iGEM 2022 — Team hk-cpu-wyy

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{% block title %}Notebook{% endblock %}
{% block lead %}Documents of the dates we worked on our project.{% endblock %}
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#### June

* Literature review
* programmatically finding target sequences from NCBI
* manually screening sequences, creating gene constructs and primer pairs (for screening and PCR) in Benchling
* order reagents

#### August

## 1st-7th

---

* Cloning of DNA 1-9 with SacI and SalI
* Colony PCR screening

## 8th-14th

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* Cloning of DNA 1-9 into pET-SUMO
* Colony PCR screening

## 15th-21th

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* Cloning of DNA 1-9 into pET-SUMO
* Colony PCR screening
* Mini-prep and sequencing for DNA 6

## 22nd-28th

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* Cloning of DNA 1-9 into pET-SUMO and pBAD-HisB
* Colony PCR screening
* Expression and SDS-PAGE of DNA 6

## 29th-4thSept

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* Cloning of DNA 1-9 into pET-SUMO and pBAD-HisB
* Colony PCR screening

#### September

## 5th-11th

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* Cloning of DNA 1-9 into pET-SUMO and pBAD-HisB
* Colony PCR screening
* Mini-prep for DNA 2 and 9

## 12th-18th

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* UST Lab visit

## 19th-25th

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* Cloning of DNA 1-10 into pET-SUMO, pBAD-HisB and pET-22b
* Colony PCR screening
* Mini-prep for DNA 10
* Expression and SDS-PAGE of DNA 6

## 26th-2ndOct

---

* Expression and SDS-PAGE of DNA 6 and 10
* Building a silk collection model

#### October

## 3rd-9th

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* Expression and SDS-PAGE of DNA 6 and 10
* Purification of DNA 10
* Protein concentrating
* silk fabrication

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| Samples | Treatment | Content | Predicted results |
| --- | --- | --- | --- |
| 1 | Culture solution after sonication | Lysed cell with all cell content | Lots of bands and should include the target band |
| 2 | Supernatant after centrifuge | Cell pellet and insoluble proteins are removed | Less bands than sample 1 and should include the target band |
| 3 | The flow out after adding wash buffer | Soluble protein w/o His-tag | Similar to sample 2, but doesn’t include target band |
| 4 | The flow out eluted by buffer B | Our target protein | Show target band |

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{% block title %}Experiments{% endblock %}
{% block lead %}Description of our research, experiments, and protocols we used in our iGEM project.{% endblock %}
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#### Plasmids design

---

Three types of expression plasmids are involved in our project, they are pET-SUMO, pBAD-HisB and pET-22b.

## pET-SUMO

---

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/pETSUMO.jpeg)

The plasmid consists of 3 parts: the origin of replication, kanamycin resistance gene and the expression system. The origin of replication is needed for the propagation of the plasmid in *E. coli*. The kanamycin resistance gene gives transformants resistance to kanamycin for selection purposes. The expression system consists of an inducible promoter called lacI promoter, restriction sites for enzymes SacI and SalI, the gene of interest and the T7 terminator. The inducible promoter and the terminator combined allows for the regulation of the gene of interest using IPTG as the inducer. The restriction sites are used to clone the gene of interest into the plasmid backbone. The gene of interest encodes the spidroin that we want to express. The expression system expresses the spidroin with a his-tag which helps in the isolation and purification of the desired spidroin.

## pBAD-HisB

---

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/pBADhisB.jpeg)

Compared with pET-sumo, the most essential differences of pBAD-HisB plasmid are inducer promoter, antibiotic resistance gene and restriction sites. L-arabinose operon is the inducer promoter instead of lacl. Thus, arabinose is used instead to allow the expression. The Ampicillin resistance gene allows the transformants to resist another antibiotic, ampicillin. The enzymes used for digestion are EcoRI and HindIII.

## pET-22b(+)

---

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/pet22b3.jpeg)

This is a common plasmid with high copy plasmid. The enzymes for restriction digestion are XbaI and Xhol. The inducer used for expression is IPTG, same as pET-SUMO. The plasmid contains an ampicillin resistance gene which allows the transformants to resist ampicillin.

After modelling, we found that the minimal concentration of IPTG for protein expression is around 0.001M. In order to lower the cost of spridroin production, we will choose 0.001M for protein expression. Also, the modelling results show that proportional expression is the most suitable way to get and adjust the properties of spider silk made.

#### Replication

---

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/replication2.jpeg.jpeg)

Limited by financial burden, we could only order a small amount of DNA from gene companies, but it is impossible for us to succeed by just doing a few experiments, so we had to replicate the amount of DNA in order to allow us to perform more experiments until we succeeded.

## 1. Transformation

---

Before transformation, the competent cells were prepared by mixing the cultured Top10, which is designed for copying plasmids, calcium chloride and calcium chloride with glycerol in different processes. This made the cells become positively charged so that they were easier to take up plasmids which were negatively charged. Since the competent cells were highly unstable, they were stored at -80°C before use.

Firstly, we transformed the plasmids backbones and plasmids containing target genes which were ordered from IDT into the component cells, *E. coli* Top10 by undergoing heat shock. After that, we recovered and plated the cells on the LB agar plate with kanamycin or ampicillin. Only the *E. coli* that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

## 2. Liquid culture

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We selected the colonies from the agar plate, put them in LB broth with proportional amounts of antibiotics to ensure only the cells which have received expected plasmids can replicate in the culture tube. Then, we put the LB broth in a shaking incubator for incubation for around 24 hours.

## 3. Miniprep

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After culture, we pelted the cells, resuspended them with the resuspension buffer, broke the cells with the lysis buffer and neutralised the solution with the neutralisation buffer.

Then, we added the binding buffer and transferred the solution into spin miniprep kits, centrifuged the kits. The plasmids were in the column now. Moreover, we washed away the unwanted materials with the wash buffer twice. Finally, we extracted the plasmid by adding the elution buffer and centrifuging them.

## Final prducts

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* large amount of plasmids containing target genes
* large amount of plasmid backbones

#### Cloning

---

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/cloning2.jpeg.jpeg)

## 1.Digestion

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Firstly, we digested the plasmids containing target genes with corresponding restriction enzymes. Sall and Sacl for pET-SUMO plasmid, HindIII and EcoRI for pBAD-HisB plasmid and XbaI and Xhol for pET-22b(+).

After digestion, we ran gel to ensure the target genes were digested, two bands should be formed, and we cut out the band with lower size which matches the band size of the genes coding spidroin from the agarose gel. Then, we carried out the gene clean, dissolved the gel with the binding buffer, washed away the impurities with the washing buffer and extracted the genes with the elution buffer.

The next step was digesting the plasmid backbones with the same enzymes we mentioned. As a result, both inserts and backbones were digested to create complementary sticky ends for ligation.

## 2. Ligation

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The complementary sticky ends on the backbone and inserts were ligated to create the desired recombinant plasmid.

## 3. Transformation

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In this process, we used the same method we mentioned before. Recombinant plasmid would be transformed into *E. coli* Top10.

After that, recovered and plated the cells on the LB agar plate with kanamycin or ampicillin. Only the *E. coli* that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

## 4. Colony PCR

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After incubation, the colonies were picked to perform colony PCR in order to distinguish between undigested plasmid transformants and recombinant plasmids transformants.

The colonies with the correct band size were extracted from the plate and cultured by liquid culture in order to get a large amount of plasmids for further analysis.

## 5. Gel electrophoresis and restriction digestion

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In order to check the recombinant plasmid, we digested the plasmids with corresponding restriction enzymes and carried out gel electrophoresis. If the insert and plasmid ligated successfully, two bands should be formed and the band size should match the size of backbones and the target gene.

## 6. Sanger sequence

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To verify the recombinant plasmid actually contains the anticipated inserts, we sent the plasmids to the companies to check the sequence of the recombinant plasmids.

#### Expression

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![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/experiments2.jpeg.jpeg)

## 1.Transformation

---

In this process, we used the same method we mentioned before. However, we should transform the recombinant plasmid into *E. coli* BL21 this time.

After that, recovered and plated the cells on the LB agar plate with kanamycin or ampicillin. Only the **E. coli** that had taken up the plasmids could grow and be selected.

Incubated the cells at 37°C for 24-28 hours in order to form colonies for further selection.

## 2. Liquid culture

---

Firstly, we prepared seed culture because freshy culture is recommended for large culture and thus inducer incubation. We selected one of the colonies from the agar plate and cultured by LB both, corresponding antibiotics were added to ensure only the cells which received the plasmids to grow and underwent cell division. We inoculated the **E. coli** overnight.

The next morning, a certain amount of seed culture solution was transferred into a large amount of LB (5ml seed culture solution : 1L LB culture solution) at 37°C until the OD600 reached 0.8. (It should be around 4 hrs for BL21.)

## IPTG incubation

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After the OD600 reached 0.8, we induced the protein expression with a corresponding amount of inducers. The LB culture would be incubated with 0.001M IPTG/arabinose in room temperature for around 12hrs.

## Protein purification

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After IPTG/arabinose incubation, we harvested the cells by centrifugation.Then we discarded the supernatant, resuspended the cell pellet with bufferA (300mM NaCl, 50mM Tris, pH8.0), and added 1mM Phenylmethylsulphonyl fluoride (PMSF). Moreover, we lysed the cells by sonication on ice and collected the supernatant by centrifugation. Finally, we loaded the collected supernatant onto Ni-NTA column, used 5% bufferB (300mM NaCl, 50mM Tris, 250mM imidazole, pH 8.0) and 95% buffer A to wash out unwanted protein at least twice, and use 100% buffer B to elute target protein.

#### Sonicating

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/sonicating2.jpeg.jpeg)

#### Spidroin in gravity column was being eluted bu buffer B(300mM NaCl, 50mM Tris, 250mM imidazole, pH 8.0)

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/spiderioningravity2.jpeg.jpeg)

#### Centrifuge Filter(1000MWCO)

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/filter.jpeg.jpeg)

## SDS-PAGE

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To verify the protein extracted was what we wanted, we had to perform the SDS-PAGE to check the length of the proteins. To ensure the protein detected was not contaminated, we also ran negative samples.

## Protein concentration

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After elution, we filtered the eluted protein solution through the 0.45μm syringe filter to remove insoluble impurities. Then we measured the concentration of the protein which has size around 35kDA using Nanodrop. After that, we transferred the solution into centrifugal concentrators with 10000 MWCO, centrifuged the solution and discarded the filtrate. And then, we measured the concentration of the protein solution. We repeated the whole process until the protein concentration reached at least 200 mg/ml.

## Silk Fabrication

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The concentrated protein is transferred into a syringe and stood overnight to ensure no gas bubbles in the solution. The next day, the spider silk was ejected from the syringe into the mixture of NaAc and NaCl with pH5.0 using a syringe pump. The spider silk should be immersed in a buffer for at least two days to ensure it is fully solidified.

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{% block title %}Results{% endblock %}
{% block lead %}the results of our project{% endblock %}
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#### Overview

## This year, we have successfully:

* replicated Masp2\_2, Masp2\_1, Masp\_1\_102, Masp1\_1 and NT-2Rep-CT-SpyTag.
* made two recombinant plasmids, Masp1\_102 with pET-SUMO and NT-2Rep-CT-SpyTag with pET-22b(+)
* expressed proteins from NT-2Rep-CT-SpyTag
* purified and concentrated the spidroins
* synthesised spider silk from the spidroins expressed

## Replication

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Five DNA including Masp2\_2, Masp2\_1, Masp1\_102, Masp1\_1 and NT-2Rep-CT-SpyTag and three plasmids, including pET-SUMP, pET-HisB and pET-22b(+) are successfully replicated.

## Cloning

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To make the recombinant plasmids, we carried out digestion and ligation. And the recombinant plasmids were transformed in *E. coli* Top10. Colony PCR were carried out for those five plasmids. All plasmids have at least one colony that shows the correct band. Liquid culture and miniprep were carried out for those colonies with positive results in colony PCR. All show correct bands in gel electrophoresis. We then sent all of the samples to the gene company to do sequencing. Only Masp1\_102 and NT-2Rep-CT-SpyTag have ideal sequencing results. For those two recombinant plasmids, transformation has been done one more time, but the *E. coli* should be BL21 for better expression.

#### XbaI and HindIII digestion check for Masp1\_102(~1.2kb)

Here shows eight digestion products of Masp1\_102 samples

Four of them (lines 2,4,6 and 7) shows positive results

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/wyygel3.jpeg)

#### XbaI and HindIII digestion check for Masp1\_1(~1.2kb)

lane 1 is digested and lane 2 does not

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/gel1_2.jpeg)

#### T7 primers for screening PCR

with size of ~1.2kb

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/gel_1-13_.jpeg)
![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/masp.jpeg)
![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/ntrep.jpeg)

## Expression

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After transformation, we selected the colonies to carry out protein expression with inducer IPTG. After that, we did SDS-PAGE for both samples. Both results are not good.

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/sdspage.jpeg)
![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/spytag.jpeg)

As the protein samples used for SDS-PAGE is only from 2ml of LB culture solution, the spridrion concentration is too low and undetectable. And then, the 35kDa band in the NT-2 Rep-CT-SpyTag sample may be caused by non-target protein. In order to figure out the problem, the NT-2Rep-CT-SpyTag sample proceeded to protein purification. The 2L LB culture solution was then purificated. Around 60ml of protein solution is finally produced and another SDS-PAGE has been done.

For the NT-2Rep-CT-SpyTag, we proceed to protein purification. And run SDS-PAGE. The results are satisfactory.

![](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_images/sonication.jpeg)

During purification we collected the samples at different steps for SDS-PAGE. Compared to the other line, the last two lines obviously show less proteins and bands with 35kDa are formed again. This indicates that His-tag gene is work and the 35kDa band should represent the presence of spridroin.

## Silk Fabrication

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The spider silk was successfully formed from the spridroin by pushing the syringer.

[

](https://gitlab.igem.org/2022/hk-cpu-wyy/-/raw/main/wiki/pages/wiki_videos/49s_video.mp4)
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