for each plasmid sample.
5. Streak on LB + Kan plates; grow overnight and pick colonies for colony PCR to confirm transfection.
6. If level 1 assembly is successful, miniprep the four plasmids and proceed to level 2 assembly
7. Mix all level 1 plasmids with the appropriate backbone for level 2 assembly of the final expression vector.
8. Perform colony PCR to check success of level 2 assembly.
9. Perform plasmid miniprep with streaked cells made during PCR step and store plasmids in -20C˚

Expression and Protein Purification

#### Protein Expression in ShuffleT cells

1. **Transform SHuffle T7 competent *E. coli*** with the level 2 plasmid prepared earlier, using above transformation protocol.
2. Streak the bacteria on LB + Kan plates to induce expression of plasmid and ensure successful transformation, selecting for transformed cells.
3. After individual colonies appear, **perform colony PCR (see above)** to ensure plasmid presence
4. If PCR successfully confirms transformation, streak out cells on LB + Kan plates again and add some colonies to five test tubes of 1 L LB + Kan solution for overnight growth
5. Ideally, the SHuffle T7 cells will correctly fold the silk protein with disulfide bonds as they grow and express the genes in the plasmid.

#### Cell Lysis and Preparing for Protein Purification

1. While cells grow in the liquid culture, prepare for protein purification by making **lysis buffer** and **sec buffer**.

* Cell lysis buffer: 25 µM Tris, 500 µM NaCl, 5% glycerol, Beta-me
* Sec buffer for gel chromatography: 500 µM NaCl, 50µM Tris buffer, 5% glycerol (5% in terms of volume), ultrapure water, 2µM DTT

2. Measure cell growth in spectrophotometer. After cell growth reaches the desired OD600, centrifuge in plasmid bottles and discard supernatant
3. Resuspend cell pellets in prepared lysis buffer (15 ml per 3L samples) and pass through hydraulic grinder

* Wash grinder first with anti-bacterial fluids to clean out any potential contamination
* Add some lysis buffer and some sec buffer to completely covert the internal environment of the tubes
* (After wash) flow resuspended cell solution into the grinder and increase pressure slowly until 580 - 800 bar
* Harvest solution after cell solution appear less viscous

4. Collect solution of lysed cells and add to high strength test tubes for high speed centrifuge (13500 g for 1-2 hr)
5. While centrifuging samples, prepare nickel beads by washing beads 3 times in sec buffer

#### Protein Purification (Must be performed in 4-degrees Celsius)

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.

## Relevant Protocols

High Efficiency Transformation for NEB® Stable Competent E. coli (C3040)

#### Materials

* NEB StableCompetent E. coli
* Ice in ice bucket
* Microcentrifuge Tubes
* Heat Block
* Ethanol benchtop

#### Procedure

1. Warm heat block to 42°C
2. Calculate how many tubes of NEB Stable Competent E. coli cells needed. (minimum 25uL of cells needed per reaction) (they come in 200uL / 50uL tubes)
3. Thaw a tube of NEB Stable Competent E. coli cells on ice for 10 minutes. Once the stock tube is thawed, you cannot re-freeze it or the cells will explode and not be competent. So each tube is 1 time use only
4. Add 1 -2 µl containing 100 pg to 100 ng of plasmid DNA to the cell mixture. (unless you have a ridiculously low DNA concentration, 2 uL will usually be sufficient.) Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex or pipette.
5. Place the mixture on ice for 30 minutes.
6. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix. Place on ice for 5 minutes. Do not mix.
7. Pipette 475 µl of room temperature NEB 10-beta/Stable Outgrowth Medium into the mixture and place at 30°C for 60 minutes. Shake the tube horizontally at 250 rpm or rotate.
8. Warm selection plates to 30°C.
9. Mix the cells thoroughly by flicking the tube and inverting. Then spread 50-100 µl of cells or diluted cells onto a selection plate. Incubate plates 24 hrs at 30°C or overnight at 37°C.

Colony PCR (ThermoFisher RepliQ A Kit)

1. 10µl ultra pure water, 2.5µl forward primer, 2.5 µl reverse primer,
   10µl enzyme mixture for each sample
2. Pick individual colonies and steak them out first on a separate LB + Kan plate before adding colonies into solution mixture
3. Run in thermocycler using the right settings (I will check Benchling to see what our actual cycle is so I can put it in here)
4. Add loading dye (5µl of 5x purple loading dye)
5. Make 100ml 1x TAE + 1mg agarose mixture, heat and mix until homogenous, and pour into mold with appropriate well divider
6. After gel solidifies, pour 1x TAE buffer to max line and add 5µl of the appropriate ladder on the far left; then add 5µl of each sample to subsequent wells
7. Run at 110V until leading edge almost reaches the end; then image with available machines

ProMega PureYield Miniprep

#### Preparing Cells and Lysis

1. Make sure that the **Column Wash Solution** has been prepared by adding ethanol.
2. Preparing Cells:

1. Centrifuge 1.5ml of bacterial culture for 30 seconds at maximum speed in a microcentrifuge. Discard the supernatant.
2. Add an additional 1.5ml of bacterial culture to the same tube and repeat Step 1.
3. Add 600μl of TE buffer or water to the cell pellet, and resuspend completely.

3. Add 100μl of Cell Lysis Buffer (Blue), and mix by inverting the tube 6 times.

* The solution should change from opaque to clear blue, indicating complete lysis.
* Note: Proceed to Step 3 within 2 minutes. Excessive lysis can result in denatured plasmid DNA.
* If processing a large number of samples, process samples in groups of ten or less.
* Continue with the next set of ten samples after the first set has been neutralized and mixed thoroughly.

4. Add 350μl of cold (4–8°C) Neutralization Solution, and mix thoroughly by inverting.
5. The sample will turn yellow when neutralization is complete, and a yellow precipitate will form. Invert the sample an additional 3 times to ensure complete neutralization
6. Centrifuge at 20000g in a microcentrifuge for 3 minutes.
7. Transfer the supernatant (~800 to 900μl) to a PureYield™ Minicolumn with a pipette. Do not disturb the cell debris pellet.
8. Place the minicolumn into a Collection Tube, and centrifuge at maximum speed (20,000 g) in a microcentrifuge for 30 seconds.
9. Discard the flowthrough, and place the minicolumn into the same Collection Tube.

#### Washing and Elution

1. Add 200μl of Endotoxin Removal Wash (ERB) to the minicolumn. Centrifuge at 20000g for 30 seconds.
2. Add 400μl of Column Wash Solution (CWC) to the minicolumn. Centrifuge at 20000g for 30 seconds.
3. Transfer the minicolumn to a clean 1.5ml microcentrifuge tube, then add 30μl of elution buffer to the column.
4. Let the column sit for 1 minute.
5. Centrifuge at 20000g for 30 seconds.
6. Store the eluted purified plasmids in -20˚C.

You can also find this protocol on the [ProMega website.](https://www.promega.com/resources/protocols/technical-bulletins/101/pureyield-plasmid-miniprep-system-protocol/)

Resuspension of Dry DNA Products

###### iGEM Distribution Kit Parts:

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that you want. Make sure you have properly oriented the plate. Do not remove the foil cover, as it could lead to cross contamination between the wells
2. Pipette 10µL of dH2O (distilled water) into the well. Pipette up and down a few times and let sit for 5 minutes to make sure the dried DNA is fully resuspended. The resuspension will be red, as the dried DNA has cresol red dye. We recommend that you do not use TE to resuspend the dried DNA.
3. Transform 1µL of the resuspended DNA into your desired competent cells, plate your transformation with the appropriate antibiotic\* and grow overnight.
4. Pick a single colony and inoculate broth (again, with the correct antibiotic) and grow for 16 hours.
5. Use the resulting culture to miniprep the DNA AND make your own glycerol stock (for further instruction on making a glycerol see this page). We recommend using the miniprepped DNA to run QC tests, such as restriction digests and sequencing.

###### Twist Biosciences:

* For resuspension, briefly centrifuge the tube or plate before opening and resuspend in nuclease free Tris-EDTA (TE) buffer, pH 8.0 or 10 mM Tris-HCl, pH 8.0 to the desired concentration. A concentration of at least 10 ng/μL is recommended for the stock dilution

###### IDT Products:

* Use the same protocol as with iGEM Distribution Kit parts, but make sure to use molecular biology / PCR-grade water as millipore DI water can be too acidic.

Inoculating a Bacterial Culture

1. Prepare liquid LB. For example, to make 400 mL of LB, weigh out the following into a 500 mL glass bottle:

* 4g NaCl
* 4 g Tryptone
* 2 g Yeast Extract
* Add dH2O to 400 mL
* Note: If your lab has pre-mixed LB agar powder, use the suggested amount, instead of the other dry ingredients above.

2. Loosely close the cap on the bottle (do NOT close all the way or the bottle may explode!) and then loosely cover the entire top of the bottle with aluminum foil. Autoclave and allow to cool to room temperature.
3. Now screw on the top of the bottle and store the LB at room temperature.

* Note: If you intend to do a mini-prep you will usually want to start 3 mL in a falcon tube, but for larger preps you might want to use as much as a liter of LB in a 2 L Erlenmeyer flask.

4. Using a sterile pipette tip or toothpick, select a single colony from your LB agar plate.
5. Drop the tip or toothpick into the liquid LB + antibiotic and swirl.
6. Loosely cover the culture with sterile aluminum foil or a cap that is not air tight.
7. Incubate bacterial culture at 37°C for 12-18 hr in a shaking incubator.

* Note: Some plasmids or strains require growth at 30°C. If so, you will likely need to grow for a longer time to get the correct density of bacteria since they will grow more slowly at lower temperatures

8. After incubation, check for growth, which is characterized by a cloudy haze in the media.
9. For long term storage, you can suspend bacteria in a [glycerol stock](https://www.addgene.org/protocols/create-glycerol-stock/).

## Parts List

| Part Number | Part Name | Part Source | Selection Antibiotic |
| --- | --- | --- | --- |
| BBa\_K4676000 | Truncated Control H-Fibroin of G. Pellucidus | IDT | Kanamycin |
| BBa\_K4676001 | L-Fibroin of G. Pellucidus | IDT | Kanamycin |
| BBa\_K4676002 | PDI-GPx7 Fusion Protein Gene | IDT | Kanamycin |
| BBa\_K4676003 | Truncated Control H-Fibroin Transcriptional Unit | New | Kanamycin |
| BBa\_K4676004 | L-Fibroin Transcriptional Unit | New | Kanamycin |
| BBa\_K4676005 | PDI-GPx7 Fusion Protein Transcriptional Unit | New | Kanamycin |
| BBa\_K4676006 | mCherry Transcriptional Unit | New | Kanamycin |
| BBa\_K4676007 | Final Level 2 Expression Vector | New | Spectinomycin |
| BBa\_J428326 | pJUMP29-1A(sfGFP) backbone | iGEM | Kanamycin |
| BBa\_J428326 | pJUMP29-1B(sfGFP) backbone | iGEM | Kanamycin |
| BBa\_J428326 | pJUMP29-1C(sfGFP) backbone | iGEM | Kanamycin |
| BBa\_J428326 | pJUMP29-1D(sfGFP) backbone | iGEM | Kanamycin |
| BBa\_J428327 | pJUMP49-2A(sfGFP) backbone | iGEM | Spectinomycin |
| BBa\_J428032 | RBS: BBa\_B0030 | iGEM | Chloramphenicol |
| BBa\_J428092 | Terminator: BBa\_B0015 | iGEM | Chloramphenicol |
| BBa\_J435340 | Promoter: AB\_T7 | iGEM | Ampicillin |
| BBa\_J4208079 | mCherry | iGEM | Chloramphenicol |

## Photos

![Image 1](https://static.igem.wiki/teams/4676/wiki/img-5735.jpg)

![Image 2](https://static.igem.wiki/teams/4676/wiki/img-5737.jpg)

![Image 3](https://static.igem.wiki/teams/4676/wiki/img-5731.jpg)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% 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](http://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. 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.

## 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 %}