iGEM 2023 — Team sustech-china

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{% block title %}Notebook{% endblock %}
{% block lead %}On this page, we show all our notebooks, please click on the relevant date to view according to the timeline and need. According to the timeline, on the last day of each section, we put up results that prove our success, for a more detailed description of the progress of the project, please check out our engineering page{% endblock %}
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##### Menu

* [2023 June](#june)
* [2023 July](#july)
* [2023 August](#august)
* [2023 September](#september)
* [7.13~7.17](#section1)
* [8.03~8.13](#section4)

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![...](https://static.igem.wiki/teams/4785/wiki/notebook/notebook.png)

## 2023 May

* 2023. 05. 09

  Gain hmgb1-FL DNA fragment and pET28a plasmid

* 2023. 05. 10

  Gain hmgb1-FL, -AB box, -A box, -B box primer. Gene fragments were amplified using
  PCR. And the
  linearized carrier was prepared. Plasmids were constructed using homologous
  recombination.

* 2023. 05. 11

  Plasmids were expanded in DH5α and the plasmids were sequenced.

* 2023. 05. 12

  Plasmids with the correct sequence were recovered and stored in a -20 ° C
  refrigerator.

* 2023. 05. 15

  Gain pslg DNA fragment and primer.

* 2023. 05. 16

  Gene fragments were amplified using PCR. And the linearized carrier was prepared.
  Plasmids were
  constructed using homologous recombination.

* 2023. 05. 17

  Plasmids were expanded in DH5α and the plasmids were sequenced.

* 2023 .05. 18

  Plasmids with the correct sequence were recovered and stored in a -20 ° C
  refrigerator.

* 2023. 05. 22

  Plasmid “pET28a-6×His-hmgb1-FL” with ampicillin resistance was transferred into
  BL21.
  And BL21 was coated on LB plates containing ampicillin.

* 2023. 05. 23

  Single colonies were selected on LB plates and inoculated into 10mL of LB liquid
  medium containing
  ampicillin for initial enrichment culture.

* 2023. 05. 24

  10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid
  medium containing
  ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was
  added to 100 μM
  to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a
  shaker.

* 2023. 05. 25

  The bacteria that fully expressed the target protein were recovered and stored in a
  -80 ° C
  refrigerator for further use.

## 2023 June

* 2023. 06. 20

  Plasmid “pET28a-6×His-hmgb1-AB” with ampicillin resistance was transferred into
  BL21.
  And BL21 was coated on LB plates containing ampicillin.

* 2023. 06. 21

  Single colonies were selected on LB plates and inoculated into 10mL of LB liquid
  medium containing
  ampicillin for initial enrichment culture.

* 2023. 06. 22

  10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid
  medium containing
  ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was
  added to 100 μM
  to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a
  shaker.

* 2023. 06. 23

  The bacteria that fully expressed the target protein were recovered and stored in a
  -80 ° C
  refrigerator for further use.

* 2023. 06. 25

  Plasmid “pET28a-6×His-hmgb1-A” with ampicillin resistance was transferred into BL21
  was transferred
  into BL21. And BL21 was coated on LB plates containing ampicillin.

* 2023. 06. 26

  Single colonies were selected on LB plates and inoculated into 10mL of LB liquid
  medium containing
  ampicillin for initial enrichment culture.

* 2023. 06. 27

  10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid
  medium containing
  ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was
  added to 100 μM
  to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a
  shaker.

* 2023. 06. 28

  The bacteria that fully expressed the target protein were recovered and stored in a
  -80 ° C
  refrigerator for further use.

* 2023. 06. 29

  Plasmid “pET28a-6×His-hmgb1-B” with ampicillin resistance was transferred into BL21.
  And BL21 was coated on LB plates containing ampicillin.

* 2023. 06. 30

  Single colonies were selected on LB plates and inoculated into 10mL of LB liquid
  medium containing
  ampicillin for initial enrichment culture.

## 2023 July

* 2023. 07. 01

  10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid
  medium containing
  ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was
  added to 100 μM
  to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a
  shaker.Thaw and
  recover the HT-29 cells, seed them in a 60 mm petri dish, and begin cell culturing.

* 2023. 07. 02

  The bacteria that fully expressed the target protein were recovered and stored in a
  -80 ° C
  refrigerator for further use.

* 2023. 07. 03

  Subculture cells at the 1:2 ratio.Filter the LPS solution through a 0.22 μm syringe
  filter, and
  dilute the original solution to 1.0 mg/mL for experiment use. Store it at -20℃.

* 2023. 07. 04

  Plasmid “pET28a-6×His-pslg” with ampicillin resistance was transferred into BL21 was
  transferred
  into BL21. And BL21 was coated on LB plates containing ampicillin.Subculture again,
  and adjust the
  subculture ratio to 1:4.

* 2023. 07. 05

  Single colonies were selected on LB plates and inoculated into 10mL of LB liquid
  medium containing
  ampicillin for initial enrichment culture.

* 2023. 07. 06

  10 mL of LB liquid medium containing enriched BL21 was poured into 1L of LB liquid
  medium containing
  ampicillin and incubated for 4 h at 37 ° C at 180 rpm on a shaker. And then IPTG was
  added to 100 μM
  to induce protein expression. And incubate it for 16 h at 16 ° C at 180 rpm on a
  shaker.Subculture
  the cells at the 1:4 ratio into 2 dishes.

* 2023. 07. 07

  The bacteria that fully expressed the target protein were recovered and stored in a
  -80 ° C
  refrigerator for further use.

* 2023. 07. 09

  The BL21 containing HMGB1-FL and HMGB1-AB box was broken, and the supernatant was
  collected after
  ultra-high speed centrifugation. The target proteins HMGB1-FL and HMGB1-AB box were
  obtained after
  the supernatant was purified by Ni beads.Subculture the cells in one dish at the 1:4
  ratio. Freeze
  cells in the other dish, and preserve it at -80℃ first, then store in liquid
  nitrogen.

* 2023. 07. 10

  The BL21 containing HMGB1-A box and HMGB1-B box was broken, and the supernatant was
  collected after
  ultra-high speed centrifugation. The target proteins HMGB1-FL and HMGB1-AB box were
  obtained after
  the supernatant was purified by Ni beads.

* 2023. 07. 11

  The BL21 containing PslG was broken, and the supernatant was collected after
  ultra-high speed
  centrifugation. The target proteins HMGB1-FL and HMGB1-AB box were obtained after
  the supernatant
  was purified by Ni beads. Observed many floating dead cells, and cells grew slowly
  at
  the same
  time.Wash with PBS twice, then subculture the cells at the 1:4 ratio.

* 2023. 07. 13

  The phenomenon remained still, so it was assumed contaminated by mycoplasmas. Wash
  with PBS twice,
  then add Mycoplasma Elimination Reagent (C0292, Beyotime) to the culture medium and
  subculture the
  cells at the 1:4 ratio. Resuscitate PAO1.

* 2023. 07. 16

  Dead cells became less, but its growth rate was still slow. Subculture the cells at
  the 1:4 ratio. Do
  inhibition of biofilm growth experiment using FL. Collect the data of 0h, 6h and
  12h. Add different
  concentrations of 3OC12HSL to the medium in the 96-well plate and incubate at 37℃,
  detecting
  fluorescence every 10 minutes, lasting 3 hours.

* 2023. 07. 18

  Few dead cells existed, and the growth rate gradually recovered. Subculture the cells
  at the 1:4
  ratio. Do inhibition of biofilm growth experiment using FL. Collect the data of
  24h. Add different
  concentrations (adjusted after the first experiment) of 3OC12HSL to the medium in
  the 96-well plate
  and incubate at 37℃, detecting fluorescence every 10 minutes, lasting 3 hours.

* 2023. 07. 20

  Cells returned normal. Subculture the cells at the 1:4 ratio. Resuscitate PAO1. Repeat
  the Quorum
  Sensing experiment. Shake before each detection.

* 2023. 07. 22

  Add 100 ng/mL LPS into one cell culture (positive control) and an equal amount of
  PBS to the other
  (negative control), then incubate for an hour. Extract RNA, and conduct reverse
  transcription
  followed by qPCR. Store RNA at -20℃. Trypsinize cells from one cell culture to make
  the cell
  suspension for cytotoxicity test (control). Stain with trypan blue and calculate
  survival
  rate. Subculture the cells at the 1:4 ratio. Inoculate PAO1 into 96-well plates (100ul
  per well),
  culture at 37℃ for 24h. Repeat the Quorum Sensing experiment.

* 2023. 07. 23

  Use the remaining extracted RNA again, and adjust the dilution factor of products of
  reverse
  transcription to twofold. Then rerun qPCR. Do biofilm disassembly experiment using
  PslG and FL.

* 2023. 07. 24

  Subculture the cells at the 1:4 ratio. Prepare M9 medium.

* 2023. 07. 25

  Resuscitate PAO1. *Pseudomonas aeruginosa* were resuscitated and spread onto LB
  solid
  medium.

* 2023. 07. 26

  Add 2,000 ng/mL PslG, 500 ng/mL and 2,000 ng/mL HMGB1 (fl) into 3 cell cultures
  respectively, then
  incubate for an hour. Add 100 ng/mL LPS, 2,000 ng/mL PslG, 500 ng/mL and 2,000 ng/mL
  HMGB1 (fl) into
  4 cell cultures respectively, then incubate for 2 hours. Extract RNA, and conduct
  reverse
  transcription followed by qPCR using the twofold dilution factor. Store RNA at
  -20℃. Repeat the
  cytotoxicity test for the control group. Add 1 - 6 μg/mL LPS into 6 cell cultures and
  incubate for 24
  hours for cytotoxicity test. Subculture the cells at the 1:4 ratio. Inoculate PAO1
  into 96-well plates
  (100ul per well), culture at 37℃ for 24h.

* 2023. 07. 27

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (1 - 6 μg/mL
  LPS). Stain with trypan blue and calculate survival rate. Do biofilm disassembly
  experiment using FL,
  AB, A, B, PslG.

* 2023. 07. 28

  Subculture the cells at the 1:4 ratio. Repeat the Quorum Sensing experiment.

* 2023. 07. 30

  Add 1 - 6 μg/mL HMGB1 (fl) and 1 - 6 μg/mL PslG into cell cultures and incubate for
  24 hours for
  cytotoxicity test. Subculture the cells at the 1:4 ratio. Resuscitate PAO1, prepare
  300nM imidazole.

* 2023. 07. 31

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (1 - 6 μg/mL
  HMGB1 (fl) and 1 - 6 μg/mL PslG). Stain with trypan blue and calculate survival
  rate. Do inhibition
  of biofilm growth experiment using FL, AB, A, B, PslG. Collect the data of 0h, 6h
  and 12h.

## 2023 August

* 2023. 08. 01

  Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment
  using FL, AB, A, B,
  PslG. Collect the data of 24h. *Pseudomonas aeruginosa* were resuscitated and
  spread
  onto LB solid
  medium.

* 2023. 08. 02

  Single colonies were picked on LB plates, inoculated into LB liquid medium, and
  incubated overnight
  at 37 ° C in a shaker.

* 2023. 08. 03

  Repeat inflammation verification for the negative control group, the positive
  control group (1 h and
  2 h), 500 ng/mL and 2,000 ng/mL HMGB1 (fl) group (1 h and 2 h), and 2,000 ng/mL PslG
  group (1 h and
  2 h)R epeat cytotoxicity test for the LPS groups (1 - 6 μg/mL LPS). Subculture the
  cells at the 1:4
  ratio. Prepare GB buffer. Resuscitate PAO1. The medium was diluted and inoculated into
  cell culture
  dishes and incubated for 48 hours in a constant temperature incubator at 37 °
  C. Repeat the Quorum
  Sensing experiment. Add bacteria sample as representative of 3OC12HSL near the
  pathogenic bacteria.

* 2023. 08. 04

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (1 - 6 μg/mL
  LPS). Stain with trypan blue and calculate survival rate. Do inhibition of biofilm
  growth experiment
  using FL, AB, A, B, PslG. Collect the data of 0h, 6h and 12h.
  HMGB1\_A box and PslG
  proteins were also
  added and treated for 4 hours in a constant temperature incubator at 37 ° C. After
  treatment,
  staining was performed, followed by imaging under a confocal microscope.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig2.png)

  Figure 2. HMGB1 and PslG contribute to
  conspicuous clearance of biofilm.
  DNA is stained by SYTO9 (green), and polysaccharide is stained by HHL (red).

* 2023. 08. 05

  Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment
  using FL, AB, A, B,
  PslG. Collect the data of 24h. Repeat the Quorum Sensing experiment.

* 2023. 08. 06

  Resuscitate PAO1.

* 2023. 08. 07

  Repeat inflammation verification for the negative control group, the positive
  control group (1 h and
  2 h), 500 ng/mL and 2,000 ng/mL HMGB1 (fl) group (1 h and 2 h), and 2,000 ng/mL PslG
  group (1 h and
  2 h). Repeat cytotoxicity test for the HMGB1 (fl) groups (1 - 6 μg/mL HMGB1
  (fl)). Subculture the
  cells at the 1:4 ratio. Inoculate PAO1 into 96-well plates (100ul per well), culture
  at 37℃ for
  24h. Repeat the Quorum Sensing experiment to revise some extreme outliers.

* 2023. 08. 08

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (1 - 6 μg/mL
  HMGB1 (fl)). Stain with trypan blue and calculate survival rate. Do biofilm
  disassembly experiment
  using FL, AB, A, B, PslG. Resuscitate PAO1.

* 2023. 08. 09

  Subculture the cells at the 1:4 ratio. Do inhibition of biofilm growth experiment
  using FL, AB, A, B,
  PslG. Collect the data of 0h, 6h and 12h. Repeat the Quorum Sensing experiment to
  revise some extreme
  outliers.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig6-1.png)

  Figure 6. Characterization results of
  quorum
  sensing device.
  a, GFP production rate per cell over time at different concentration of
  3OC12HSL.
  b, Time-average GFP production rate per cell over time at different
  concentration of
  3OC12HSL.
  Error bar represents s.d.

* 2023. 08. 10

  Do inhibition of biofilm growth experiment using FL, AB, A, B, PslG. Collect the
  data of 24h.

* 2023. 08. 11

  Repeat cytotoxicity test for the LPS groups (1 - 6 μg/mL LPS), and PslG groups (1 -
  6 μg/mL
  PslG). Subculture the cells at the 1:4 ratio. Resuscitate PAO1.

* 2023. 08. 12

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (1 - 6 μg/mL
  LPS and 1 - 6 μg/mL PslG). Stain with trypan blue and calculate survival
  rate. Inoculate PAO1 into
  96-well plates (100ul per well), culture at 37℃ for 24h.

* 2023. 08. 13

  Subculture the cells at the 1:4 ratio. Do biofilm disassembly experiment using FL,
  AB, A, B, PslG.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig1.png)

  Figure 1. a, Time course of
  PslG，FL，AB，A，B
  effect on biofilm disassembly. The biofilm biomass was measured by CV assay.

   b,
  Time
  course of PslG，FL，AB，A，B effect on biofilm growth. The biofilm biomass was
  measured by
  CV assay.

* 2023. 08. 15

  Repeat cytotoxicity test for the PslG groups (1 - 6 μg/mL PslG), and HMGB1 (fl)
  groups (1 - 6 μg/mL
  HMGB1 (fl)). Subculture the cells at the 1:4 ratio.

* 2023. 08. 16

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (1 - 6 μg/mL
  PslG, and 1 - 6 μg/mL HMGB1 (fl)). Stain with trypan blue and calculate survival
  rate.

* 2023. 08. 17

  Subculture the cells at the 1:4 ratio.

* 2023. 08. 19

  Repeat cytotoxicity test for the 2 μg/mL and 6 μg/mL HMGB1 (fl) groups, and 2 μg/mL,
  5 μg/mL, and 6
  μg/mL PslG groups to revise extreme data outliers. Repeat inflammation verification
  for the 2,000
  ng/mL HMGB1 (fl) group (2 h), and 2,000 ng/mL PslG groups (1 h and 2 h) to revise
  extreme data
  outliers.

* 2023. 08. 20

  Trypsinize cells from cell cultures to make the cell suspension for cytotoxicity
  test (2 μg/mL and 6
  μg/mL HMGB1 (fl), and 2 μg/mL, 5 μg/mL, and 6 μg/mL PslG). Stain with trypan blue
  and calculate
  survival rate.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig3.png)

  Figure 3. Results of cytotoxicity
  test.
  Different curves indicate the toxicity of different substances for cells.
  Error bar represents s.d.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig4.png)

  Figure 4. Engineered HMGB1 (full
  length)
  exhibits an alleviated pro-inflammatory effect.
  Cells are treated with HMGB1 (fl) for (a) an hour and (b) 2 hours (n = 3).
  \*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA). Error bar represents s.d.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig5.png)

  Figure 5. PslG exhibits little
  pro-inflammatory effect on human intestinal cells.
  Cells are treated with PslG for (a) an hour and (b) 2 hours (n = 3).
  \*P < 0.05, \*\*P < 0.01 (One-way ANOVA). Error bar represents s.d.

## 2023 September

* 2023. 09. 10

  construct the plasmid

* 2023. 09. 11

  construct the plasmid

* 2023. 09. 17

  transform into BL21, spread a plate.

* 2023. 09. 19

  Do the experiment: The growth curve induced by arabinose (M9&LB-ECN&BL21)

* 2023. 09. 21

  Do the experiment: The growth curve induced by arabinose (LB-BL21)

* 2023. 09. 23

  Do the experiment: DNA concentration detection

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig7.png)

  Figure 7. Upon arabinose induction,
  Lysis E7
  is expressed followed by massive death of engineered bacteria.
  Error bar represents s.d.

  ![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig8.png)

  Figure 8. Lysis E7 induces lysis of
  bacteria.
  Error bar represents s.d.

---

### In the protein function validation experiment, we also did the following exploratory experiments, which we hope will be helpful to future teams

## 7.13~7.17

### Design

In order to verify the disassembly ability of PslG to biofilm and to find out the
protein with the strongest biofilm disassembly ability among the four HMGB1-based
modified proteins, we designed a crystal violet staining experiment with reference to an
article on the disassembly of biofilm using PslG and selected *Pseudomonas
aeruginosa*
PAO1 (abbreviated to PAO1 in the following part), which is a gram-negative bacterium
that can and easily form biofilm, as the experimental strain. The experiment was divided
into two parts: inhibition of biofilm growth and disassembly of biofilm. In the
experiment of inhibiting biofilm growth, we inoculated PAO1 and added the proteins to
the mediums at the same time, and the ability of the proteins to disassemble the biofilm
is reflected by measuring the growth status of the biofilm; In the biofilm disassembly
experiment, we inoculated PAO1 and cultured it first, and then added proteins after it
formed a sufficient amount of biofilm, so as to reflect the ability of protein
disassembly of biofilm by measuring the biofilm disassembly status. The biomass of
biofilm in both experiments was measured by crystal violet staining assay.

### Experiment & Result

Inhibition of biofilm growth experiment: (1) Use LB medium to resuscitate PAO1; (2) The
resuscitated PAO1 bacterial solution was diluted to OD600=0.001 using LB medium, and
inoculated into 96-well plates (100ul per well). FL (final concentration 200nM) was
added to the treatment group, while no protein was added to the control group, and
cultured at 37℃; (3) When the culture time reaches 0h, 6h, 12h, 24h, the following
operations are carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out
(twice), add 150ul 0.1% crystal violet solution for dark dyeing for 15min and sucking
out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice), add 200ul
anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was detected
with an enzyme-labeler and the data was recorded.

![...](https://static.igem.wiki/teams/4785/wiki/notebook/1.png)

Note: Time course of FL effects on biofilm growth. The biofilm biomass was measured by CV
assay.

Biofilm disassembly experiment: (1) PAO1 was resuscitated using LB medium; (2) The
recovered PAO1 solution was diluted to OD600=0.001 using LB medium, inoculated into
96-well plates (100ul per well), and cultured at 37℃ for 24h; (3) In the treatment
group, FL (final concentration 200nM) and PslG (final concentration 50nM) were added
respectively and cultured at 37℃; (4) When the culture time reaches 0min, 1h, 2h, 4h,
8h, the following operations are carried out: Add 150ul ddH2O for oscillatory cleaning
and sucking out (twice), add 150ul 0.1% crystal violet solution for dark dyeing for
15min and sucking out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice),
add 200ul anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was
detected with an enzyme-labeler and the data was recorded.

![...](https://static.igem.wiki/teams/4785/wiki/notebook/2.png)

Note: Time course of FL effects on biofilm disassembly. The biofilm biomass was measured
by CV assay.

### Analysis & Plan

In the experiment of inhibiting biofilm growth, the absorbance of OD550
corresponding to biofilm biomass in the control group reached about 2.0 at 24h, while
the absorbance of FL treatment group was only about 1.3 at the same time, which
effectively inhibited the growth of biofilm. In the biofilm disassembly experiment, the
FL or PslG treatment groups reduced the OD550 absorbance corresponding to the
biofilm biomass from about 1.5 to about 0.5 in the first two hours. From the results of
the two groups of experiments, it can be concluded that FL and PslG have significant
disintegration effects on biofilm, which provides a theoretical basis for us to select
these two proteins and their modified proteins to destroy biofilm.

Since the purpose of our experiment was only to illustrate and measure the disassembly
ability of various proteins to the biofilm, protein concentration gradient was not
established, and the concentration used in the literature was directly adopted. These
two sets of results show that both proteins can produce significant effects at the
concentrations provided in the literature, so we will continue to use these two
concentrations in subsequent experiments.

In the following experiments, we need to further compare the ability of the four modified
proteins of HMGB1 (FL, AB, A, B) to disassemble the biofilm, and select the most
functional protein as the secreted protein of the engineered bacteria. Considering that
LB medium is relatively rich in nutrients, which may not be conducive to the formation
of biofilm, we plan to replace LB medium with chemical defined M9 medium (with
relatively less nutrients and closer to the intestinal environment) in subsequent
experiments. In order to make the experiment more convincing, we also need to supplement
the protein-free group in the biofilm disassembly experiment.

## 8.03~8.13

### Design

To further determine the function of the various proteins, we performed biofilm growth
inhibition and biofilm disassembly experiments below in more detail.

### Experiment & Result

Buffer replacement: (1) Prepare a new buffer GB (50mM Tris, 150mM NaCl); (2) Add the
protein into a suitable type of concentration tube and centrifuge it to the limit volume
by 2300g; (3) Discard the waste liquid, add 2ml buffer solution GB to the concentration
tube, and centrifuge 2300g to the limit volume; (4) Repeat step 3 twice; (5) Add 2ml
buffer GB to the concentration tube, mix with the protein, suck out and store.

Inhibition of biofilm growth experiment: (1) Use LB medium to resuscitate PAO1; (2) The
resuscitated PAO1 bacterial solution was diluted to OD600=0.001 using M9 medium and
inoculated into 96-well plates (100ul per well). FL (final concentration 200nM), AB
(final concentration 200nM), A (final concentration 200nM), B (final concentration
200nM) were added to the treatment group, respectively. PslG (final concentration 50nM)
was used to supplement the total volume of solution in each hole to 130ul using buffer
GB (50mM Tris, 150mM NaCl), and the control group was directly added with 30ul buffer GB
and cultured at 37℃. (3) When the culture time reaches 0h, 6h, 12h, 24h, the following
operations are carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out
(twice), add 150ul 0.1% crystal violet solution for dark dyeing for 15min and sucking
out, add 200ul ddH2O for oscillatory cleaning and sucking out (twice), add 200ul
anhydrous ethanol for dark dissolution for 15min. The OD550 of the sample was detected
with an enzyme-labeler and the data was recorded.

![...](https://static.igem.wiki/teams/4785/wiki/notebook/5.png)

Note: Time course of PslG，FL，AB，A，B effect on biofilm growth. The biofilm biomass was
measured by CV assay.

Biofilm disassembly experiment: (1) PAO1 was resuscitated using LB medium; (2) The
recovered PAO1 solution was diluted to OD600=0.001 using M9 medium, inoculated into
96-well plates (100ul per well), and cultured at 37℃ for 24h; (3) FL (final
concentration 200nM), AB (final concentration 200nM), A (final concentration 200nM), B
(final concentration 200nM), PslG (final concentration 50nM) were added to the treatment
group, and the buffer solution GB (50mM Tris) was used. 150mM NaCl) was used to
supplement the total volume of solution in each hole to 130ul, and the control group was
directly added with 30ul buffer GB and cultured at 37℃. (4) When the culture time
reaches 0min, 30min, 1h, 2h, 4h, 8h, the following operations are carried out: Add 150ul
ddH2O for oscillatory cleaning and sucking out (twice), add 150ul 0.1% crystal violet
solution for dark dyeing for 15min and sucking out, add 200ul ddH2O for oscillatory
cleaning and sucking out (twice), add 200ul anhydrous ethanol for dark dissolution for
15min. The OD550 of the sample was detected with an enzyme-labeler and the data was
recorded.

![...](https://static.igem.wiki/teams/4785/wiki/notebook/6.png)

Note: Time course of PslG，FL，AB，A，B effect on biofilm disassembly. The biofilm biomass
was measured by CV assay.

### Analysis & Plan

In disassembly assay, the biomass of the biofilm decreases sharply in the first 30min and
keep reducing for a few hours. PslG, HMGB1-B box, HMGB1-FL have better effect than
others. In growth inhibition assay, all the protein treatment groups have almost the
same result on growth inhibition. Through these assays, we proof PslG indeed have an
effective ability to disassemble biofilm. We illustrated that the HMGB1-FL (full
length) is the relatively efficient one among four HMGB1 modified proteins to
disassemble biofilm. Those two proteins will be chosen to be our functional protein.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}On this page, we show the results of our many iterations and the progress of our project{% endblock %}
{% block offcanvas %}

##### Menu

* [What is "biofilm"?](#section1)
* [Arabinose-induced cell lysis](#section2)
* [Intuitive observation by confocal microscopy](#section3)
* [Cytotoxicity Test and Inflammation Verification](#section4)
* [Quorum sensing system](#section5)
* [Arabinose-induced cell lysis](#section6)

{% endblock %}
{% block page\_content %}

![...](https://static.igem.wiki/teams/4785/wiki/engineering/2.svg)

### Overview

In order to successfully construct engineered bacteria that can effectively remove biofilm,
we need to iterate the experiment according to the experimental results during the whole
project. We follow the cycle of "Design → Build → Test → Learn" and continuously
improve the experimental conditions to achieve better experimental results.

We designed the experiments in four aspects: target protein purification and function test,
cytotoxicity test, quorum sensing system test, lysis system test and secretion system test.
The target proteins were first purified *in vitro* to test their ability to disrupt
biofilm
formation. Secondly, cytotoxicity tests were performed with purified proteins to observe in
what concentration range our target proteins were harmless to humans. After determining the
concentration of the protein that would be safe, we targeted the concentration of the
protein by measuring the expression potency of the quorum-sensing system.
Finally, we will release the target proteins through the lysis system and secretion system
to destroy the biofilm.

### Quantitative analysis by crystal violet

#### Design

In order to verify the disassembly ability of PslG to biofilm and to find out the protein
with the strongest biofilm disassembly ability among the four HMGB1-based modified proteins
(including HMGB1\_Full Length, HMGB1\_AB box, HMGB1\_A box and HMGB1\_B box), we use crystal
violet staining method.

#### Build

Five kinds of plasmids were constructed to express PslG, HMGB1\_FL, HMGB1\_AB box, HMGB1\_A box
and HMGB1\_B box, respectively. After transferring the plasmid into DH5α for amplification,
transferring into BL21 to express the target protein, cleaving BL21 and purifying using
nickel column and molecular sieve, we obtained high purity of the target protein.
The experiment was divided into two parts: inhibition of biofilm growth and disassembly of
biofilm. In the experiment of inhibiting biofilm growth, we inoculated PAO1 and added the
proteins to the mediums at the same time, and the ability of the proteins to disassemble the
biofilm is reflected by measuring the growth status of the biofilm; In the biofilm
disassembly experiment, we inoculated PAO1 and cultured it first, and then added proteins
after it formed a sufficient amount of biofilm, so as to reflect the ability of protein
disassembly of biofilm by measuring the biofilm disassembly status. The biomass of biofilm
in both experiments was measured by crystal violet staining assay.

#### Test

In both experiments, we treated the samples with PslG, HMGB1\_FL, HMGB1\_AB box, HMGB1\_A box
and HMGB1\_B box, respectively. The group of control is protein free.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig1.png)

Figure 1. a, Time course of PslG，FL，AB，A，B
effect on biofilm disassembly. The biofilm biomass was measured by CV assay.
 b,
Time
course of PslG，FL，AB，A，B effect on biofilm growth. The biofilm biomass was measured by
CV assay.

#### Learn

PslG indeed have an effective ability to disassemble biofilm. We illustrated that the
HMGB1\_FL box is the most efficient one among four HMGB1 modified proteins to disassemble
biofilm. Those two proteins will be chosen to be our functional protein, and we also need a
more visualized method to prove that our idea is successful. Confocal microscopy is a great
choice.

### Intuitive observation by confocal microscopy

#### Design

The HMGB1\_FL was found to be the most effective part of HMGB1 for biofilm disassembling. And
also PslG is effective. We have confirmed that our target protein can effectively remove
biofilm through the previous experiment, but we wanted to observe the biofilm state before
and after treatment more intuitively, so we designed a more detailed observation using
confocal microscopy

#### Build

The HMGB1\_FL was mixed with PslG to co-treat the biofilm. After co-treatment with HMGB1\_FL
and PSLG, the grid structure of biofilm was observed by confocal microscopy after
staining with SYTO9 and HHL.

#### Test

Imaging was performed using confocal microscopy after treatment with protein for 24 h and
co-staining with the staining solution for 4 h.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig2.png)

Figure 2. HMGB1 and PslG contribute to
conspicuous clearance of biofilm.
DNA is stained by SYTO9 (green), and polysaccharide is stained by HHL (red).

#### Learn

Compared with the control group, most of the eDNA and polysaccharides in the experimental
group were digested. At the same time, the biofilm thickness was significantly reduced and
the grid structure was completely destroyed, indicating that our idea was successful. The
co-treatment of PslG and HMGB1 indeed had a strong ring breaking effect on biofilm .

### Cytotoxicity Test and Inflammation Verification

#### Design

After the successful expression of these effector proteins and examination of their effect
on eliminating the biofilm, it is necessary that they should be validated for safety, given
that we want these proteins to function in the intestines. There are two main approaches to
verifying the safety of the protein: cytotoxicity and pro-inflammatory activity, that is,
the protein will not cause a large number of cells to undergo cell death within a certain
range of concentration, and will not stimulate the relevant pathways of intestinal
epithelial cells to cause inflammation. To this end, we designed cytotoxicity tests and
inflammation verification experiments. We first added different proteins to the culture
medium for a certain period of time and then calculated the cell survival rate to
characterize the toxic and side effects of the protein on the cells. In addition, we also
examined the effect of different proteins on the expression of cytokines in cells. If there
is an up-regulation, it indicates that the pro-inflammatory activity is obvious. If there is
no significant difference from the control group, it means that there is almost no obvious
pro-inflammatory activity.

#### Build

To accomplish our objective, we cultured HT-29 cells to mimic the intestinal epithelium and
added different proteins into the medium fora certain period of time. For example, in terms
of validation of the cytotoxicity, proteins were added to the culture medium and incubated
for 24 hours. After that, we employed trypan blue staining to calculate the cell survival
rate because trypan blue only stains dead cells blue but not living cells. For experiments
related to inflammation verification, it turned out that one hour or two hours of incubation
was sufficient, as this is about how long it takes from the activation of the relevant
receptor by the protein to the transcription of the gene of the cytokine, which is what we
want to measure. Therefore, we first lysed the cells, extracted total RNA, reverse
transcribed them, and then subjected them to real-time PCR by adding primers for the
specified cytokine genes. Thus, we could examine the pro-inflammatory activity of different
proteins in terms of the amount of transcription.

#### Test

Different concentrations of engineered HMGB1 (full length) and PslG were added into the cell
culture medium and we incubated the cells for a certain period of time. After several
attempts, we found that those proteins would not impose a fatal influence on intestinal
cells in humans considering they would only cause strong toxic effects at very high
concentrations (over 2 μg/mL). What’s more, they are unlikely to exert a lethal impact
concerning inflammation of human intestinal cells according to the results.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig3.png)

Figure 3. Results of cytotoxicity test.
Different curves indicate the toxicity of different substances for cells.
Error bar represents s.d.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig4.png)

Figure 4. Engineered HMGB1 (full length)
exhibits an alleviated pro-inflammatory effect.
Cells are treated with HMGB1 (fl) for (a) an hour and (b) 2 hours (n = 3).
\*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA). Error bar represents s.d.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig5.png)

Figure 5. PslG exhibits little
pro-inflammatory effect on human intestinal cells.
Cells are treated with PslG for (a) an hour and (b) 2 hours (n = 3).
\*P < 0.05, \*\*P < 0.01 (One-way ANOVA). Error bar represents s.d.

#### Learn

The safe concentration range of the effector proteins was explored by cytotoxicity
experiments, and it was verified that the concentration range did not cause inflammatory
responses in intestinal cells. After discussion, we believed that it was necessary to
explore the secretion potency of the secretion system of the engineered bacteria, so as to
ensure that the concentration of secreted proteins would not have a serious effect on human
intestinal cells.

### Quorum sensing system

#### Design

The sensing device was designed based on the Type I quorum sensing mechanism of
*P. aeruginosa* to effectively produce PslG and HMGB1 only in response to the presence
of
*P. aeruginosa*. It is important to figure the relationship between target protein
expression
rate and induction concentration of 3OC12HSL. However, production of target
protein is hard to measure. In this work, we use green fluorescent protein (GFP) signal as a
reporter to
quantificationally characterize the quorum sensing device by flux gene that encoding GFP to
sensing device to build a test circuit.

#### Build

As described above, we build the plasmid: ptetR-LasR-pluxR-GFP. The tetR promoter
constitutively enhances expression of transcriptional factor LasR, which can bind to AHL
3OC12HSL.Then, LasR-3OC12HSL activator complex can open luxR promoter,
leading to production
of GFP which can be quantificationally measured by microplate reader.

#### Test

Single colonies of ptetR-LasR-pluxR-GFP (BL21) were induced with 3OC12HSL at
varying molar
concentration (5e-9,1e-8, 2.5e-8, 5e-8, 1e-7, 5e-7, 1e-6, 5e-6, 1e-5). Time-series
fluorescence and OD600 data were obtained at intervals of 10m for a total run time of 3h. We
observed a basal expression level of 0.427 RFU per OD per minute with lower concentration (<
5.0E-9M) of 3OC12HSL induction, followed by a sharp increase in GFP production
rate as the
concentration of 3OC12HSL was increased beyond1.0E-8M. GFP expression achieved a
peak at
1.0E-7M. These results announced that the optimal detection range of the sensing device was
higher than 1.0E-7M3OC12HSL. At the same time, we also used Hill equation to
curve fit the relationship between the time-average GFP expression rate and 3OC12HSL
concentration.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig6-1.png)

Figure 6. Characterization results of quorum
sensing device.
a, GFP production rate per cell over time at different concentration of 3OC12HSL.
b, Time-average GFP production rate per cell over time at different concentration of
3OC12HSL.
Error bar represents s.d.

To verify that the sensing device would be able to sense the natively produced
3OC12HSL by
*P. aeruginosa*, the sensing device was induced by the filtered culture of *P.
aeruginosa*. And
the result show that GFP expression rate was 1.159 RFU per OD per minute so that we can
estimate the concentration of natively produced 3OC12HSL was 5.0E-7M.This value
is higher
than 1.0E-7 which confirmed that the sensing device was able to detect the natively produced
3OC12HSL.

#### Learn

We confirmed that the sensing device have a lower leakage expression and a sharp induced
response curve that can effective induce production of target protein with natively
produced 3OC12HSL. It is excited that GFP expression rate reach the peak in 40min
(> 2.5 RFU
per OD per minute) and then become lower with a rapid decline, which means the quorum
sensing device has a very fast response rate and the regulation of 3OC12HSL is
instantaneous. However, the overall induced expression of GFP remained unsatisfactory,
increasing the number of copies of pluxR may be absolution. At the same time, how to secrete
the protein we induce to extracellular expression is also a problem we need to consider
next.

### Arabinose-induced cell lysis

#### Design

Our previous experiments demonstrated that the quorum sensing factor of Pseudomonas
aeruginosa can successfully activate the expression of downstream proteins. In a practical
application scenario, we need the engineered bacteria to express two proteins and release
them to the outside world to act on the biofilm. We tried to let bacteria lyse itself by
expressing lysis E7 protein, in order to release a large number of disassembly proteins in a
short time near the biofilm like a "demolition truck". We also tried to insert lysis E7
into arabinose operon, so as to achieve artificial regulation of cell lysis. Here we need to
verify whether this lysing system can function properly.

#### Build

We first construct the plasmid in which lysis E7 is inserted into Arabinose operon by
homologous recombination and transfer it to BL21. Next, we designed two detection methods:
First, 1%, 0.1% and 0.01% arabinose were used for induction, while a control group inhibited
by 0.2% glucose was added (theoretically, the function of the arabinose operon could be
effectively inhibited), and growth curves were drawn to detect the induction effect and
lysis status. Second, arabinose induced group, non-induced group and non-induced physical
lysing group were set up. After the bacterial solution of the three groups was cultured for
a period of time, the supernatant was centrifuged to detect the DNA content, and the lysing
effect was measured by comparing the DNA concentration with each other.

#### Test

Arabinose-induced growth curve: The bacterial solution with an OD600 absorbance of 2
was diluted 1:100 and cultured until the OD600 absorbance reached 0.5. 1%, 0.1% or
0.01% arabinose was added for induction, while the control group inhibited by 0.2% glucose
was
added. The OD600 absorbance was detected every 15min and a curve was drawn.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig7.png)

Figure 7. Upon arabinose induction, Lysis E7
is expressed followed by massive death of engineered bacteria.
Error bar represents s.d.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig8.png)

Figure 8. Lysis E7 induces lysis of bacteria.
Error bar represents s.d.

DNA concentration detection of supernatant: The bacterial solution with an OD600 absorption
value of 2 was diluted 1:100 and cultured until the OD600 absorbance reached 0.5. The
treatment group was induced by adding 1% arabinose, while the two control groups were
arabinose free. After culturing for 5h, we collect the supernatant of three groups by
centrifuging (one of the two control groups was cracked by freezing and thawing repeatedly
before centrifuging). Test their DNA concentrations.

#### Learn

After adding arabinose of different concentrations, the growth of bacterial solution
concentration slowed down and began to decline at 50-100 minutes. The induction effect of
1% arabinose and 0.1% arabinose is basically the same, and both can greatly reduce the
concentration of fine liquid. However, the induction effect of 0.01% arabinose was
relatively
weak, and the concentration of bacterial solution showed a rising trend when it was about
4h20min. The curve of the control group treated with 0.2% glucose reached a plateau at about
3h, and there was no downward trend. The DNA content of the bacterial supernatant after
1% arabinose induction was basically the same as that of the physical cracking group, both
of
which were significantly higher than that of the non-induction group.

The results of these two sets of experiments indicate that arabinose operon can function
normally, and the cracking system we constructed can also be artificially regulated. It
means we have at least one way to ensure a relatively sufficient proteins can reach the
biofilm to serve the function of disassembly. Based on this, we can add some signal peptides
to help the functional proteins be secreted out of the cell. Under this condition, lysis E7
can become a backup solution which will only used when there is a large amount of biofilm.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}On this page, we show all the experimental steps and protocols that we did{% endblock %}
{% block offcanvas %}

##### Menu

* [Overview](#overview)
* [Plasmid construction](#section1)
* [Protein purification](#section2)
* [Protein function verification](#section3)
* [Confocal microscopy imaging](#section4)
* [Cytotoxicity Test and Inflammation Verification](#section5)
* [Arabinose-induced cell lysis](#section6)
* [Future Plans](#future)
* [Reference](#reference)

{% endblock %}
{% block page\_content %}

![...](https://static.igem.wiki/teams/4785/wiki/experiments/head.png)

Experiment Design

### Plasmid construction

#### Reagents:

pET28a plasmid, DNA fragment, primer, Phanta Max Super-Fidelity
DNA
Polymerase Kit, DNA Loading Buffer, FastPure Gel DNA Extraction Mini Kit, 1×TAE Buffer

#### Steps:

##### PCR amplification of gene fragments/preparation of linearized vector

1. Primer design: According to the sequence information of the target gene, the
   upstream
   and downstream primers should be designed respectively. The primer should have 15-20
   bp
   on the primer to pair with the target gene template, and the Tm value of the primer
   should be close to 58℃. When designing the primers for homologous recombination, the
   homology arm sequence of 15-20 bp should be added to the 5 'end of the primer.
2. Configuration and procedures of the PCR reaction system: Phanta Max Super-Fidelity
   DNA Polymerase kit from Vazyme was used for PCR in this study. The PCR reaction
   system and reaction procedures were as follows
![...](https://static.igem.wiki/teams/4785/wiki/experiments/2.png)

##### Agarose gel electrophoresis

The PCR products were confirmed by agarose gel electrophoresis to confirm whether the
molecular weight was consistent with the target amplification fragment, and the enzymes
and other reagents in the PCR system were separated to avoid affecting the subsequent
experiments. Here's how:

1. Prepare 1% agarose gel: Weigh 0.5 g agarose powder, add 50 mL 1×TAE Buffer, dissolve
   by heating, add nucleic acid dye and pour into the mold to let cool.
2. 24 μL of PCR product was added to 3 μL of 10×DNA Loading Buffer and subjected to
   electrophoresis at 150 V for 20 min. DNA markers were used as molecular weight
   controls during electrophoresis.
3. The luminous DNA bands were observed under ultraviolet light and their molecular
   weights were compared.

##### Gel recycling

After confirmation by electrophoresis, the DNA bands could be recovered by gel recovery.
FastPure Gel DNA Extraction Mini Kit from Vazyme was used for glue recovery in this
study. The steps are as follows:

1. After DNA electrophoresis, the gel containing the target DNA fragment was quickly
   cut out under UV lamp, and the weight of the gel was weighed. 100 mg of gel was
   equivalent to 100 μL volume, which was regarded as one gel volume.
2. Equal volume of Buffer GDP was added. A water bath at 55 ° C for 10 min was used to
   dissolve the gel blocks.
3. The sol solution was transferred to the adsorption column and centrifuged at 13400×g
   for 1 min.
4. Discard the filtrate, add 300 μL Buffer GDP, and let stand for 1 min. The samples
   were centrifuged at 13400×g for 1 min.
5. Discard the filtrate and add 700 μL Buffer GW. The samples were centrifuged at
   13400×g for 1 min.
6. Repeat step (e), discard the filtrate and centrifuged at 13400×g for 2 min.
7. 30 μL Elution Buffer was added to the center of the adsorption column and left for 2
   min. DNA fragments were recovered by centrifugation at 13400×g for 1 min and used
   for subsequent experiments.

##### homologous recombination connection

1. The amount of DNA required for recombination reaction was calculated as follows: the
   optimal amount of linearized vector X = [0.02× linearized vector base pairs] ng; The
   optimal amount of target gene fragment Y = [0.04× number of base pairs of target
   gene fragmenting
2. The homologous recombination ligation reaction system was prepared as follows
![...](https://static.igem.wiki/teams/4785/wiki/experiments/4.png)

The reaction was performed at 37 ° C for 30 min to obtain homologous recombination
products.

### Protein purification

#### Reagents:

LB medium, ampicillin, IPTG, PMSF, ddH2O, binding buffer, Washing buffer,
Elution buffer, Ni NTA Beads 6FF beads.

#### Steps:

##### Protein expression in **E. coli* BL21* cell line

The prokaryotic expression system is the most convenient and economical expression
system, which does not require the construction of complex expression vectors or
purification of a large number of expression vectors. However, the prokaryotic
expression system has some disadvantages such as the inability to modify proteins and
secrete proteins. In this study, the **E. coli* BL21* cell line was used to express
intracellular proteins. The procedure is as follows:

1. The constructed expression vector was transfected into BL21 competent cells, and
   the monoclonal colonies were inoculated into 10 mL ampicillin resistant LB medium
   and cultured overnight.
2. The overnight culture solution was added to 1 L ampicillin resistant LB medium,
   incubated at 37 ℃ for 3-4 h in shaking table, and then cooled to 16 ℃.
3. IPTG was added to 100 μmol/L to induce protein expression, and the protein was
   expressed at 16 ℃ for 16-20 h.

The bacteria were collected by centrifugation of the overnight expressed bacteria
solution, and the bacteria were frozen at -80 ℃ for precipitation until use

4. Purification by affinity chromatography

In this study, PslG protein and HMGB1 series protein sequences were constructed in
pET28a-6 ×His vector and expressed and purified using *Escherichia coli* BL21 cell line.
The cell precipitation containing the expressed proteins was obtained in the above
experiments. In this study, Ni NTA Beads 6FF were used to purify proteins with His tag.
The adsorption beads, that is, nickel columns, can bind recombinant proteins with His
tag by chelating nickel ions. The combination of recombinant proteins with nickel
columns can be competitively eluted by imidazole.

Lysis of bacterial cells: 1×Binding Buffer was added to the collected cell
precipitate, cells were lysed and fixed to 40 mL, 400 μL of 100 mM PMSF protease
inhibitor was added, and resuspended with shaking until no clumps occurred. The
bacterial solution was poured into the material cup of the high-pressure homogenate
crusher at 4 ℃ for 500 mPa until the bacterial solution became clear from turbidity. At
this time, the intracellular proteins were lysed into the solution. The broken bacterial
solution was collected and centrifuged at 18000 rpm for 50 min at 4 ° C to remove bulk
cell debris.

Pretreatment of Ni beads: 5 mL NTA wall was added to the gravity chromatography
column, rinsed 3-5 times with 5 times the column volume of ultrapure water, and rinsed
1-2 times with 1×Binding Buffer.

Ni beads affinity chromatography: After filtration, the supernatant of the bacterial
lysate was added to the chromatography column at room temperature for 30 min to bind the
wall. After elution of unbound lysate, the walls were rinsed with 10 times the column
volume of 1×Binding Buffer, and after rinsing, the walls were rinsed with 20 times the
column volume of nickel column wash litter. After washing, the protein was eluted by 1
times nickel column eluent, which contained the eluted recombinant protein with His tag.
The eluent was stored at 4 ℃.

### Protein function verification

#### Reagent & Materials:

1. Proteins from [Protein Purification] part, including A, B, AB, FL, PslG;
2. GB buffer (50mM Tris,150mM NaCl), LB medium, M9 medium;
3. Strain PAO1;
4. Incubator(Thermofisher), enzyme-labeler(Tecan).

##### [Part1] Buffer replacement

1. Prepare a new buffer GB (50mM Tris,150mM NaCl);
2. Add the protein into a suitable type of concentration tube and centrifuge it to the
   limit volume by 2300g;
3. Discard the waste liquid, add2ml buffer solution GB to the concentration tube, and
   centrifuge 2300g to the limit volume;
4. Repeat step 3 twice;
5. Add 2ml buffer GB to the concentration tube, mix with the protein, suck out and
   store.

##### [Part2] Inhibition of biofilm growth experiment

Use LB medium to resuscitate PAO1;

The resuscitated PAO1 bacterial solution was diluted to OD600=0.001 using M9 medium and
inoculated into 96-wellplates (100ul per well). FL (final concentration 200nM), AB (final
concentration 200nM), A (final concentration 200nM), B (final concentration200nM) were added
to the treatment group, respectively. PslG (final concentration 50nM) was used to supplement
the total volume of solution in each hole to 130ul using buffer GB (50mM Tris, 150mM NaCl),
and the control group was directly added with 30ul buffer GB. Cultured at 37℃.

When the culture time reaches 0h, 6h, 12h, 24h, the following operations are carried
out: Add 150ul ddH2O for oscillatory cleaning and sucking out (twice), add150ul 0.1% crystal
violet solution for dark dyeing for 15min and sucking out, add 200ul ddH2O for oscillatory
cleaning and sucking out (twice), add 200ulanhydrous ethanol for dark dissolution for 15min.
The OD550 of the sample was detected with an enzyme-labeler and the data was recorded.

##### [Part3] Biofilm disassembly experiment

1. PAO1was resuscitated using LB medium;
2. The recovered PAO1 solution was diluted to OD600=0.001 using M9 medium, inoculated
   into 96-well plates (100ul per well), and cultured at 37℃ for 24h;
3. FL (final concentration 200nM), AB (final concentration 200nM), A (final
   concentration 200nM), B (final concentration 200nM), PslG (final concentration 50nM)
   were added to the treatment group, and the buffer GB (50mM Tris, 150mM NaCl) was used to
   supplement the total volume of solution in each hole to 130ul, and the control group was
   directly added with 30ul buffer GB. Cultured at 37℃.
4. When the culture time reaches 0min, 1h, 2h, 4h, 8h, the following operations are
   carried out: Add 150ul ddH2O for oscillatory cleaning and sucking out (twice), add 150ul
   0.1% crystal violet solution for dark dyeing for15min and sucking out, add 200ul ddH2O
   for oscillatory cleaning and sucking out(twice), add 200ul anhydrous ethanol for dark
   dissolution for 15min. The OD550 of the sample was detected with an enzyme-labeler and
   the data was recorded. a

### Confocal microscopy imaging

#### Reagents:

syto9 stain, HHL stain, M9 medium, LB medium, purified protein, GB buffer,
ddH2O.

#### Steps:

1. *Pseudomonas aeruginosa* (PAO1) was resuscitated and spread on LB plates. After culturing
   in a 37 ° C constant temperature incubator for one day, monoclonal colonies were
   selected and inoculated into LB liquid medium. After culturing in a 37 ° C shaker, the
   colonies were grown to OD greater than 1, and diluted with M9 liquid medium for a
   thousand times, 3ml of the diluent was inoculated into cell culture dishes. Mature
   biofilm was obtained by incubating the cells in an oven at 37 ° C for 48h
2. In the experimental group, 1mL HMGB1\_A box (200nM) and 1mL PslG (50nM) (dissolved in GB
   buffer) were added into the mature biofilm cell culture dish, and then returned to the
   37 ° C constant temperature incubator for 24 hours

In the control group, 2mL GB buffer was added into the mature biofilm cell culture dish,
and then returned to the 37 ° constant temperature incubator for 24 hours

3. After the treatment, the biofilm was carefully sucked out and discarded, and the
   biofilm was cleaned with ddH2O. syto9 (staining for eDNA) and HHL (staining of Psl)
   staining solution were diluted to the working concentration, respectively, and 1mL syto9
   and 1mL HHL staining solution were added to each dish at the same time and treated in
   the dark for 4h.
4. At the end of the treatment, the liquid in the Petri dish was carefully aspirated and
   discarded, and the biofilm was washed twice with ddH2O.
5. Observation under confocal microscope

### Cytotoxicity Test and Inflammation Verification

To verify the safety of the two proteins, we designed two experiments in this section,
cytotoxicity test and inflammation verification. The cell line we used was HT-29 (American
Type Culture Collection, ATCC). LPS was purified from *E. coli* O111:B4 (Beyotime). The
technique we used to test toxicity was trypan blue staining. In terms of the inflammation
verification, we first extracted total RNA from the cells and then carried out the reverse
transcription followed by qPCR, in order to detect the expression of 2 major
pro-inflammatory cytokines - TNF-α and IL-1β.

#### Section 1: Cell Culture

1. Take out a 60mm cell culture dish, and seed 5×106 HT-29 cells in 5 mL McCoy's 5A
   complete medium (Components: McCoy's 5A + 10% fetal bovine serum + 1%
   Penicillin/Streptomycin).
2. Incubate at 37°C with 5% CO2 until the confluence reaches 90%. All reagents should
   be pre-warmed at room temperature.
3. Afterward, wash the cells three times with 2 mL phosphate buffer solution (PBS) in
   preparation for the experiment.
4. In terms of subculture, add 1 mL 0.25% Trypsin-0.02% EDTA (mixed in PBS with phenol
   red) and wait for 5 minutes.
5. Use 3 mL complete medium to terminate digestion, and collect all the cell suspension
   into a 15 mL centrifuge.
6. Centrifuge at 1,000 rpm lasting 5 minutes.
7. Remove the supernatant and add 1 mL complete medium to resuspend the cells. Then
   transfer cell suspension to another petri dish at a specific ratio.

#### Section 2: Cytotoxicity Test

1. Incubate HT-29 cells until the confluence reaches 90%.
2. Add specific substances to the medium and incubate at 37°C with 5% CO2 for 24
   hours.
3. Trypsinize the cells and make the cell suspension according to Section 1.
4. Mix 10 μL of the cell suspension (diluted if needed) with 10 μL 0.4% trypan blue
   solution in a 1.5 mL centrifuge tube.
5. Mix the suspension, add 10 μL mixture to the hemocytometer, and count the cells.
   Count both the live cells and dead cells, then calculate the survival rate.

#### Section 3: Inflammation Verification

##### Extract Total RNA

1. Incubate HT-29 cells until the confluence reaches 90%.
2. Cell Lysis:

   1. Discard the culture medium and wash once with 1 × PBS.
   2. Add 1-2 mL of RNAiso Plus (TaKaRa) to each 10 cm2 of the cell growth area,
      gently shake to ensure even distribution of the lysis solution on the cell
      surface (a 60 mm culture dish has a growth area of 21 cm2 and can use 3 mL).
   3. Transfer the lysate-containing cells to a centrifuge tube, and repeatedly
      pipette up and down until there is no obvious precipitation in the lysis
      solution.
   4. Allow it to stand at room temperature (15-30°C) for 5 minutes.
3. Add chloroform (1/5 volume of RNAiso Plus) to the homogenized lysate from the above
   step. Seal the centrifuge tube tightly, mix until the solution becomes milky white, and
   let it stand at room temperature for 5 minutes.
4. Centrifuge with 12,000 g at 4°C for 15 minutes. Carefully remove the centrifuge tube
   from the centrifuge. At this point, the homogenate separates into three layers: a
   colorless upper aqueous layer (containing RNA), a white middle protein layer (mostly
   DNA), and a colored lower organic phase.
5. Aspirate the upper aqueous layer and transfer it to another clean centrifuge tube
   (do not aspirate the white middle layer).
6. Add isopropanol at 0.5-1 times the volume of RNAiso Plus to the upper aqueous layer.
   Invert the centrifuge tube several times to mix thoroughly and let it stand at room
   temperature for 10 minutes (if RNA quantity is low, 1 μL of glycogen dye can be added
   after adding isopropanol).
7. Centrifuge with 12,000 g at 4°C for 10 minutes. After centrifugation, RNA will
   precipitate at the bottom of the tube.
8. Carefully discard the supernatant without disturbing the precipitate. A small amount
   of residual isopropanol is acceptable. Add an equal volume of 75% ethanol to the
   precipitate, gently invert the centrifuge tube to wash the tube wall, and centrifuge
   with 7,500 g at 4°C for 5 minutes. Carefully discard the supernatant without disturbing
   the precipitate.
9. Remove the centrifuge tube cap and air-dry the precipitate at room temperature for a
   few minutes. Once the precipitate is dry, add an appropriate volume (20 μL) of
   RNase-free water (DEPC water) to dissolve the precipitate.
10. Measure the RNA concentration, loading 1 μL each time for analysis on the
    instrument (NanoDrop).

##### Reverse Transcription

1. Removal of Residual Genomic DNA
   Prepare the following mixture in an RNase-free centrifuge tube and gently mix by
   pipetting up and down. Incubate at 42°C for 2 minutes.
   ![...](https://static.igem.wiki/teams/4785/wiki/experiments/5.png)
2. Reverse Transcription Reaction Setup (20 μL reaction)
   Directly add 4 × Hifair III SuperMix plus (YEASEN) to the reaction tube from Step 1 and
   gently mix by pipetting up and down.
   ![...](https://static.igem.wiki/teams/4785/wiki/experiments/6.png)
3. Reverse Transcription Program Settings

   ![...](https://static.igem.wiki/teams/4785/wiki/experiments/7.png)

[Note]: Recommended reverse transcription temperature is 55°C. For templates with a high GC
content or complex templates, the reverse transcription temperature can be increased to
60°C. Reverse transcription products can be used immediately for qPCR reactions or stored at
-20°C for short-term use. For long-term storage, it is recommended to aliquot and store at
-80°C to avoid repeated freeze-thaw cycles.

##### Real-time Fluorescent Quantitative PCR

1. Design the primers for human IL-1β and TNF-α genes. The HT-29 cell cDNA obtained in the
   above steps was used as the template for real-time fluorescence quantitative PCR
   experiments.
2. Reaction System:

   ![...](https://static.igem.wiki/teams/4785/wiki/experiments/8.png)
3. Reaction Program Settings:

   ![...](https://static.igem.wiki/teams/4785/wiki/experiments/9.png)
4. The relative mRNA expression was calculated by the 2-ΔΔCt method to
   demonstrate the
   amplification ratio of inflammatory cytokine genes in different cell treatment groups
   relative to the internal reference genes.

##### Quorum sensing system

###### Characterization of pTetR-LasR-pLuxR-GFP with 3OC12HSL

Single colonies of pTetR-LasR-pLuxR-GFP (BL21) were each inoculated into 5 ml of prewarmed
supplemented M9 ampicillin for overnight culture in a shaking incubator at 37℃. After
overnight growth, the cultures were diluted to OD600 of 0.002 and allowed to incubate
further to OD600 of 0.5 under the same condition. Cultures were then transferred into a
transparent, flat-bottom 96-well plate in triplicate aliquots of 200 ml for induction with
3OC12HSL at varying molar concentrations (5e-9, 1e-8, 2.5e-8, 5e-8, 1e-7, 5e-7, 1e-6, 5e-6,
1e-5). The plate was incubated at 37℃ with rapid shaking in a microplate reader (Biotek) and
assayed for green fluorescence. Time-series fluorescence and OD600 data were obtained at
intervals of 10 m fora total run time of 3 h. The result was zeroed with supplemented M9 to
remove background fluorescence and OD600. A relative GFP production rate was derived as a
ratio of background subtracted green fluorescence to OD600 value. A time-averaged GFP
synthesis rate was obtained by averaging the relative GFP production rates between 20 and 80
m after induction with 3OC12HSL. The experimental results were fitted using an empirical
mathematical model (Hill equation),

![...](https://static.igem.wiki/teams/4785/wiki/experiments/10.png)

The equation models GFP synthesis rate (y) as a function of input concentration of 3OC12HSL
([C12]). The four parameters (A, B, C, n) were estimated to obtain the best fit curve by
performing a non-linear curve fitting using the experimental results. This curve fitting was
performed using Origin.

### Arabinose-induced cell lysis

#### Reagent & Materials:

1. Arabinose, glucose;
2. Strain BL21;
3. Incubator (Thermofisher).

#### [Part1] The growth curve induced by arabinose

1. The bacterial solution with OD600 absorbance of 2 was diluted 1:100 and divided
   into four groups;
2. Cultured at 220 RPM at 37℃ until theOD600 absorbance reached 0.5, three groups
   were induced by adding 1%,0.1% or 0.01% arabinose, while the control group was inhibited
   by 0.2% glucose;
3. Put it back to 37℃ for cultivation at 220rpm, detect the OD600 absorbance
   every 15min, and draw a curve.

#### [Part2] DNA concentration detection of supernatant

1. The bacterial solution with OD600 absorbance of 2 was diluted 1:100 and divided
   into three groups (induction group, non-induction group, physical cracking group);
2. Cultured at 220 RPM at 37℃ until theOD600 absorbance reached 0.5, the
   induction group was induced by adding1% arabinose, and the supernatant of the induction
   group and the non-induction group was centrifuged after 5h;
3. Repeated freeze-thaw of the physical cracking group: liquid nitrogen is frozen,
   stored at -80℃ for 30min, and thawed with 99℃ metal baths after removal;
4. Repeat step 3 twice;
5. Physical cracking group centrifuge supernatant. Three DNA concentrations were
   measured, data collected and compared.

### Future Plans

#### Introduction

Many Gram-negative bacteria have a macromolecular complex called the type II secretion system
(T2SS) that spans the cell envelope. It belongs to a much larger superfamily of systems that
comprise type IV filaments and share homologous parts and conserved mechanistic principles.
These include the archaeal T4 pilus, the type 4a and 4b pilus, the tight adherence (Tad)
pilus, the competence pilus, the mannose-sensitive hemagglutinin pilus, and the flagellum.
Phylogenetic investigations indicate a lineage split from the last universal common ancestor
(LUCA), and these systems are ancient and crucial to both the bacterial and archaeal
kingdoms. The T2SS is definitely present in ECN and is particularly concentrated in the
Alpha-, Beta-, Gamma-, and Delta-proteobacteria, the Bacteroidetes, and the Deferribacteres.
The T2SS substrate transport pathway consists of two phases, the first of which is carried
by the Sec or Tat protein export pathway from the cytoplasm to the periplasmic space and
stays there for a while until a signal is received that activates the secondary export of
the substrate. To target to either the Sec or the Tat (twin-arginine translocation) protein
export pathway, proteins that are found outside the cytoplasm must be produced with
amino-terminal signal peptides. The Tat pathway transports proteins that have already
folded, whereas the Sec apparatus translocate polypeptides in an unstructured state. This is
the primary functional distinction between these two export systems. The Sec pathway is
fundamental, universally conserved, and typically the primary channel for protein export.
Contrarily, only a small number of organisms have been discovered to require the Tat route,
at least under normal laboratory settings. It is present in some bacteria and archaea but
not all. The Tat pathway, which enables the effective export of tailored proteins to
microvirus/biofilm lesion sites, is fortunately present in ECN by natural means.

#### Steps

##### 1. Design the primers

The T2SS is a macromolecular complex that spans the cell envelope of many Gram-negative
bacteria. It is part of a much larger superfamily of type IV filament containing systems all
of which share homologous components and conserved mechanistic principles. These include the
type 4a and 4b pilus, tight adherence (Tad) pilus, mannose-sensitive hemagglutinin pilus,
competence pilus and archaeal T4 pilus and flagellum. These systems are ancient and
fundamental to both bacterial and archaeal kingdoms with phylogeny analyses suggesting a
lineage split from within the last universal common ancestor (LUCA). The T2SS is
particularly concentrated in the Alpha-, Beta-, Gamma- and Delta-proteobacteria, the
Bacteroidetes and Deferribacteres and for sure, this secretory system exists in ECN. In
T2SS, the substrate transport pathway consists of two phases, the first of which is
transported from the cytoplasm to the periplasmic space via the Sec or Tat protein export
pathway and remains there for a period of time until a signal is obtained that activates the
secondary export of the substrate.

![...](https://static.igem.wiki/teams/4785/wiki/future-plan/1.png)

Costa, T., Felisberto-Rodrigues, C., Meir, A.
et al. Secretion systems in Gram-negative bacteria: structural and mechanistic insights.
Nat Rev Microbiol 13, 343–359 (2015). https://doi.org/10.1038/nrmicro3456

It is necessary for proteins that are located outside the cytoplasm to be synthesized with
amino-terminal signal peptides so that they can target to either the Sec or the Tat
(twin-arginine translocation) protein export pathway. The key functional difference between
these two export systems is that the Sec apparatus translocates polypeptides in an
unstructured state, whereas the Tat pathway transports proteins that have already folded.
The Sec pathway is universally conserved, essential and normally the main route of protein
export. The Tat pathway, by contrast, is found in some, but not all, bacteria and archaea
and has been identified as essential (at least, under standard laboratory conditions) in
only a few organisms. We are fortunate that ECN naturally possesses the Tat pathway, which
allows for the successful export of designed proteins to microvirus/biofilm lesion
sites.

After researching the typical signal sequence of Tat export pathway, according to the Amino
Acid Codon, we simply designed two signal sequences for exporting and processed codon
optimization that would increase translational efficiency via website.

![...](https://static.igem.wiki/teams/4785/wiki/future-plan/2.png)

Seq 1:
AUGTTTCAGGCCAGCCGCCGTCGCTTCATCAAAGCGAGCCCGATCGCACTGTGCGCTGGTGCGGGTGCAGTTCCGCTGCCACGTGCTACCGCG

Seq 2:
AUGTCCCTGTCTCGCCGTCAGTTCCTGCAGGCATCCCCGATTGCACTGTGTGCTGGCGCTGGTGCAGTACCGCTGAAGAGCAAGGC
Considering the length of the primer, we choose the shorter one as our final signal
sequence.

Primer F: Tm = 51℃
AAAAGGATCTTCACCTAGATC
Primer R: Tm = 48℃
TGATAATCTCATGACGTGTTCCAGGCAGGTAGGTGGCGGTTCGTTCAGACGAGCCCAGTTGCGCTGGGTACACGGACCTGGGCAGTTAC

GTTCCAGGGCGAAGCTCAAAATCCCTTAACGTGA

##### 2. PCR

![...](https://static.igem.wiki/teams/4785/wiki/future-plan/3.png)

We simulated the results of PCR and showed that this design is feasible.
After PCR, we send the plasmid for sequencing to make sure that there’s no mistake on base
pairs.

##### 3. Protein purification

We will conduct transformation in DH5α competent cells to get more engineered plasmids.
Meanwhile, BL21C is used to purify proteins. After incubation, we collect suspension(because
our targeting proteins will be secreted outside the cells according to the signal peptide),
which contains our proteins.
The protein also has GST tag. Thus we use GST beads to purify the proteins.

##### 4. Test

SDS-PAGE and Western blotting are used to examine whether the proteins are secreted. By test the
mRNA quantities, we can calculate the secretion efficiency.

### Reference

1. *Costa, T. R. D., Felisberto-Rodrigues, C., Meir, A., Prevost, M. S., Redzej, A.,
   Trokter, M., & Waksman, G. (2015). Secretion systems in Gram-negative bacteria:
   Structural and mechanistic insights. Nature Reviews Microbiology, 13(6), Article 6.
   https://doi.org/10.1038/nrmicro3456*

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}On this page, we describe all our results in detail{% endblock %}
{% block offcanvas %}

##### Menu

* [Protein Expression and Purification](#overview)
* [Quantitative Analysis by Crystal Violet
  Staining](#section1)
* [Intuitive Observation by Confocal Microscopy](#section2)
* [Side Effects Examination and Sensor
  Validation](#section3)
* [Arabinose-Induced Cell Lysis](#section4)
* [Future Plan](#section5)
* [Considerations for replicating the experiments](#section6)

{% endblock %}
{% block page\_content %}

## Protein Expression and Purification

### Plasmid design, construction

In order to achieve the purification of the target proteins, a total of five plasmids
were constructed. These plasmids were named as follows: pET28a-6×His-HMGB1\_FL,
pET28a-6×His-HMGB1\_AB, pET28a-6×His-HMGB1\_A, pET28a-6×His-HMGB1\_B, and
pET28a-6×His-PslG. Upon completion of the sequencing process, it was ascertained that
the plasmid had been accurately sequenced and was deemed suitable for utilization.

![...](https://static.igem.wiki/teams/4785/wiki/result/1.png)

Figure 1. Schematic of protein expression
plasmids. a, plasmid pET-28a-6×His-HMGB1\_A. b, plasmid pET-28a-6×His-HMGB1\_B. c,
plasmid pET-28a-6×His-HMGB1\_AB. d, plasmid pET-28a-6×His-HMGB1\_FL. e, plasmid
pET-28a-6×His-PSLG\_FL.

### Expression and purification of proteins

The plasmids exhibiting accurate sequencing outcomes were subsequently introduced into
*Escherichia coli* BL21 for the purpose of inducing expression. Following this,
*Escherichia coli* cells were disrupted, and the desired protein was isolated by
affinity
chromatography and molecular sieve techniques, resulting in the acquisition of a very
pure form of the target protein.

![...](https://static.igem.wiki/teams/4785/wiki/result/2.png)

Figure 2. SDS-PAGE of protein expression
and Ni-NTA purification. a, HMGB1\_A box. b, HMGB1\_B box. c, HMGB1\_AB box. d,
HMGB1\_FL. e, PslG\_FL

## Section 1: Quantitative Analysis by Crystal Violet Staining

### 1.1 Biofilm Disassembly assay

Crystal violet is a viable option for the quantitative analysis of biofilm quantities.
Through the application of several proteins, we have successfully proved their capacity
to mitigate the formation of biofilms. One notable observation is that the presence of
these proteins in the surrounding environment has the capacity to impede the formation
of biofilms. Moreover, with respect to a fully developed biofilm, these proteins possess
the capability to degrade its architecture within a specific temporal duration.

In the experiment involving the disassembly of biofilms, the treatment group using PslG
demonstrated a reduction in the absorbance of OD550, which corresponds to the
biomass of the biofilm, from approximately 1.6 to approximately 1.0 within a one-hour
timeframe. On the other hand, among the four HMGB1 modified proteins, HMGB1\_FL and
HMGB1\_B box exhibited a stronger ability to disassemble the biofilm. Based on these
findings, we have decided to select PslG and HMGB1\_FL as our functional proteins.

![...](https://static.igem.wiki/teams/4785/wiki/result/3.png)

Figure 3. Time course of PslG，FL，AB，A，B
effect on biofilm disassembly. The biofilm biomass was measured by CV assay.

### 1.2 Biofilm inhibition assay

In the inhibition of biofilm growth experiment, it was observed that the group treated
with PslG efficiently suppressed the growth of biofilm in comparison to the control
group. Additionally, four proteins modified with HMGB1 exhibited similar efficacy in
inhibiting biofilm growth.

![...](https://static.igem.wiki/teams/4785/wiki/result/4.png)

Figure 4. Time course of PslG，FL，AB，A，B
effect on biofilm growth. The biofilm biomass was measured by CV assay.

## Section 2: Intuitive Observation by Confocal Microscopy

In our experimental approaches, we employed confocal microscopy to enable the visual
depiction of changes occurring within the biofilm. Through the utilization of this
imaging method, we have successfully proved the complete elimination of fully grown
biofilms through the action exerted by these proteins. The use of these proteins led to
a significant reduction in biofilm biomass as compared to the control group. The absence
of the DNA and polysaccharide network is apparent, yet the persistent presence of
fluorescent dots, which serve as representations of *P. aeruginosa*, confirms the
effectiveness of our strategy in degrading the biofilm.

![...](https://static.igem.wiki/teams/4785/wiki/result/5.png)

Figure 5. Direct observation of biofilm
with SYTO9 and HHL staining.

![...](https://static.igem.wiki/teams/4785/wiki/result/6.png)

Figure 6. HMGB1 and PslG contribute to
conspicuous clearance of biofilm.
DNA is stained by SYTO9 (green), and polysaccharide is stained by HHL (red).

The comparative study of the two sets of trials allows for the observation of changes in
biofilm thickness before and after the treatment. The experimental group demonstrated a
statistically significant drop in extracellular DNA (eDNA) and polysaccharide content,
together with a reduction in biofilm thickness, in comparison to the control group.
These findings provide more evidence to substantiate the influence of the target
protein.

![...](https://static.igem.wiki/teams/4785/wiki/result/7.png)

Figure 7. HMGB1 and PslG contribute to
conspicuous reduction of biofilm thickness.
DNA is stained by SYTO9 (green), and polysaccharide is stained by HHL (red).

## Section 3: Side Effects Examination and Sensor Validation

### 3.1 Cytotoxicity Test and Inflammation Verification

Given the expected release of these proteins by genetically engineered bacteria in the
human gastrointestinal tract, it is crucial to establish that these proteins do not
induce any adverse effects on human cells. The experimental results suggest that the
proteins being studied exhibit low levels of toxicity, as they only cause cell death at
very higher concentrations. Moreover, it is important to acknowledge that these proteins
do not induce a significant inflammatory response.

![...](https://static.igem.wiki/teams/4785/wiki/result/8.png)

Figure 8. Results of cytotoxicity test.
Different curves indicate the toxicity of different substances for cells.
Error bar represents s.d.

![...](https://static.igem.wiki/teams/4785/wiki/result/9.png)

Figure 9. Engineered HMGB1 (full length)
exhibits an alleviated pro-inflammatory effect.
Cells are treated with HMGB1 (fl) for (a) an hour and (b) 2 hours (n = 3).
\*\*P < 0.01, \*\*\*P < 0.001 (One-way ANOVA). Error bar represents s.d.

![...](https://static.igem.wiki/teams/4785/wiki/result/10.png)

Figure 10. PslG exhibits little
pro-inflammatory effect on human intestinal cells.
Cells are treated with PslG for (a) an hour and (b) 2 hours (n = 3).
\*P < 0.05, \*\*P < 0.01 (One-way ANOVA). Error bar represents s.d.

### 3.2 Quorum Sensing System

The quorum sensing system was selected as the sensor, and afterwards, our system's
ability to detect the presence of pathogenic bacteria was demonstrated. Initially, we
conducted an investigation to determine the ideal concentration range for the
functionality of the quorum-sensing system. Subsequently, we effectively illustrated
that the system is capable of detecting the presence of pathogenic bacteria (namely,
*Pseudomonas aeruginosa*) with high efficiency.

In this study, green fluorescent protein (GFP) was employed as a surrogate for the
downstream target protein. The fluorescence intensity was quantified as a measure of the
relative expression level of the target protein. Initially, the concentration of a
quorum-sensing effector molecule (3OC12HSL) surrounding *P. aeruginosa* was
quantified,
yielding a value of approximately 0.1μM. We proceeded to forward the process. The
experiment was initially conducted on two gradients. There are two factors to consider
in this context: the concentration of quorum-sensing effector molecules and the duration
of the induction period. Ultimately, it was determined that our system achieved optimal
expression efficiency at approximately 0.1μM over a duration of 30 minutes, so showing
the effectiveness of the system. The average rate of GFP production per minute per cell
was subsequently computed over a period of 3 hours. This calculation was performed under
conditions where the concentrations of quorum-sensing effector molecules were
approximately 0.1μM, representing the peak potency of the expression system.

![...](https://static.igem.wiki/teams/4785/wiki/result/11.png)

Figure 11. Schematic of quorum sensing
device.

a, Promoter luxR was activated by LasR-3OC12HSL complex, leading to produce HMGB1
and PslG.

b, pTetR-LasR-pluxR-GFP was used to characterize the sensing device.

![...](https://static.igem.wiki/teams/4785/wiki/engineering/fig6-1.png)

Figure 12. Characterization results of
quorum sensing device.

a, GFP production rate per cell over time at different concentration of 3OC12HSL.

b, Time-average GFP production rate per cell over time at different concentration of
3OC12HSL.
Error bar represents s.d.

![...](https://static.igem.wiki/teams/4785/wiki/result/13.png)

Figure 13. Schematic of test quorum
sensing device (ptetR-LasR-pluxR-GFP).
Promoter luxR was activated by LasR-3OC12HSL complex, leading to produce GFP.

## Section 4: Arabinose-Induced Cell Lysis

In the end, the proteins can be liberated by the modified bacteria following their
stimulation via the presence of arabinose. In the beginning, we established that the
introduction of arabinose resulted in the successful activation of Lysis E7, hence
leading to a notable decrease in the survival rate of genetically engineered bacteria.
Following this, an experimental verification was performed in order to demonstrate that
the reason for this decline was associated with bacterial lysis generated by Lysis E7.

In the first phase of our study, we conducted an analysis of the protein expression
levels of the downstream targets controlled by the arabinose promoter under different
levels of arabinose induction. Upon the introduction of arabinose, successful expression
of Lysis E7 was attained, leading to the observed phenomenon of a decline in optical
density (OD) and eventual lysis of *Escherichia coli* as the duration of induction
increased.

![...](https://static.igem.wiki/teams/4785/wiki/result/14.png)

Figure 14. Upon arabinose induction,
Lysis E7 is expressed followed by massive death of engineered bacteria.
Error bar represents s.d.

Furthermore, we conducted a comparative analysis of the DNA content in the lysate of
*Escherichia coli* that was lysed using Lysis E7, in comparison to the bacterial
solution
and the lysate obtained using physical fragmentation. Ultimately, it was determined that
the DNA composition of Lysis E7 lysate closely resembled that of physical Lysis lysate,
although exhibited notable dissimilarity from that of the bacterial solution. This
observation suggests that Lysis E7 possesses a commendable capacity for lysing.

![...](https://static.igem.wiki/teams/4785/wiki/result/15.png)

Figure 15. Lysis E7 induces lysis of
bacteria. Error bar represents s.d.

## Future Plan

Following the completion of the aforementioned investigations, we devised the following
future plan:

1. To increase protein expression and investigate improved expression conditions for
   HMGB1 and PslG.
2. In order to facilitate the regulation of protein expression, ascertain the
   correlation between the expression of GFP and that of our target protein.
3. Constructing a secretion system that facilitates the regulation of target protein
   expression and enables the engineered bacteria to secrete proteins continuously.

## Considerations for replicating the experiments

For repeating our experiments, we strongly recommend that future iGEM teams refer to our
experiment page, which details what should be noted in each part of the [experiment](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27experiments%27%29%20%7D%7D).

{% endblock %}