iGEM 2021 — Team HUST2-China

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# Team:HUST2-China/Notebook

Notebook | iGEM HUST2-China

[HUST2-China](https://2021.igem.org/Team%3AHUST2-China)

* Team

  [Team](https://2021.igem.org/Team%3AHUST2-China/Team)[Attributions](https://2021.igem.org/Team%3AHUST2-China/Attributions)[Partnership](https://2021.igem.org/Team%3AHUST2-China/Partnership)
* Project

  [Description](https://2021.igem.org/Team%3AHUST2-China/Description)[Design](https://2021.igem.org/Team%3AHUST2-China/Design)[Safety](https://2021.igem.org/Team%3AHUST2-China/Safety)[Implementation](https://2021.igem.org/Team%3AHUST2-China/Implementation)
* Lab

  [Notebook](https://2021.igem.org/Team%3AHUST2-China/Notebook)[Protocols](https://2021.igem.org/Team%3AHUST2-China/Protocols)[Engineering](https://2021.igem.org/Team%3AHUST2-China/Engineering)
* HP

  [Human Practices](https://2021.igem.org/Team%3AHUST2-China/Human_Practices)[Communication](https://2021.igem.org/Team%3AHUST2-China/Communication)[Collaborations](https://2021.igem.org/Team%3AHUST2-China/Collaborations)
* Model

  [Model](https://2021.igem.org/Team%3AHUST2-China/Model)[Genetic Expression](https://2021.igem.org/Team%3AHUST2-China/Genetic_Expression)[TLR2 Antagonist](https://2021.igem.org/Team%3AHUST2-China/TLR2_Antagonist)[Bacteriostasis of BLP-7](https://2021.igem.org/Team%3AHUST2-China/Bacteriostasis_of_BLP-7)[ELP Aggregation](https://2021.igem.org/Team%3AHUST2-China/ELP_Aggregation)
* Parts

  [Contribution](https://2021.igem.org/Team%3AHUST2-China/Contribution)

# Notebook

A sample page for the theme.

---

##### Contents

# July 2021

## WEEK3

**7.13**

* Prepare for plasmid transformation.

**7.14**

* Plasmid transformation of Part4(wild-type BLP-7 + linker +27\*ELP) into E.coli.
* Culture the transformed bacteria on the plate with ampicillin added and liquid medium LB with ampicillin.

**7.15**

* Culture the transformed bacteria on the plate with ampicillin.

**7.16**

* Plasmid transformation of Part2(optimized-type BLP-7 + linker +27\*ELP) into E.coli.

**7.17**

* Plasmid transformation of Part 2, 7(wild-type TLR-2 competitive inhibitor + linker + 27*ELP), 8(mutational TLR-2 competitive inhibitor + linker + 27*ELP) into E.coli. Pick monoclones from plate of Part 4.

## WEEK4

**7.18**

* Pick monoclones from plates of Part 2, 4, 7, 8.
* Keep 4 tubes of monoclones(Part 2, 4, 7, 8) in shaker at 45 centigrade to induce E.colito express the target protein.

**7.19**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and analysis the samples with SDS-PAGE.

**7.20**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and analysis the samples with Western Blot.

**7.21**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and analysis the samples with Western Blot.

**7.22**

* Prepare 4L LB medium.

**7.23**

* Induce E.coli to express the target protein by infrared light.
* Induce E.coli to express the target protein at 45 centigrade, and purify the protein by Ni-column.

**7.24**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.

## WEEK5

**7.25**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
* Purify the target protein by Ni-column.

**7.26**

* Purify the target protein E.coil by Ni-column.
* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8, and perform Western Blot.

**7.27**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform Western Blot.
* Induce E.coli to express the target protein at 45 centigrade.
* Prepare 3L LB medium.

**7.28**

* Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
* Analysis the target protein on SDS-PAGE.

**7.29**

* Induce E.coli to express the target protein at 45 centigrade for 3, 6, 9, 12 hours.
* Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.

**7.30**

* Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2,4,7,8 and perform SDS-PAGE and Western Blot.
* Perform SDS-PAGE of the target protein.

**7.31**

* Lyse the induced(by infrared light for 40, 50, 60, 70 minutes) and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.

# August 2021

## WEEK6

**8.1**

* Prepare solution needed.

**8.2**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
* Purify protein with FPLC.
* Purify the target protein.

**8.3**

* Induce E.coli to express target protein at 45 centigrade.
* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.
* Perform SDS-PAGE of the purified target protein.
* Prepare for experiment of ELP assembly.

**8.4**

* Test the anaerobic bag.
* Purify the sample of Part2 and 4 with FPLC.
* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8 and perform SDS-PAGE and Western Blot.

**8.5**

* Lyse the induced and non-induced E.coli with Part2, 4, 7, 8.
* Purify protein with FPLC.

**8.6**

* Perform SDS-PAGE with sample collected yesterday.
* Purify the sample with FPLC.

**8.7**

* Purify the sample of Part4 with FPLC.
* Culture the transfected bacteria on the plate with ampicillin.
* Perform BCA and SDS-PAGE to determine the concentration of E.coli induced (temperature at 45 centigrade for 24､27､30 and 33 hours) protein.

## WEEK7

**8.8**

* Perform SDS-PAGE to determine the concentration of sample.
* Induce Part2 by infrared light for 40, 50, 60, 70 minutes and then Perform SDS-PAGE, and get a unclear band between 10KDa and 15KDa marker.

**8.9**

* Purify the sample of Part7 with FPLC.
* Perform SDS-PAGE and Western Blot of Part2 induced by infrared light for 40,50,60,70 minutes.

**8.10**

* Perform BCA test and Western Blot.
* Perform SDS-PAGE of samples of Part4 and Part7 again.
* Test the anaerobic and prepare and culture the P.acnes on the plate with heart and brain extract.
* Pick monoclones from the plates of Part2.
* Induce E.coli to express the target protein at 45 centigrade for 24, 27, 30 and 33 hours.

**8.11**

* Purify the sample of Part8 with FPLC.
* Pick monoclones from the plates of Part8.
* Culture the transfected bacteria on the plate with ampicillin and induce E.coli to express target protein at 45 centigrade.
* Perform Western Blot of samples of Part 2.

**8.12**

* Perform SDS-PAGE and Western Blot of Part 2 induced by temperature.
* Test ELP aggregation.
* Prepare solution needed.

**8.13**

* Purify the sample of Part2 with FPLC.
* Prepare solution needed.
* Culture the transfected bacteria on the plate with ampicillin and induce E.coli to express target protein at 45 centigrade.
* Pick monoclones from plate of Part 2 and culture at 37 centigrade.

**8.14**

* Perform SDS-PAGE of purified Part 2.

## WEEK8

**8.15**

* Purify the sample of Part7 with FPLC.
* Perform Western Blot.
* Learn about protein purification processes.

**8.16**

* Perform SDS-PAGE of purified Part 7 yesterday.
* Dynamic light scattering experiment ELP aggregation pre-experiment,size between 10 and 1000nm, Zeta potential from defeat 1.6 volts to defeat 9.8 volts,forms a visible precipitate.
* Prepare LB medium.
* Pick monoclones from plates.

**8.17**

* Culture the Part4 transfected bacteria,spread plate with Part1､2､4､7 and 8 transfected bacteria, failed.
* Purify the sample of Part7 with FPLC.
* Inoculate the Part8 transfected bacteria.
* Renaturation of ELP, failed.
* Dynamic light scattering experiment ELP aggregation, no visible precipitate.
* Induce Part4 expression and preserve P.acnes.

**8.18**

* Perform SDS-PAGE of the purified Part7,unclear stripe.
* Renaturation of ELP, failed.
* Through A350 to test ELP aggregation, obvious effect.
* Induce Part4 to express.

**8.19**

* Purify the sample of Part2 and 8 with FPLC.
* Prepare 4L LB medium.
* Preserve P.acnes.

**8.20**

* Purify the sample of Part8 with FPLC.
* Prepare 4L LB medium and inoculate Part7.
* Spread plate.

**8.21**

* Induce Part2 and 7 at temperature of 45 centigrade.
* Perform SDS-PAGE of Part7 and 8.
* Inoculate P.acnes.
* Prepare solution needed.

## WEEK9

**8.22**

* Purify the sample of Part7 with FPLC.

**8.23**

* Perform BCA to test the concentration of protein.
* Culture P.acnes and Part4.
* Purify the sample of Part2 with FPLC.
* Perform SDS-PAGE of Part7 and 2.
* Transfect part3, activate the ECN and cleanout the nickel column.

**8.24**

* Transfect Part3.
* Purify the sample of Part7 with FPLC.
* Inoculate Part8 in the LB medium.
* Prepare solution needed.
* Pick monoclones from plate of Part2 and 4.
* Induce Part 4 at temperature of 45 centigrade.

**8.25**

* Purify the sample of Part4 with FPLC.
* Inoculate Part4 in the LB medium and culture.
* Prepare LB medium.

**8.26**

* Purify the sample of Part8 with FPLC.
* Inoculate Part8 in the LB medium and culture.
* Learn about ITC, culture P.acnes.

**8.27**

* Perform BCA and SDS-PAGE to test the concentration of Part7 and 8.
* Learn about ITC.
* Repair the nickel column.

**8.28**

* Purify the sample with FPLC.
* BCA test.

## WEEK10

**8.30**

* Perform BCA and SDS-PAGE to test the concentration of samples collected before.
* Try new ways of culturing P.acnes.

**8.31**

* Purify the sample of Part3 with FPLC and Perform SDS-PAGE.
* Induce Part 2 at temperature of 45 centigrade, culture P.acnes.
* Pick monoclones from plate of Part7 and 8.

# September 2021

**9.1**

* Transfect the mutational-27 ELP.
* Sample sequencing, transmission electron microscopy.
* Purification of Part2.
* Culture Part4 sample.

**9.2**

* Perform SDS-PAGE of Part2 and 3.
* Test the ITC condition of PBS.
* Keep Part4 at 45 centigrade to induce E.coli express target protein.
* Culture Propionibacterium acnes.
* Perform BCA to test the concentration of protein in lysed E.coli with Part3(8.30) and collected Part2(9.1).

**9.3**

* Purify the sample of Part4 with FPLC.
* Observe P.acnes.
* Test and finalize the ITC conditions.

**9.4**

* Purification of Part4 sample.

## WEEK11

**9.5**

* Induce Part2 at temperature of 45 centigrade.

**9.6**

* Purify the sample of Part7 with FPLC.

**9.7**

* Perform SDS-PAGE of Part2, 3 and 7, perform part of DMS.

**9.8**

* Continue practicing DMS.

**9.9**

* Pick monoclones from the plates.
* Transmission electron microscopy.

**9.10**

* Culture Part4 sample.
* Perform antibacterial test.
* Perform PCR of Part7 and 8.
* Perform SDS-PAGE, purification and measure the concentration.

**9.11**

* Prepare 4L LB medium and solution needed.
* Transfect the Part7.

## WEEK12

**9.12**

* Transfect Part7’ and 8’, culture them on the shaking table as well as culture by plate scribing method.
* Inoculate Part 7’ in the LB medium and culture it at 37 centigrade.
* Prepare 4L LB medium.
* Purification of Part4 sample.
* Frozen Part4 protein.

**9.13**

* Perform antibacterial test (use E.coli and P.acnes).

**9.14**

* Purify the sample of Part7’ with FPLC.
* Observe the results of antibacterial test.

**9.15**

* AFM sample diluted 10 times in the condition of heated or not heated.

**9.16**

* AFM sample in the condition of heated for 20 mins.

**9.17**

* Transfect the wild-type TLR2/Part5.

**9.18**

* Prepare 4L LB medium.

## WEEK13

**9.19**

* Transfect the Part5, 6 and culture on the shaking table and incubator.

**9.20**

* Transfect the Part5, 6.

**9.22**

* Prepare 4L LB medium.

**9.23**

* Pick monoclones from plate of Part5 and 6.
* Inoculate Part6 in the LB medium and culture it at temperature of 37 centigrade.

**9.24**

* Induce Part6 at temperature of 45 centigrade.
* Purify the sample of Part5,6 with FPLC.

**9.25**

* Inoculate Part5 in the LB medium and culture it at temperature of 37 centigrade.
* Prepare 4L LB medium.
* Antibacterial test.

## WEEK14

**9.26**

* Perform BCA and SDS-PAGE to test the concentration of Part2 and Part4 protein.

**9.27**

* Observe the results of antibacterial test and SDS-PAGE.

**9.28**

* Perform SDS-PAGE of samples collected before.
* Inoculate Part6 in the LB medium and culture it at the temperature of 37 centigrade.

**9.29**

* Prepare 4L LB medium.
* Inoculate Part5 and 6 in the LB medium and induce at temperature of 45 centigrade.

**9.30**

* Purify Part2 protein again.
* Purify the sample of Part5,6 with FPLC.
* Perform SDS-PAGE of samples of Part5, 6 induced or not.

# October 2021

**10.1**

* Perform BCA and SDS-PAGE to test the concentration of Part2 Purify(9.30).
* Prepare 4L LB medium.
* Pick monoclones from plate of Part5 and 6.
* Induce Part 5 at temperature of 45 centigrade.

**10.2**

* Purify the sample of Part5, 6 with FPLC.
* Perform SDS-PAGE of samples of Part5, 6 induced or not.

## WEEK15

**10.3**

* Inoculate monoclones Part5 and 6.
* Prepare solutions needed.

**10.4**

* Pick monoclones from plate of Part5, 6 and culture at temperature of 37 centigrade.

**10.6**

* Inoculate Part5, 6 and culture at temperature of 37 centigrade, Perform SDS-PAGE of samples of Part5,6 induced or not.
* Prepare solutions needed.

**10.7**

* Perform SDS-PAGE to test the concentration of Part2 purified (9.30) again.
* Induce Part5 and 6 at temperature of 45 centigrade, perform SDS-PAGE of samples induced at different temperature, Part5 at 37 centigrade, Part5 at 45 centigrade, Part6 at 37 centigrade and Part6 at 45 centigrade.
* Prepare solutions needed.

**10.8**

* Perform SDS-PAGE of samples collected before.
* Inoculate monoclones Part5, 6 and culture at temperature of 37 centigrade.

**10.9**

* Induce Part5 and 6 at temperature of 45 centigrade.
* Perform SDS-PAGE of samples collected before.
* Inoculate monoclones Part5, 6.

## WEEK16

**10.10**

* Inoculate monoclones Part5, 6.
* Prepare the samples induced at different temperature, part 5 at 37 centigrade, Part5 at 45 centigrade, Part6 at 37 centigrade and Part6 at 45 centigrade, ECN for SDS-PAGE.
* Transfect ECN.
* Check the results of SDS-PAGE in 10.9.
* Prepare solutions needed.

**10.11**

* Check the results of SDS-PAGE in 10.10.
* Part5, 6 samples sequencing.

**10.12**

* Culture the Part5 transfected bacteria.
* TransfectPart5, 6.
* Inoculate monoclines Part5, 6.
* The sequencing in 10.11 has no signal.

**10.13**

* Induce Part5 at temperature of 45 centigrade.
* Transfect Part5, 6.

**10.14**

* Purify the sample of Part5 with FPLC.
* Induce Part5 and 6 at temperature of 45 centigrade.

**10.15**

* Perform SDS-PAGE of samples collected in 10.14.

**10.16**

* Prepare the samples induced at different temperature, Part5 at 37 centigrade, Part5 at 45 centigrade, Part6 at 37 centigrade and Part6 at 45 centigrade and perform SDS-PAGE.
* Check the results of SDS-PAGE in 10.15.

## WEEK17

**10.17**

* Check the results of SDS-PAGE in 10.16.

[College of Life Science & Technology](http://life.hust.edu.cn/english/index.htm)

[Huazhong University of Science and Technology](http://english.hust.edu.cn/)

Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C:430074

Copyright © 2021 Huazhong University of Science & Technology. Produced By HUST2-China

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# Team:HUST2-China/Engineering

Engineering Success | iGEM HUST2-China

[HUST2-China](https://2021.igem.org/Team%3AHUST2-China)

* Team

  [Team](https://2021.igem.org/Team%3AHUST2-China/Team)[Attributions](https://2021.igem.org/Team%3AHUST2-China/Attributions)[Partnership](https://2021.igem.org/Team%3AHUST2-China/Partnership)
* Project

  [Description](https://2021.igem.org/Team%3AHUST2-China/Description)[Design](https://2021.igem.org/Team%3AHUST2-China/Design)[Safety](https://2021.igem.org/Team%3AHUST2-China/Safety)[Implementation](https://2021.igem.org/Team%3AHUST2-China/Implementation)
* Lab

  [Notebook](https://2021.igem.org/Team%3AHUST2-China/Notebook)[Protocols](https://2021.igem.org/Team%3AHUST2-China/Protocols)[Engineering](https://2021.igem.org/Team%3AHUST2-China/Engineering)
* HP

  [Human Practices](https://2021.igem.org/Team%3AHUST2-China/Human_Practices)[Communication](https://2021.igem.org/Team%3AHUST2-China/Communication)[Collaborations](https://2021.igem.org/Team%3AHUST2-China/Collaborations)
* Model

  [Model](https://2021.igem.org/Team%3AHUST2-China/Model)[Genetic
  Expression](https://2021.igem.org/Team%3AHUST2-China/Genetic_Expression)[TLR2 Antagonist](https://2021.igem.org/Team%3AHUST2-China/TLR2_Antagonist)[Bacteriostasis of BLP-7](https://2021.igem.org/Team%3AHUST2-China/Bacteriostasis_of_BLP-7)[ELP Aggregation](https://2021.igem.org/Team%3AHUST2-China/ELP_Aggregation)
* Parts

  [Contribution](https://2021.igem.org/Team%3AHUST2-China/Contribution)

# Engineering Success

---

##### Contents

# Construction of NANO-AI-Cleaner

### Overview:

we have already successfully constructed the plasmid ([BBa K3712015](https://parts.igem.org/Part%3ABBa_K3712015)) and engineered an
intelligent temperature-controlled and user-friendly expression system to express two small
drug molecules BLP-7 and TLR2 antagonist with ELP to achieve in situ response at the lesion
,verifying the feasibility of four molecules expression Wild-type BLP-7-27 ELP ([BBa K3712023](https://parts.igem.org/Part%3ABBa_K3712023)), Optimized BLP-7-27
ELP ([BBa K3712018](https://parts.igem.org/Part%3ABBa_K3712018)), Wild-type TLR2
antagonist-27ELP ([BBa K3712022](https://parts.igem.org/Part%3ABBa_K3712022)), and
Optimized TLR2 antagonist-27 ELP ([BBa
K3712008](https://parts.igem.org/Part%3ABBa_K3712008)).

### Methods:

After GenScript synthesized the gene fragments, we use in-fusion cloning method to construct
them in the pVB plasmid respectively, which is transformed into E. coli Nissle 1917, then
culture the strains at 37\(^\circ\mathrm C\) for 12 hours, and induce at 45\(^\circ\mathrm
C\) for one day. Then, SDS-PAGE is performed to prove the successful expression by molecular
weight, and Western Blot experiment is conducted to confirm the expression results.

### Results:

The gene fragments of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP,
Optimized TLR2-27 ELP were synthesized at GenScript, and was constructed in the pVB plasmid
(Figure1).

![The agarose gel electrophoresis map of Wild-type BLP-7-27 ELP (BBa K3712023), Optimized BLP-7-27 ELP (BBa K3712018), Wild-type TLR2 antagonist-27ELP (BBa K3712022), and Optimized TLR2 antagonist-27 ELP (BBa K3712008) respectively constructed in pR/pL expression system. About 200-1000 ng of plasmid was digested at 37 degree centigrade for 30-60 minutes and analyzed on 1% Agarose Gel. Lane1: circular plasmid (brighter) and supercoiled structure; Lane2: plasmid (brighter) digested by EcoRI and BamHI and target gene fragment.](https://static.igem.org/mediawiki/2021/e/e9/T--HUST2-China--img--Results-image--Fig1.png)

Then we transform the constructed plasmids into E. coli Nissle 1917 to express the target
proteins. All transformed E. coli Nissle1917 are cultured at 37\(^\circ\mathrm C\) for 12
hours and induced at 45\(^\circ\mathrm C\) for one day. The bacterial that were only
cultured at 37\(^\circ\mathrm C\) for 12 hours without transformation and the bacterial
transformed without induction were used as the control group. The experimental group is
induced at 45\(^\circ\mathrm C\) for one day after transformation and culturation. The
standard curve was made by BCA assay. By determinating the appropriate loading
concentration, the loading amount of each channel was controlled to 30\(\mu\)g, and then
perform SDS-PAGE to verify the expression of our product.

![SDS-PAGE of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2 antagonist-27ELP, Optimized TLR2 antagonist-27 ELP expression results. Near the 25kDa marker, the binds of Wild-type TLR2-27ELP and Optimized TLR2-27 ELP after induction are darker than the expression system without induction. Near the 15kDa marker, the bands of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP after induction are darker than the expression system without induction, which indicates that our constructed pR/pL temperature control system is able to express our product in E. coli Nissle1917.](https://static.igem.org/mediawiki/2021/8/8f/T--HUST2-China--img--Results-image--Fig2.jpg)

Figure 2: SDS-PAGE of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2
antagonist-27ELP, Optimized TLR2 antagonist-27 ELP expression results. Near the 25kDa
marker, the binds of Wild-type TLR2-27ELP and Optimized TLR2-27 ELP after induction are
darker than the expression system without induction. Near the 15kDa marker, the bands of
Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP after induction are darker than the
expression system without induction, which indicates that our constructed pR/pL
temperature control system is able to express our product in E. coli Nissle1917.

Western Blot is conducted to further verify that the molecule near the marker is the right
target protein we want to express. After transmembrane, we incubate overnight with the
primary antibody against His-tag, and then the secondary antibody for development. After
induction, there are obvious bands, as shown in Figure 3, which further proves the success
of vector construction and expression of our target protein.

![Western blot results of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP. A) In the vicinity of 25kDa, the color of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without induction have obvious color differences. B) Around 25kDa, the color development of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without induction is different obviously.](https://static.igem.org/mediawiki/2021/b/bb/T--HUST2-China--img--Results-image--Fig3.jpg)

Figure 3: Western blot results of Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP,
Wild-type TLR2-27ELP, and Optimized TLR2-27 ELP. A) In the vicinity of 25kDa, the color
of Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without
induction have obvious color differences. B) Around 25kDa, the color development of
Wild-type TLR2-27ELP, Optimized TLR2-27 ELP after induction and the one without
induction is different obviously.

### Future direction:

(1) Due to time and guidance issues, we could not make up the Western blot with internal
parameters, but we can guarantee it is our target protein. Because after the transformation
was successful, we stained with Ponceau red to observe obvious bands. Later, we will
complete the Western blot with internal parameters as a control to further illustrate the
expression of the four proteins Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type
TLR2-27ELP, and Optimized TLR2-27 ELP level.

(2) In order to better verify the aggregation properties of ELP, the bactericidal properties
of antimicrobial peptides and the anti-inflammatory effects of antagonists, we will continue
to express the products we need in the future to further verify the functions of the
proteins we need.

# Purification

### Overview:

After clarifying that the above four kinds of proteins successfully expressed in *E. coli
Nissle 1917*, we successfully purified a large amount of Wild-type BLP-7-27 ELP and
Optimized BLP-7-27 ELP. Unfortunately, we have not been able to purify Wild-type TLR2-27ELP
and Optimized TLR2-27 ELP. The results of SDS-PAGE only show the molecular weight of 27ELP.
Therefore, we speculated that passing through the nickel column caused a break between
Wild-type/Optimized TLR2 antagonist and 27ELP.

### Methods:

The above-mentioned strains that can successfully express products are cultured in LB medium
at 37\(^\circ\mathrm C\) for 12 hours, and induced at 45\(^\circ\mathrm C\) for 24 hours.
Then the cultures are centrifuged at 6000 rpm to collect the bacteria that are suspended and
concentrated with PBS. After that, add *lysozyme* at 1:100 and PMSF at 1:1000 when
lysing on ice for 30 minutes, and then disrupt ultrasonically at 400W. Centrifuge the turbid
liquid again to collect the supernatant, filter and sterilize, and pass the sterilized
liquid through FPLC for nickel column affinity chromatography. Then we use BCA to measure
the concentration of the product, and perform SDS-PAGE for product conformation.

### Results:

After culturing, induction and sterilization, the results of nickel column affinity
chromatography show that both of Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP elution
have single peaks at 80 mM with good symmetry, which preliminarily indicates that the target
protein is purified. Then we take the BCA method to determine the concentration (Table1) and
SDS-PAGE to verify the existence of product (Figure4).

**Table1. BCA tests the concentration of Wild-type BLP-7-27 ELP and Optimized
BLP-7-27 ELP after purification.**

| sample | concentration |
| --- | --- |
| Wild-type BLP-7-27 ELP | 1.5mg/ml |
| Optimized BLP-7-27 ELP | 1.2mg/ml |

![The elution peak height of Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP and the running gel spectrum after purification.  A) The elution peak height of Wild-type BLP-7-27 ELP is 1.41; B) The height of the Optimized BLP-7-27 ELP elution peak is 1.26; C) SDS-PAGE, lane 1/2 is two-tube stream, lane 3 For Wild-type BLP-7-27 ELP, a thicker band appears near the 15kDa marker; lane 4/5/6 is a three-pipe stream that collects Optimized BLP-7-27 ELP, and a thicker band appears near the 15kDa marker.](https://static.igem.org/mediawiki/2021/f/fc/T--HUST2-China--img--Results-image--Fig4.png)

Figure 4: The elution peak height of Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP
and the running gel spectrum after purification. A) The elution peak height of Wild-type
BLP-7-27 ELP is 1.41; B) The height of the Optimized BLP-7-27 ELP elution peak is 1.26;
C) SDS-PAGE, lane 1/2 is two-tube stream, lane 3 For Wild-type BLP-7-27 ELP, a thicker
band appears near the 15kDa marker; lane 4/5/6 is a three-pipe stream that collects
Optimized BLP-7-27 ELP, and a thicker band appears near the 15kDa marker.

### Future direction:

(1) Unfortunately, due to time issues, we have no way to complete the purification of TLR2
antagonist. In the future, we will try different linkers to connect Wild-type/Optimized TLR2
antagonist and 27ELP.

(2) Although our purified samples will not affect the subsequent functional verification, it
can be seen from SDS-PAGE that even the purified bands will also have miscellaneous bands.
Therefore, we want to use molecular sieves and freeze dryers for further purification,
attempting to obtain purer samples and make our experiment more successful.

# Functional testing

## ELP

### Overview:

We use Wild-type BLP-7-27 ELP to verify the aggregation function of ELP, because we want to
measure the aggregation ability if linked with another protein, but another protein can't be
too large to guarantee the aggregation of ELP, so we use the ELP connected with the
Wild-type BLP-7 for ELP functional verification.

We first verify that the molecular size of ELP has changed under the heating condition of
30~50\(^\circ\mathrm C\) with dynamic light scattering. At the same time, we also measure
the change of zeta potential of the ELP during the aggregation process. An inflection point
appears at 44\(^\circ\mathrm C\), which preliminarily indicates that the ELP aggregates at
44\(^\circ\mathrm C\). Furthermore, we use transmission electron microscopy and atomic force
microscope. We have observed an ellipsoid structure. This is because the energy obtained by
the ELP in the aggregation process is not complete and the Wild-type BLP-7 connected to the
end of the ELP. As a result, ELP only assemblies into a spherical shape and it is difficult
to form a perfect spherical structure.

However, this still fulfills our requirement for temperature-controlled aggregation, skin
repair, and increasing drug concentration at lesion and prolonging administration time.

### Methods:

We use Wild-type BLP-7-27 ELP (1.5mg/ml) to measure its aggregation ability by dynamic light
scattering. Under the condition of 30~50\(^\circ\mathrm C\), equilibrate for 5 minutes and
increase the temperature by 1\(^\circ\mathrm C\) to obtain continuous change of the size and
zeta potential of ELP aggregation under this temperature gradient. Then, we use the same
batch of samples to incubate at a constant temperature of 44\(^\circ\mathrm C\) for 20
minutes. Then we use transmission electron microscope and atomic force microscope to observe
the morphology.

### Results:

Under the temperature gradient of 30~50\(^\circ\mathrm C\), the particle size measurement is
carried out for every 1\(^\circ\mathrm C\) increase. We find that the main peak of ELP
aggregates distribution (more than 70%) gradually shifted to the larger particle size as the
temperature increased. Location gathering. We averaged the main peaks of the three particle
diameter measurements, and then plotted the average value as shown in Figure 5.A. We found
that there is an inflection point at 44\(^\circ\mathrm C\), which means that
44\(^\circ\mathrm C\) is the transition temperature of Wild-type BLP-7-27 ELP. At the same
time, we found that at 44\(^\circ\mathrm C\) there is a rising inflection point for the Zeta
potential, which is caused by rich positive charges of the Wild-type BLP-7 connected with
the ELP aggregate.

![Changes in particle size and zeta potential during ELP aggregation. A) The particle size of the ELP aggregate changes. The particle size is around 10nm before 44 degree centigrade, then it reaches 100nm or more at 44 degree centigrade, and thereafter it rises sharply. B) Changes in zeta potential of ELP aggregates. The change is relatively flat before 40 degree centigrade, and the value is around -5mV; at 40 degree centigrade, there is a sharp drop. That's because ELP has accumulated locally, but no aggregates are formed. The positive charge of ELP attracts anions in the solution environment, so it appears as a anionic enhancement; the inflection point appears at 43 degree centigrade, where the ELP has been aggregated, and the relative density of the surface charge of the aggregate is lower than the charge density of the dispersed state. Therefore, the absolute value of the charge is reduced, but it will not cause electrical reversal and a particularly drastic change in the amount of charge occur.](https://static.igem.org/mediawiki/2021/5/50/T--HUST2-China--img--Results-image--Fig5.png)

Figure 5: Changes in particle size and zeta potential during ELP aggregation. A) The
particle size of the ELP aggregate changes. The particle size is around 10nm before 44
degree centigrade, then it reaches 100nm or more at 44 degree centigrade, and thereafter
it rises sharply. B) Changes in zeta potential of ELP aggregates. The change is
relatively flat before 40 degree centigrade, and the value is around -5mV; at 40 degree
centigrade, there is a sharp drop. That's because ELP has accumulated locally, but no
aggregates are formed. The positive charge of ELP attracts anions in the solution
environment, so it appears as a anionic enhancement; the inflection point appears at 43
degree centigrade, where the ELP has been aggregated, and the relative density of the
surface charge of the aggregate is lower than the charge density of the dispersed state.
Therefore, the absolute value of the charge is reduced, but it will not cause electrical
reversal and a particularly drastic change in the amount of charge occur.

We use the same batch of Wild-type BLP-7-27 ELP samples (1.5mg/ml) for the transmission
electron microscopy test with the dynamic light scattering test. What stimulates us is that
we find there are more spherical structures, but there are some contiguous structures. The
reason for these phenomena is that (Figure 6D), these ball-like structures have quantity of
positive charges, attracting the negative charges materials around them, and these materials
attract positive charges spherical structures again, thus forming some darker adhesions. For
the lighter colored adhesions. (Figure 6C) Although the ELP aggregates at 44\(^\circ\mathrm
C\), it doesn't completely aggregate. The ELP molecules that do not have enough energy to
aggregate a relatively loose adhesion structure with a lower density, so it looks lighter
under the transmission electron microscope.

![Transmission electron microscope image of ELP aggregates. A) Ball-like structure, the size of ball a is about 500nm, the size of ball b is about 400nm, the scale bar is 50nm, B) the ball-like structure, the size of ball c is about 200nm, the scale bar is 50nm; C) The darker adhesive object is about 2.5 micra, in which the size of the ball-like structure is between 100~300nm, bar 200nm; D) The lighter adhesive object, the whole is about 6 micra, bar 600nm.](https://static.igem.org/mediawiki/2021/f/f9/T--HUST2-China--img--Results-image--Fig6.jpg)

Figure 6: Transmission electron microscope image of ELP aggregates. A) Ball-like
structure, the size of ball a is about 500nm, the size of ball b is about 400nm, the
scale bar is 50nm, B) the ball-like structure, the size of ball c is about 200nm, the
scale bar is 50nm; C) The darker adhesive object is about 2.5 micra, in which the size
of the ball-like structure is between 100~300nm, bar 200nm; D) The lighter adhesive
object, the whole is about 6 micra, bar 600nm.

After knowing the two-dimensional morphology of the ELP aggregation, we want to see the
three-dimensional structure further, so we use the atomic force microscope to observe the
surface morphology. What makes us strange is that we have observed many ellipsoids, so we
feed back this phenomenon to the modeling team, which gives a good explanation from a
mathematical point of view: although the most stable structure is spherical theoretically,
but there is not enough energy, so it forms an ellipsoidal structure with incomplete
aggregation. (more details [here](https://2021.igem.org/Team%3AHUST2-China/ELP_Aggregation#DvalueVerificationandRationalInterpretationofExperimentalData):
D value verification and rational interpretation of experimental data.)

![Atomic force microscope image of ELP aggregates. The brighter means the higher the depth and the darker the color means the greater the degree of depression, bar means the depth of the height map. A) After incubating at 25 degree centigrade for 20 minutes, there are few ELP aggregates, dispersed. B). Figure A is 3D image of the field of view. C) After incubating at 44 degree centigrade for 20 minutes, there are more ELP aggregates, dense. D) Figure C is 3D image of the field of view. Since the shooting is in a liquid phase environment, the depth is not large and the shape looks relatively flat.](https://static.igem.org/mediawiki/2021/7/70/T--HUST2-China--img--Results-image--Fig7.jpg)

Figure 7: Atomic force microscope image of ELP aggregates. The brighter means the higher
the depth and the darker the color means the greater the degree of depression, bar means
the depth of the height map. A) After incubating at 25 degree centigrade for 20 minutes,
there are few ELP aggregates, dispersed. B). Figure A is 3D image of the field of view.
C) After incubating at 44 degree centigrade for 20 minutes, there are more ELP
aggregates, dense. D) Figure C is 3D image of the field of view. Since the shooting is
in a liquid phase environment, the depth is not large and the shape looks relatively
flat.

### Future direction:

(1) Although the modeling team has given a good verification that 27 ELP has the temperature
control effect we expect, in the future we will verify the temperature control aggregation
ability of 54 ELP through experiments to further support the ELP aggregation temperature of
the modeling group. The applicability of the control aggregation model.

(2) After we purify the Wild-type/optimized TL2 antagonist-27 ELP, we will verify that this
product has a similar transition temperature with Wild-type BLP-7-27 ELP within
\(\pm\)0.5~1\(^\circ\mathrm C\). (Link: Prediction: ELP with chain length=54)

## BLP-7

### Overview:

We carry out antibacterial experiments respectively with purified Wild-type BLP-7-27 ELP,
optimized BLP-7-27 ELP. Firstly, we build an anaerobic environment for *Propionibacterium
acnes* with Anaerobic bag indicator and hermetic bag and cultured at 180 rpm,
37\(^\circ\mathrm C\). Then we use the inhibition zone method and absorbance method to
measure the bacteriostatic ability of the antimicrobial peptides. Unfortunately, due to the
preliminary exploration of anaerobic culture and lack of time, we fail to measure the
minimum bacteriostatic concentration of the antimicrobial peptides. We will explore the
conditions of bacteriostatic experiments in the future.

### Methods:

### Bacteriostatic experiment one: inhibition zone experiment

Preparing solid medium: Heat and melt the BHI medium, then cool it to about 50\(^\circ\mathrm
C\). Add 1 ml of bacterial suspension to every 100 ml medium. Shake to ensure that the
bacteria distribute evenly and pour into a large culture dish. Place Oxford-cups on the
surface of the plate corresponding to the marked number, and dropwise add antimicrobial
peptide sample with concentration gradient to Oxford-cups. Incubate together for a period of
time and measure the diameter of inhibition zone.

### Bacteriostatic experiment two: absorbance method

Add BLP-7 at concentrations ranging from 1 to 1000\(\mu\)M to the culture medium and incubate
anaerobically at 37\(^\circ\mathrm C\) for 24 hours using antibiotics as positive control
and sterile water as negative control. The MIC value of BLP-7 is clarified by measuring the
absorbance at 630 nm using a microplate reader. Assume the concentration with no significant
difference from the positive control group MIC.

### Future direction:

(1) Due to the lack of experience and method of *Propionibacterium acnes* cultivation,
we fail to ascertain the optimal growth condition of *Propionibacterium acnes* after
a long period of exploration although we successfully culture it. Subsequent exploration
will be carried out to find the appropriate method to control the growth of
*Propionibacterium acnes*.

(2) After determining the optimal growth conditions, we will further explore the principles
of antibacterial experiments due to the uncertainty of incubation time and temperature of
antimicrobial peptides.

## TLR2 antagonist

### Overview:

A fracture happens when purifying Wild-type TLR2 antagonist-27ELP and optimizes TLR2
antagonist-27 ELP, thus, we choose to synthesize Wild-type/optimized TLR2 antagonist gene
directly and link to pVB plasmid and induce expression at 45\(^\circ\mathrm C\) to confirm
the competitive binding ability of TLR2 antagonist to triacylated/diacylated lipoproteins
compared to that of TLR2 considering the lack of time to explore the flexibility of linking
peptide.

### Methods:

Since triacyl-lipoprotein Pam3CSK4 must be dissolved in formic acid (1mg dissolved in 1ml
formic acid) and formic acid emits a lot of heat when titrated, we investigate the optimum
dilution rate ensuring that the release of heat have no apparent influence on the measure of
heat generated from combination between Wild-type TLR2 antagonist-27ELP and optimized TLR2.
So we try to titrate in PBS after diluting formic acid 50 times, 100 times, 200 times, 500
times with PBS, and titrate sodium chloride after diluting formic acid 50 times, 100 times,
200 times, 500 times with sodium chloride.

### Results:

We find that when formic acid is diluted 100 times with PBS and titrated with PBS, the heat
released is minimal and relatively stable. Under this background, the concentration of
acylated lipoprotein is 60\(\mu\)M, and the concentration of Wild-type/Optimized TLR2
antagonist is 6\(\mu\)M. Therefore, we are confident of the experimental results.

![Heat released in PBS titration with 100 times diluted formic acid. The upper Figure shows the heat released per second at a given point in the titration process. The lower Figure shows the heat released per drop of formic acid diluted 100 times with PBS. Add only 20 microliter formic acid diluted 100 times with PBS for the first drop and 40 microliter for the next 19 drops. At equilibrium, the titration heat released is about 1.50 microcalorie per second and 120kcal per mole of the injected substance.](https://static.igem.org/mediawiki/2021/0/04/T--HUST2-China--img--Results-image--Fig8.png)

Figure 8: Heat released in PBS titration with 100 times diluted formic acid. The upper
Figure shows the heat released per second at a given point in the titration process. The
lower Figure shows the heat released per drop of formic acid diluted 100 times with PBS.
Add only 20 microliter formic acid diluted 100 times with PBS for the first drop and 40
microliter for the next 19 drops. At equilibrium, the titration heat released is about
1.50 microcalorie per second and 120kcal per mole of the injected substance.

### Future direction:

(1) A purer Wild-type/Optimized TLR2 antagonist should be obtained through molecular sieve
and concentrated to conduct ITC experiment.

(2) Dilute PBS with Pam3CSK4 dissolved in pure formic acid for 100 times and titrate
Wild-type/Optimized TLR2 antagonist dissolved PBS to prove that optimized TLR2 antagonist
has stronger combination ability than Wild-type TLR2 antagonist, which means that optimized
TLR2 antagonist has stronger combination ability than TLR2.

# Conclusion

## Construction of NANOAICleaner:

We successfully construct different expression systems with pR/pL temperature control systems
and express Wild-type BLP-7-27 ELP, Optimized BLP-7-27 ELP, Wild-type TLR2-27ELP and
Optimized TLR2-27ELP. The expression products are verified by SDS-PAGE and Western Blot. At
the same time, we also verify that the pR/pL temperature control system can work under the
condition of infrared irradiation, which provides a good prospect for the application of our
system in photodynamic therapy. Based on the solid functional data and modeling analysis,
our NANOAICleaner system has specifically smart characteristics for potential biomedical
applications.

## Purification:

We successfully purify Wild-type BLP-7-27 ELP and Optimized BLP-7-27 ELP with high
concentration and purity, providing good raw materials for subsequent functional
verification.

## Functional testing:

**ELP:** We preliminarily prove the aggregation of ELP by measuring the changes
of particle size and Zeta potential in the ELP aggregation process by dynamic light
scattering, and observe the morphology of ELP aggregates by transmission electron microscopy
and atomic force microscopy. Combined with the verification of the molecular modeling, we
successfully prove the phase transition temperature and morphological characteristics of ELP
aggregates from both aspects of experiment and modeling.

**BLP-7:** We have figured out a method to culture *Propionibacterium
acnes*, and we are exploring condition to conduct antibacterial experiments.

**TLR2 antagonist:** We have found out the optimal solution environment for
titration, laying a good foundation for the follow-up isothermal titration experiment.

[College of Life Science &
Technology](http://life.hust.edu.cn/english/index.htm)

[Huazhong University of Science and Technology](http://english.hust.edu.cn/)

Add: 1037 Luoyu Road, Wuhan, Hubei, China; P.C:430074

Copyright © 2021 Huazhong University of Science & Technology. Produced By
HUST2-China

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HUST2-China/Experiments

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### Inspiration

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

[![](https://static.igem.org/mediawiki/2021/8/8b/HQ_page_logo.jpg)](https://2021.igem.org)

# Team:HUST2-China/Results

# Results

You can describe the results of your project and your future plans here.

### 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.

### Inspiration

See how other teams presented their results.

* [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)