iGEM 2024 — Team cug-china

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## Index

[1](#title1)
[2](#title2)
[3](#title3)
[4](#title4)
[5](#title5)
[6](#title6)
[7](#title7)
[8](#title8)
[9](#title9)
[10](#title10)

## I.Conjugative Transfer

### Materials：9k-S0 plates, LB plates, Starkey-Na2S203 solid medium,1.5 mL centrifuge tube, PBS buffer, 9k buffer

1.Collection of Bacterial Cells: *Acidithiobacillus ferrooxidans* ATCC 23270, serving as the recipient
strain, was
cultivated in 9K-S0 medium for three days until it reached the logarithmic growth phase. The
culture
contained 1% (w/v)
elemental sulfur, and the cells were collected by centrifugation at 6000 rpm for 3 minutes when the
OD600
reached 0.4.
*E. coli SM10*, carrying the recombinant plasmid and acting as the donor strain, was subcultured from an
overnight culture
into LB medium supplemented with 100 μg/mL streptomycin and grown to an OD600 of 0.6 to 0.8.
The cells
were
then
collected by centrifugation at 7000 rpm for 3 minutes.
2.Washing of Bacterial Cells: The recipient cells were washed with a 2:2 wash solution and centrifuged
at
2000 rpm for 1
minute to remove elemental sulfur. The supernatant was decanted as the cell suspension.
3.Conjugation: Both the recipient and donor cells were concentrated to an OD600 of 1. They
were mixed
in a
1:2 ratio
(recipient:donor), and 100 μL of the mixture was evenly spread onto a 2:2 solid conjugation plate
containing
a 0.45 μm
filter membrane. The plates were incubated at 30℃ for 5 days in an upright position.
4.Membrane Washing: The 0.45μm membrane was removed from the plate and placed into a sterile 50 mL EP
tube.
The cells on
the membrane were washed off with 5 mL of 2:2 wash solution, collected by centrifugation, and
concentrated
into a 1.5 mL
EP tube. Serial dilutions were made at 10-fold, 50-fold, and 100-fold. An aliquot of 100 μL of the
concentrated cell
suspension was spread onto Starkey-Na2S203 solid medium supplemented
with 100 μg/mL streptomycin and
incubated at 30℃
for 15-20 days, inverted, until colonies were observed.
5.Verification: Single colonies were first inoculated into 10 mL of 9K liquid medium containing 10%
Fe2+
(49.64 g/L
FeSO4·7H2O) without antibiotics and cultivated for 3 days until the culture turned
red. The culture was
then
transferred
into 30 mL of 9K-S0 liquid medium containing 200 μg/mL streptomycin for further cultivation
for 6 to 10
days. The sulfur
particles were observed to be fine and did not clump together, settling at the bottom with a turbid
supernatant upon
standing. A 20 mL aliquot of the culture was used to extract plasmid DNA, and PCR was performed for
verification. If
successful, subsequent experiments were conducted.

## II. Determination of the Growth Curve of Engineered *E. coli* BL21

### Materials：LB plates, 1.5 mL centrifuge tube, 96-well plate, Micro plate reader (produced by gene5®)

1.Preparation of LB Liquid Medium: The engineered *E.coli* BL21 strain, cultured to the mid-log phase
(OD600 ~ 0.5), was
used to inoculate fresh LB medium at a 1% inoculation ratio.
2.Cultivation: The culture was allowed to stand at 30 ℃ for 2 days and then transferred to a shaking
incubator
set at 180
rpm for a period of 24 hours. Samples were collected at 3, 10, 15, 21, and 25 hours post-inoculation to
monitor the OD
changes. Three parallels were set up for the experiment.
3.Sampling Method: Aliquots of 200 μL of the supernatant were taken to measure the OD600. The
growth
curve
was plotted
based on the changes in OD600. Each sample was tested in triplicate to ensure accuracy and
consistency.

## III. Determination of the Growth Curve of A. ferrooxidans Conjugants

### Materials：9k-S0 plates, 9k-Fe2+ plates, 1.5 mL centrifuge tube, 96-well plate

1. Preparation of 9k-S0 Liquid Medium: The engineered A. ferrooxidans strain, cultured to the
mid-log
phase (OD600 ~
0.5), was used to inoculate fresh 9k-S0 medium at a 1% inoculation ratio.
2. Cultivation: The culture was allowed to stand at 30 ℃ for 2 days and then transferred to a shaking
incubator set at 180
rpm for a period of 14 days. Samples were collected starting at the time of inoculation, with one sample
taken every 24
hours to monitor OD changes. Three parallels were set up for the experiment.
3. Sampling Method: The samples were centrifuged at 2000 rpm for 30 seconds to precipitate elemental
sulfur.
Then, 200
μL of the supernatant was taken to measure the OD600. The growth curve was plotted based on
the changes
in
OD600. Each
sample was tested in triplicate to ensure accuracy and consistency.

## IV. Determination of Biofilm Formation by Engineered *E. coli* BL21

### Materials：LB plates, 1.5 mL centrifuge tube, PBS buffer, Crystalline Violet Stain, 95% ethanol, 24-well plate, Micro plate reader (produced by gene5®)

1. Activation and Cultivation: The engineered *E. coli* BL21 strain was streaked from a glycerol
stock
onto an LB agar
plate and incubated to activate the culture. A single colony was then transferred to liquid LB medium
and
grown to the
late logarithmic phase. The culture was centrifuged at 5000 rpm for 10 minutes to pellet the cells. The
supernatant was
discarded, and the cells were resuspended in an equal volume of sterile PBS buffer. This washing step
was
repeated.
2. Inoculation and Incubation: One hundred microliters of the resuspended cells were diluted in 900 μL
of
PBS to achieve
a 10-fold dilution. This diluted cell suspension was mixed evenly and added to a 24-well plate at a 1%
inoculation
ratio, with 1 mL of the diluted cell suspension per well. Each strain was tested in quadruplicate, and
the
plate was
incubated at 30℃ for 12h, 24h, 48h, and 72h to allow biofilm formation.
3. Cryo Violet Staining: After the specified incubation periods, the culture medium was carefully
aspirated
from each
well using a 1 mL pipette, leaving the biofilm intact. One milliliter of crystal violet staining
solution
was added to
each well, and the plate was incubated for 15 minutes to stain the biofilms. The staining solution was
then
aspirated,
and each well was gently rinsed twice with 1 mL of sterile PBS buffer to remove unbound stain. Finally,
add
1 mL of 95%
ethanol solution to resuspend the biofilm, place it at 37 ℃ for 10 minutes to fully dissolve the biofilm,
and
use a
microplate reader at 570 nm to measure the biofilm data.

## V. Determination of Biofilm Formation by *A. ferrooxidans* Conjugants

### Materials：9k-S0 plates, 1.5 mL centrifuge tube, PBS buffer, Crystalline Violet Stain, 95% ethanol, 24-well plate, Micro plate reader (produced by gene5®)

1.Activation and Cultivation of Engineered Bacteria: The engineered *A. ferrooxidans* strain is streaked
from a glycerol
stock onto 9k-S0 medium for strain activation and subcultured to the late logarithmic phase.
The culture
is then
centrifuged at 5000 rpm for 10 minutes to pellet the cells. The supernatant is discarded, and an equal
volume of sterile
PBS buffer is added to resuspend the cells. This washing step is repeated.
2. Preparation and Inoculation: One hundred microliters of the resuspended cell solution is mixed with
900 μL of PBS to
create a 10-fold dilution. This diluted cell solution is mixed evenly and its OD600 is
measured using a
UV-Vis
spectrophotometer. The culture is diluted with 9K medium to adjust the cell density to an
OD600 of 0.05.
The diluted
cell solution is then added to a 24-well plate, with 1 mL of the cell solution per well. Each strain is
tested in
triplicate, with six sets in total, and the plate is incubated at 30℃ for 12h, 24h, 36h, 48h, 60h, and
72h to allow
biofilm formation, after which the biofilms are stained with Crystal Violet.
3. Crystal Violet Staining Method: The staining procedure is performed as described previously.

## VI. Determination of c-di-GMP Concentration

### Materials：Enzyme-Linked Immunosorbent Assay (ELISA) Kit, BCA kit

#### Assay Kit

We employ an enzyme-linked immunosorbent assay (ELISA) kit specific for cyclic di-GMP (c-di-GMP) to
measure its
concentration. Since c-di-GMP is found within cellular proteins and the quantification of c-di-GMP
requires data on the
protein content of the cells, we utilize a bacterial protein extraction kit to isolate bacterial
proteins and a BCA
protein assay kit to determine protein concentrations.

Collect the bacterial culture in the late logarithmic phase and measure the OD600. Concentrate
and
resuspend the culture
in PBS to achieve a 1 mL suspension with an OD600 of 1.8.
Bacterial Protein Extraction Kit Procedure:
① Preparation of Extraction Solution: Mix Reagent A1 and Reagent A2 in a 9:1 ratio to create Protein
Extraction Solution
A. Thoroughly mix and keep the solution on ice until use.
② Centrifugation of Bacterial Suspension: Centrifuge the bacterial suspension at 4℃ at 10,000×g for 5
minutes. Discard
the supernatant and remove any remaining liquid as much as possible to collect the bacterial cells.
③ Washing of Bacterial Cells: Wash the bacterial cells twice with PBS.
④ Protein Extraction: Add 500 uL of protein extraction solution per 50-100 mg wet weight of bacterial
sample. Vortex to
mix well and incubate at 2-8℃ with shaking for 20-30 minutes.
⑤ Sonication: Sonicate the suspension on ice under conditions of 150-300 watts, 10 seconds sonication/10
seconds
interval, until the bacterial suspension becomes clear.
⑥ Centrifugation and Collection of Supernatant: Centrifuge the bacterial suspension at 4℃ at 12,000×g
for 10 minutes.
Transfer the supernatant into a pre-chilled clean centrifuge tube. The supernatant now contains the
total bacterial
protein extract.
Determination of c-di-GMP Concentration Using a c-di-GMP Enzyme-Linked Immunosorbent Assay (ELISA) Kit
Procedure:
1.Dilution of Standards: The kit provides a concentrated standard. Prepare dilutions in small test tubes
as per the
chart below:

表格示例

|  |  |  |
| --- | --- | --- |
| 40 pg/mL | Standard number 5 | Add 150 μL of the original double standard to 150 μL of standard diluent |
| 20 pg/mL | Standard number 4 | Add 150 μL of standard 5 to 150 μL of standard diluent |
| 10 pg/mL | Standard number 3 | Add 150 μl of standard 4 to 150 μL of standard diluent |
| 5 pg/mL | Standard number 2 | Add 150 μL of standard 3 to 150 μL of standard diluent |
| 2.5 pg/mL | Standard number 1 | Add 150 μL of standard 3 to 150 μL of standard diluent |

2.Sample Application: Prepare blank wells (no samples or enzyme conjugates added, but follow the same
procedure for all
other steps), standard wells, and sample wells. Add 50 μL of each standard to the appropriate wells on
the ELISA plate.
For sample wells, first add 40 μL of sample diluent, then add 10 μL of the sample (resulting in a final
dilution of the
sample of 5 times). Apply samples to the bottom of the wells, avoiding contact with the walls, and
gently mix by
tapping.
3.Incubation: Seal the plate with adhesive film and incubate at 37 ℃ for 30 minutes.
4.Dilution of Wash Solution: Dilute the concentrate wash solution 30-fold with distilled water and keep
it ready for
use.
5.Washing: Carefully remove the adhesive film, discard the liquid, and dry by tapping. Fill each well
with the diluted
wash solution, let it sit for 30 seconds, then discard. Repeat this process five times and dry by
tapping.
6.Addition of Enzyme Conjugate: Add 50 μL of enzyme conjugate to each well, excluding the blank
wells.
7.Incubation: Follow the same procedure as step 3.
8.Washing: Follow the same procedure as step 5.
9.Color Development: To each well, first add 50 μL of substrate A, then 50μl of substrate B. Gently mix
and incubate at
37℃ in the dark for 10 minutes to develop the color.
10. Termination: Add 50 μL of stop solution to each well to stop the reaction (the color will change from
blue to yellow
immediately).
11.Measurement: Zero the ELISA reader with the blank well and measure the absorbance (OD value) at 450
nm for each well
in sequence. The measurement should be conducted within 15 minutes after the addition of the stop
solution.
Determination of Protein Concentration Using the BCA Assay Kit Procedure:
1.Dilution of Standards: The kit provides a concentrated bovine serum albumin (BSA) standard. Dilute the
standard in
small test tubes according to the chart below:

表格示例

|  |  |  |  |
| --- | --- | --- | --- |
| Name of standard sample | Diluent volume（μL） | Standard liquid volume（μL） | Final concentration（μg/mL） |
| A | 0 | 300 | 2000 |
| B | 125 | 375 | 1500 |
| C | 375 | 325 | 1000 |
| D | 175 | B tube take 175 | 750 |
| E | 325 | C tube take 325 | 500 |
| F | 325 | E tube take 325 | 250 |
| G | 325 | F tube take 325 | 125 |
| H | 400 | G tube take 325 | 25 |
| I | 400 | 0 | 0 |

2.Preparation of BCA Working Solution: Based on the number of standard samples and sample wells, prepare
a sufficient
amount of BCA working solution with a volume ratio of 50 mL of Reagent A to 1 mL of Reagent B. Mix
thoroughly.
3. Application of Standards and BCA Working Solution: Add the appropriate amount of standard samples to
a 96-well plate.
Add 200 μL of BCA working solution to each well. Measure the absorbance at 562 nm using a microplate
reader. Plot a
standard curve based on the results of the standards.
4. Sample Analysis: Take 20 μL of the prepared protein sample and add it to 200 μL of BCA working solution
in the
microplate wells. Set the microplate reader to 562 nm and measure the sample absorbance. Calculate the
protein
concentration of the sample based on the standard curve and the solution concentration. Perform each
experiment in
triplicate.

## VII. Gold Transcription Factor Specificity Verification Experiment

### Materials：LB medium, Colony template, PBS buffer, 96-well plate, Micro plate reader (produced by gene5®)

1.Activation and Inoculation of *E. coli* BL21 Cells Containing the Recombinant Plasmid: Activate *E.
coli*
BL21 cells
harboring the recombinant plasmid and inoculate them into LB medium at a ratio of 1%. Add 0.5 mM IPTG
along with varying
concentrations of HAuCl4 (0 µM, 0.25 µM, 1 µM, 5 µM, 10 µM, and 20 µM) for induction.
2.Sampling and Measurement of Fluorescence: Collect cells at 0, 6, 12, 18, and 24 hours of cultivation
by centrifugation
at 12,000 rpm for 2 minutes. Harvest the bacterial cells and wash with PBS buffer solution. Take 200 μL
of the bacterial
suspension and measure the fluorescence data versus OD600 using a microplate reader in a 96-well
plate,
with an
excitation wavelength of 485 nm and an emission wavelength of 535 nm.
3.Overnight Cultivation and Fluorescence Measurement: Cultivate the engineered *E. coli* BL21 strain in
LB
medium
containing 20 µM HAuCl4 (Au3+), 20 µM CuSO4 (Cu2+), 20 µM
CdCl2
(Cd2+), 20 µM ZnCl2 (Zn2+), or 20
µM
NiCl2 (Ni2+), with
a control group without metal ion induction. Measure the ratio of fluorescence to OD600.

## VIII. Suicide Module Verification Experiment

### Materials：LB medium, Colony template, 96-well plate, Micro plate reader (produced by gene5®)

1.Cultivation of *E. coli* BL21 Cells Transformed with the Plasmid Expression Vector: Cultivate *E.
coli* BL21
cells
harboring the plasmid expression vector for 12 hours until they reach the late logarithmic phase.
2.Addition of Inducers and Measurement of OD600: Measure the OD600 of the culture
before adding the
inducers. Set up
four control groups: one without IPTG and Au3+, one with 0.5 mM IPTG but without Au3+,
one with 20 μM Au3+
but without
IPTG, and one with both 0.5 mM IPTG and 20 μM Au3+.
3.Measurement of OD600 After Inducer Addition: After the addition of the inducers
IPTG/Au3+,
measure the
OD600 every 3
hours. Subtract the OD600 value at the 12th hour from the subsequent OD600
measurements to obtain the growth
curve data.
Plot the difference in OD600 values over time to create a growth curve.

## IX.9k liquid culturte medium

### Materials for 1 L: (NH4)2SO4 30 gKCl 1 gK2HPO4 5 gMgSO4·7H2O 5 gCa(NO3)2 0.1 g

#### Protocol:

1. Measure materials to scale that in need
2.Add the required proportion of water, shake the bottle until the solids completely dissolve.
3. Adjust pH to 2.0 using 30% concentrated sulphuric acid.
4.Place the bottles into the autoclave for sterilization, under 121°C for 30 minutes.
5.Let the bottles cool down under room temperature. The solution would preserve under room temperature for a
maximum of
7 days.
6. Add 1% sulphur monomer or 44.22 g/L ferrous sulphate heptahydrate depending on culture conditions

## X.Starkey-Na2S2O3 solid culturte medium

### Materials for 1 L: LiquidA： (NH4)2SO4 6 g KH2PO4 6 g MgSO4·7H2O 0.1 g CaCl2 0.37 g LiquidB: agar 20 g LiquidC： Na2S2O3·5H2O 3 g FeSO4·7H2O 0.012 g 20 mL H2O

#### Protocol:

1.Measure materials to scale that in need
2.Add the required proportion of water, shake the bottle until the liquid A, B, C solids completely
dissolve.
3.Place the liquid A, B bottles into the autoclave for sterilization, under 121°C for 30 minutes.Liquid C
filtrate for
sterilization
4. Mix equal volumes of liquid A and liquid B, then add 5% of liquid C of the total volume of liquid A and
B, mix well
and prepare the medium plate.

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**Tel 027-67883152**
**China University of Geosciences (Wuhan)**

---

[© 2024 -
Content on this site is licensed under a Creative Commons Attribution 4.0 International
license](https://creativecommons.org/licenses/by/4.0/)

[The repository used to create this website
is available at https://gitlab.igem.org/2024/cug-china](https://gitlab.igem.org/2024/cug-china)

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## Index

[Cycle 1](#title1)
[Cycle 2](#title2)
[Cycle 3](#title3)
[Cycle 4](#title4)

## Cycle 1:Construct the Plasmid

### 1.1 Design

To enhance the bioleaching efficiency of *Acidithiobacillus ferrooxidans*, genetic engineering is essential. However, the molecular biology manipulation of *A. ferrooxidans* has proven to be intricate. The standard genetic engineering strategies, which include transformation, transduction, and conjugation, are not straightforward for this organism. The absence of suitable phages in the *A. ferrooxidans* genome rules out transduction as a method for introducing engineered plasmids. Moreover, previous attempts to prepare competent cells and to transform *A. ferrooxidans* have been unsuccessful, and the electroporation transformation method has yielded very low efficiency and inconsistent results[1][2].

Given these challenges, conjugation stands out as the most promising approach. Despite its longer process and relatively lower efficiency, conjugation offers a simple and reliable means of plasmid transfer, making it a viable option for genetic manipulation of *A. ferrooxidans*. We have chosen *E.coli* SM10 as the donor strain for this process, leveraging its ability to transfer plasmids, such as pYYDT, which carries the mob gene, to recipient strains[2].

In the genetic engineering of *A. ferrooxidans*, careful consideration must be given to plasmid selection markers. The acidic environment in which *A. ferrooxidans* thrives limits the use of resistance markers to a few options: mercury resistance (MerR), kanamycin resistance (KmR), streptomycin resistance (SmR), and chloramphenicol resistance (CmR). This constraint necessitates the selection of appropriate design methods for plasmid modification.

The pYYDT plasmid, a derivative of the pBBR series, Is an engineered vector that aligns with the RC10 assembly standard of synthetic biology. Its design, featuring a standard biological brick structure, facilitates genetic engineering. The presence of the mob gene from the broad-host-range plasmid pBBR1-MCS2 makes it suitable for conjugation. However, the overlap of kanamycin resistance (KmR) as a selection marker in both the donor strain *E.coli* SM10 and the pYYDT plasmid complicates the screening process for successful transformations. This overlap necessitates the modification of the pYYDT plasmid to differentiate between the donor and the recipient *A. ferrooxidans* strains that have been successfully transformed. This modification is critical for the successful application of pYYDT in the genetic engineering of *A. ferrooxidans*.

### 1.2 Build

For the construction of the plasmid pYDT, we employed a seamless cloning strategy. Initially, we tailored primers to match the sequences flanking the kanamycin resistance gene within the pYYDT plasmid. PCR amplification yielded a linearized version of the vector, from which the kanamycin gene was excluded. Subsequently, we designed primers for the streptomycin resistance gene, incorporating 20 bp homology arms for the linearized pYYDT at their extremities. These primers enabled the amplification of the streptomycin resistance gene, now equipped with the necessary pYYDT homology arms.

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**Tel 027-67883152**
**China University of Geosciences (Wuhan)**

---

[© 2024 -
Content on this site is licensed under a Creative Commons Attribution 4.0 International
license](https://creativecommons.org/licenses/by/4.0/)

[The repository used to create this website
is available at https://gitlab.igem.org/2024/cug-china](https://gitlab.igem.org/2024/cug-china)


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## Index

[Modification](#title1)
[Module 1](#title2)
[Module 2](#title3)
[Module 3](#title4)

# Project Introduction

## 1. Successful Modification of Plasmids

We begin by examining the widely used pYYDT plasmid as a heterologous expression vector for *Acidithiobacillus ferrooxidans*, which serves as a versatile vector complying with the RFC10 standard. However, the *Escherichia coli* strain employed in our study, *E. coli* sm10, inherently possesses kanamycin resistance. This characteristic prevents us from using kanamycin as a selection marker for subsequent strain selection. Given that our chassis organism, *A. ferrooxidans* ATCC 23270, thrives in acidic environments, we decided to substitute kanamycin resistance with streptomycin resistance, which remains effective under acidic conditions. Details of this modification process are outlined in the engineering section.

We introduced both the unmodified pYYDT plasmid and the modified pYDT plasmid into *E. coli* DH5α, cultivating them in LB media supplemented with kanamycin and streptomycin, respectively, in order to evaluate their growth characteristics. The same experimental procedures were conducted with the *E. coli* sm10 strain. Initially, we confirmed that *E. coli* sm10 WT exhibits kanamycin resistance but is devoid of streptomycin resistance (Fig. 1A). As demonstrated in Fig. 1B, *E. coli* DH5α/pYYDT, which harbors the unmodified pYYDT plasmid, grows normally in kanamycin-supplemented media, while it fails to grow in streptomycin-supplemented media. In contrast, *E. coli* DH5α/pYDT, which contains the modified pYDT plasmid, flourishes in streptomycin-supplemented media but cannot grow in kanamycin-supplemented media (Fig. 1C).

These results confirm that we have successfully modified the vector plasmid pYYDT into the pYDT plasmid, making it suitable for our chassis cells. This foundational work paves the way for the subsequent successful conjugative transfer of the target gene into the chassis cells.

![Centered Image](https://static.igem.wiki/teams/5323/images/result/r-1.png)

Fig 1 The growth curves of *E. coli* DH5α and *E. coli* sm10 containing different plasmids. (A) The growth curve of *E. coli* sm10 WT under kanamycin and streptomycin resistance. (B) The growth curve of *E. coli* DH5α/pYYDT containing the expression vector pYYDT under kanamycin and streptomycin resistance. (C) The growth curve of *E. coli* DH5α/pYDT containing the expression vector pYDT under kanamycin and streptomycin resistance. Experiments were conducted in triplicate and the error bar represent SD.

## 2. A dynamic synthesis module for cyclic di-GMP has been constructed, successfully elucidating a highly efficient synthesis module for c-di-GMP

To facilitate the efficient formation of biofilms by engineered strains and to enhance the effectiveness of bioleaching, we have opted to overexpress the second messenger cyclic di-GMP synthase in our target strain. During the engineering phase, we constructed a recombinant vector, pYDT\_*yedQ*, which utilizes the IPTG-inducible promoter P tac to regulate the expression of the GDC gene *yedQ*, sourced from *E. coli* BL21. Additionally, we developed recombinant plasmids pYDT\_1373 and pYDT\_1379, designed to express the GDC genes AFE\_1373 and AFE\_1379, respectively, obtained from *A. ferrooxidans*.

To evaluate whether these genes could enhance the intracellular concentration of c-di-GMP and consequently improve the biofilm formation capability of the chassis cells, we initially utilized *E. coli* BL21 as the model organism. We began by investigating suitable cultivation conditions, assessing the growth curves in M9 media supplemented with 5%, 10%, and 20% LB. As shown in Fig. 2, the growth of *E. coli* BL21 with the plasmid was minimally affected at an LB concentration of 10%. Therefore, we proceeded to choose the addition of 10% LB to the M9 medium for the subsequent biofilm experimentation with the engineered strains.

![Centered Image](https://static.igem.wiki/teams/5323/images/result/r-2.png)

Fig 2 The growth curve of *E. coli* BL21 in LB medium at different concentrations. (A) The growth curve of *E. coli* BL21 is depicted at a concentration of 5% LB. (B) The growth curve of *E. coli* BL21 is depicted at a concentration of 10% LB. (C) The growth curve of *E. coli* BL21 is depicted at a concentration of 20% LB. Experiments were conducted in triplicate and the error bar represent SD.

Research has shown that biofilm formation significantly enhances leaching efficiency [1]. Consequently, it is crucial to promote substantial biofilm growth by bacteria on tailings to optimize leaching performance. In our study, we evaluated the biofilm content of *E. coli* BL21/pYDT\_*yedQ*, *E. coli* BL21/pYDT\_1373, and *E. coli* BL21/pYDT\_1379 in response to the induction by 1 mM IPTG at various time intervals. As depicted in Fig. 3, the addition of IPTG in *E. coli* BL21 led to a significant increase in biofilm formation for *E. coli* BL21/pYDT\_1373, *E. coli* BL21/pYDT\_1379, and *E. coli* BL21/pYDT\_*yedQ* when compared to the control group that did not receive IPTG, In contrast, no significant IPTG-induced biofilm increase was observed in the control strain *E. coli* BL21/pYDT.Notably, *E. coli* BL21/pYDT\_1373 demonstrated the most robust biofilm formation. Through the overexpression of DGE genes, we effectively enhanced the biofilm-forming capabilities of the engineered strains.

![Centered Image](https://static.igem.wiki/teams/5323/images/result/r-3.png)

Fig 3 (A) Bar chart of biofilm formation of *E. coli* BL21. (B) Biofilm formation of *E. coli* BL21 at 12 hours. Biofilm formation of *E. coli* BL21 at 24 hours. (C) Biofilm formation of *E. coli* BL21 at 48 hours. (D) *E. coli* BL21 at 24 hours. Biofilm formation of E. coli BL21 at 72 hours. Experiments were conducted in triplicate and the error bar represent SD.Two-sided Student’s t-test was used to analyze the statistical significance (\*0.01< P < 0.05; \*\*P < 0.01, NS: No significance).

Direct quantification of the normaliezd intracellular c-di-GMP showed that the c-di-GMP concentration in *E. coli* BL21/pYDT\_1373, *E. coli* BL21/pYDT\_1379, *E. coli* BL21/pYDT\_*yedQ*, and *E. coli* BL21/pYDT of 24-h cultures with 1 mM IPTG was significantly higher than the cells without IPTG. The presence of 1 mM IPTG successfully increased the c-di-GMP level. The c-di-GMP content was determined based on the standard curve presented in Fig. 4B. As illustrated in Fig. 4, the c-di-GMP levels in *E. coli* BL21/pAFE\_1373, *E. coli* BL21/pAFE\_1379, and *E. coli* BL21/pYedQ displayed a significant increase following the addition of the inducer IPTG compared to the control group. In contrast, the intracellular concentration of c-di-GMP in the empty vector strain did not show any significant changes.

![Centered Image](https://static.igem.wiki/teams/5323/images/result/r-4.png)

Fig 4 The schematic diagram of c-di-GMP content in *E. coli* BL21/pYDT, *E. coli* BL21/pYDT\_1373, *E. coli* BL21/pYDT\_1379, and *E. coli* BL21/pYDT\_*yedQ*. (A) The c-di-GMP content of *E. coli* BL21/pYDT, *E. coli* BL21/pYDT\_1373, *E. coli* BL21/pYDT\_1379, and *E. coli* BL21/pYDT\_*yedQ*. (B) The standard curve for c-di-GMP. (C) The total protein concentrations of *E. coli* BL21/pYDT, *E. coli* BL21/pYDT\_1373, *E. coli* BL21/pYDT\_1379, and *E. coli* BL21/pYDT\_*yedQ*. (D) The standard curve for protein concentration is depicted. Experiments were conducted in triplicate and the error bar represent SD.Two-sided Student’s t-test was used to analyze the statistical significance (\*0.01< P < 0.05; \*\*P < 0.01, NS: No significance).

*A. ferrooxidans* is a chemolithoautotrophic bacterium that oxidizes sulfide minerals to release metal ions while producing sulfuric acid, thereby facilitating further metal dissolution. Due to the difficulty in culturing this strain, we employed ferrous iron and sulfur as energy sources to more intuitively and effectively study the growth characteristics of *A. ferrooxidans*  [2] . Our observations indicate that when ferrous iron was used as the energy source, the engineered strains *A. ferrooxidans*/pYDT\_1379, *A. ferrooxidans*/pYDT\_0053, and *A. ferrooxidans*/pYDT\_1373 displayed a lag phase during the mid-growth period. We hypothesize that the enhanced biofilm formation capability may impact the growth rate of the bacteria in a planktonic state (Fig. 5A). In contrast, when cultured on sulfur, the lag phase exhibited by the three engineered strains *A. ferrooxidans*/pYDT\_1379, *A. ferrooxidans*/pYDT\_0053, and *A. ferrooxidans*/pYDT\_1373 was significantly reduced (Fig. 5B). This reduction is likely attributed to the solid powdered form of sulfur, which may mitigate the lag phenomenon associated with enhanced biofilm formation.

![Centered Image](https://static.igem.wiki/teams/5323/images/result/r-5.png)

Fig 5 The growth curve of *A. ferrooxidans*.(A) The growth characteristic curve of *A. ferrooxidans* when using ferrous iron as the energy source. (B) The growth characteristic curve of *A. ferrooxidans* using sulfur as an energy source. Experiments were conducted in triplicate and the error bar represent SD.

After assessing the growth characteristics of *A. ferrooxidans*, we proceeded to measure the biofilm content of the strains in the presence of the inducer IPTG at various time intervals. Due to the interference caused by ferrous ions with the spectrophotometry used for detecting biofilm content, we chose to culture the strains using sulfur as the energy source. At the peak of biofilm formation at 36 hours, *A. ferrooxidans*/pYDT\_0053 displayed the thickest biofilm, which was significantly different from the control group. Furthermore, *A. ferrooxidans*/pYDT\_1379 and *A. ferrooxidans*/pYDT\_1373 also contributed to biofilm formation, demonstrating significant differences (P＜0.05) that indicate a substantial enhancement in biofilm development (Fig. 6).

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