iGEM 2021 — Team SUSTech_Shenzhen

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# Team:SUSTech Shenzhen/Notebook

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# Notebook

Notebook of Project

[About Notebook](#down)

## **2020**

### July

Quorum sensing consultation (target Vibrio cholera)Salt promoter consultation (chloride induced promoter)pH promoter consultation (induced at pH5.5)Design of the circuitColonic acid assay methodologyLiterature research about antimicrobial peptides(AMPs)Purchase chemicals and genePlasmid design of LactobacillusPurification of human lactoferrin(hLF)

### Augest

Antibacterial test of hLF on V.cholera and E. coli Nissle 1917CAI-1 quorum sensing modelingCulture and preservation of LactobacillusAntibacterial Test(hLF, 7475 and kanamycin on V.cholera)Make competent cell of Nissle 1917, LactobacillusPlasmid extraction of pBbE6k and pTRKH3-slpGFPTransformation of pBbE6k into Nissle 1917Plasmid construction of RcsB – pBbE6k(digestion and ligation)Purification of hLF and 7475Transformation of pBbE6k into E. coli Top10

### September

Transformation of RcsB – pBbE6k and control into E. coli DH5α and Top10Colony PCR and sequencing of Top10+pBbE6k, pBbE6k-RcsBPurification of hLFSequencing of RcsB – pBbE6k and control into Top10Plasmid extraction of pBbE6kTransformation of pBbE6k into E. coli BL21 and E. coli AcellaAntibacterial Test

### October

IPTG induced RcsB expression, electrophoresis, western blot in Acella and BL21IPTG induced expression of RFP in Nissle 1917Transformation of pBbE6k-RcsB into Nissle 1917Staining and observation of BL21 and Acella with RcsB expressionDesign of experiment of hLF

### November~December

Transformation of RcsB into Nissle 1917the Lactoferrin testVerify the chloramphenicol function

## **2021**

### March

Western blot of cholera toxin detection in Vibric cholera BNCC 232030

### 4.1~4.9

Five schools exchange meetingCooperation negotiation with SZU-ChinaCounseling from Feiru BiotechCounseling from doctors at the First Affiliated Hospital of Xi’an Jiaotong University

### 4.10~6.16

Expression and verification of RcsB in Nissle using IPTGConstruction of plasmids (CAI-1 detector, lac-ara-induced(EsaR), Lactobacillus gadR)Counseling from doctors at the Shenzhen Second People's HospitalDeep Blue Charity SaleCommunication with Central South University (CSU)Counseling from scientistSpring buds lectureChina iGEM Online Meetup

### 6.17~7.18

Learn and practice molecular cloning methods and other methods, including PCR, Gibson Assembly, plasmid
transformation, Western Blot, HPLC etcCommunication with the National University of Singapore (NUS)Spring buds lecture"Cloud West" online volunteering teaching project

### 7.19~7.25

Preliminary purification of colanic acid in NissleTransformation EsaR plasmid into BL21

### 7.26~8.1

Further literature research about UHPLC assayPreparation of competent cell Acella and DH5αTransformation of CAI-I and EsaR plasmid into Acella and gadR plasmid into DH5αPreservation of transformed bacteria Acella and DH5α

### 8.2~8.8

Optimize the condition for UHPLC assay (different mobile phases)SUSTech Hospital ExchangeGastrointestinal meeting

### 8.23~8.30

Set up four standard curves of monosaccharides using UHPLC assayPreservation of transformed bacteria DH5α with PKRTH3-ermBPCR of arabinose promoterPCR of vector(lac-ara-induced(EsaR))CCiC

### 8.13~9.5

Optimize the condition for UHPLC assay (different flow rate, time constant, reference standards)DNA electrophoresis for fragment(arabinose promoter) and vector GibsonTransformation of the product of GibsonPreservation of transformed bacteria

### 9.6~9.12

Optimize the condition for UHPLC assay (different gradient methods)Western blot of PhlF in BL21Spring buds lecture

### 9.13~9.19

Modification to get a better separation under more detailed conditions for UHPLC assayPCR of the colony from Gibson

### 9.20~9.26

Western blot of EsaR, CqsS, LuxU, LuxO in Acella

>

## Quick access

[#### Project

Introduction and description of the team project](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Description)

[#### Human Practice

Introduction and description of HP's objective, process and achievements](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Human_Practices)

[#### Team

Introduction of the team, team members and their attributions](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Team)

[#### Experiment

Introduction and description of the experiment ideas, process and results](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Experiments)

[#### Model

Explain how we make predictions mathematically](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Model)

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# Team:SUSTech Shenzhen/Engineering

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# Engineering

Engineering process of the designed biological
system.

[About Engineering](#down)

## **Part1: Engineering bacteria with RcsB can produce colanic acid to remedy diarrhea-related dehydration**

### Design Part:

Colanic acid plays a vital role in preventing dehydration in microorganisms[1](#re_1), where its biosynthesis is regulated by *Rcs*
(regulator of capsule synthesis) phosphorelay system[2](#re_2). *Rcs* is a complex system comprising the sensory
*RcsC*, receiver/effector *RcsB*, regulator *RcsA*, and phosphotransmitter
*RcsD* [2-5](#re_2) . Leveraging on this microbial
behaviour, we intended to adapt the *Rcs* system regulation to facilitate microbial cytoplasmic
water retention to prevent water loss.

Systematic verification of the colonic acid biosynthesis is conducted using an IPTG
(Isopropyl-beta-D-thiogalactopyranoside)-inducible promoter in the plasmid pBbE6k-RFP[6](#re_6) where the induced RFP-tagged RcsB expression
is validated via western blot followed by colanic acid production quantification via staining method and
HPLC(High-performance liquid chromatography). As a proof-of-concept of this component of the overall
circuit, the RcsB expression is regulated using IPTG, where our findings suggest good water retention
supported by tunable RcsB expression using the different inducer concentrations.

In our subsequent experiments, the water storage of polysaccharides is tested using THEMT
(American Society of Experimental Materials) 570 standard and other methods in other
studies[7](#re_7).

### Build Part:

The codon-optimized sequence of the *RcsB* was synthesized and cloned into the pBbE6k vector using
an *Escherichia coli (E. coli)* host BL21. The *RcsB* gene was cloned to make use of
the LacI-promoter for gene transcription and translation.

![image](https://static.igem.org/mediawiki/2021/1/17/T--SUSTech_Shenzhen--engineering-picture-2.png)

**Figure 1|** Restriction enzyme digestion products of pBbE6k plasmid and
*RcsB* in pUC57

![image](https://static.igem.org/mediawiki/2021/c/c3/T--SUSTech_Shenzhen--engineering-picture-3.png)

**Figure 2|** *RcsB* gene cloned under *lacI* promoter in pBbE6K
plasmid

### Test Part:

1.The fluorescence curve shows that IPTG can successfully induce the expression of RFP. Each fluorescence
level is measured three times in 96-well plates.

![image](https://static.igem.org/mediawiki/2021/1/12/T--SUSTech_Shenzhen--engineering-3.png)

**Figure 3|** IPTG-induced RFP expression in *E.coli*

2.We detected the expressed *RcsB* protein via electrophoresis and western blot. The RcsB on the
western blot is indicated by the red box, showing a size of approximately 24.5kDa.

![image](https://static.igem.org/mediawiki/2021/5/5e/T--SUSTech_Shenzhen--engineering-picture-4.png)

**Figure 4|** Western blot of RcsB

3.To primarily verify whether our engineered bacteria can produce colanic acid, we conducted the staining
test to detect the polysaccharide. Bacteria cells treated with the fuchsin solution are stained red and
capsules of bacteria treated with the methylene blue are stained blue.

![image](https://static.igem.org/mediawiki/2021/8/8c/T--SUSTech_Shenzhen--engineering-picture-5.png)

**Figure 5|** Staining *BL21* cells without/with *RcsB* expressed

![image](https://static.igem.org/mediawiki/2021/6/66/T--SUSTech_Shenzhen--engineering-picture-6.png)

**Figure 6|** Staining *Accela* cells without/with *RcsB* expressed

4.4.Currently, we are quantifying the colanic acid yield using Ultra-High Pressure Liquid Chromatography
to isolate and determine its concentration. Further studies on the output would help in finetuning the
colanic acid production within the cells to facilitate proper dehydration prevention.

### Learn Part:

Due to travel restrictions during the COVID-19 pandemic and limited laboratory time, we have not
succeeded in quantifying the yield of colanic acid in probiotic strain
*E. coli* Nissle 1917 and testing the water holding capacity. Furthermore, it is difficult to
determine the exact concentration due to the overlapping emission spectra of the various sugar
components that make up colanic acid. However, we intend to adopt an alternative approach to stain
colanic acid for easier identification[8](#re_8), where
we can precisely determine the colanic acid yield via UHPLC assay or other optimized alternatives.

### **Reference**

[1]Holowko, M. B.; Wang, H.; Jayaraman, P.; Poh, C. L., Biosensing Vibrio cholerae with
Genetically Engineered Escherichia coli. ACS Synth Biol 2016, 5 (11), 1275-1283.

[2]Higgins, D. A.; Pomianek, M. E.; Kraml, C. M.; Taylor, R. K.; Semmelhack, M. F.;
Bassler, B. L., The major Vibrio cholerae autoinducer and its role in virulence factor production.
Nature 2007, 450 (7171), 883-6.

[3]Pan, Y.; Chua, N.; Lim, K.; Ho, C. L., Engineering of Human Lactoferrin for Improved
Anticancer Activity. ACS Pharmacology & Translational Science 2021.

[4]Azman, A. S.; Rudolph, K. E.; Cummings, D. A.; Lessler, J., The incubation period of
cholera: a systematic review. J Infect 2013, 66 (5), 432-8.

[5]Wagner, M.; Peterson, C. G. B.; Stolt, I.; Sangfelt, P.; Agnarsdottir, M.; Lampinen, M.;
Carlson, M., Fecal eosinophil cationic protein as a marker of active disease and treatment outcome
in collagenous colitis: A pilot study. Scandinavian Journal of Gastroenterology 2011, 46 (7-8),
849-854.

[6]Rivera, F. P.; Medina, A. M.; Bezada, S.; Valencia, R.; Bernal, M.; Meza, R.; Maves, R.
C.; Ochoa, T. J., Bovine lactoferrin decreases cholera-toxin-induced intestinal fluid accumulation
in mice by ganglioside interaction. PLoS One 2013, 8 (4), e59253.

[7]Moon, T. S.; Lou, C.; Tamsir, A.; Stanton, B. C.; Voigt, C. A., Genetic programs
constructed from layered logic gates in single cells. Nature 2012, 491 (7423), 249-53.

[8]Shong, J.; Collins, C. H., Quorum sensing-modulated AND-gate promoters control gene
expression in response to a combination of endogenous and exogenous signals. ACS Synth Biol 2014, 3
(4), 238-46.

## **Part2: Engineering pathway to detect Vibrio cholerae’s autoinducer molecule, CAI-1**

### Design Part

In efforts to develop an input sensor that is responsive to the specific *Vibrio cholerae*
quorum
sensing molecule (CAI-1), we engineered the metabolic pathways of *E. coli* to establish an
optimized
relationship between the sensing the cell density of *V. cholerae* and the promotor response
used to
regulate the *E. coli* cellular behaviour.

Previously, a team led by Poh CL., Holowko MB., and Wang H.[1](#re_9) identified three critical phosphorylation proteins
involved in the CAI-1 signalling pathway. To further optimize the phosphorylation cascade, closer
studies on the interaction between CAI-1 concentration and the promotor strength are used in
activating the downstream pathway within an *E. coli* chassis. Through modelling and
calculating the
required expression levels of the three phosphorylation proteins, we generated an optimized sequence
to facilitate the *E. coli* cellular response to CAI-1.

![image](https://static.igem.org/mediawiki/2021/e/ee/T--SUSTech_Shenzhen--engineering-picture-object.png)

**Figure 1|** CAI-1 detecting sequence design

### Build Part:

We cloned the optimized sequence containing the three phosphorylation proteins into the pBbE6k
plasmid that contains a *KanR* antibiotic resistance gene as a selection marker. Following the
original CqsS, LuxU, and LuxO protein sequences from *Vibrio cholerae*, we codon-optimized the
sequences according to the codon utilization in the *E. coli* cell. We paired the different
genes with
constitutive promotors with differing expression strengths to minimize the metabolic burden imposed
on the *E. coli* host. Additionally, we included a strep-tag sequence on either the N- or C-
terminus
of CqsS, LuxU, LuxO to track the protein expression levels in the *E. coli* cytoplasm. We
specifically
added the strep-tag to the N-terminal of CqsS as the C-terminal comprises the transmembrane domain.
We were concerned that the inclusion of an N-terminus strep-tag might interfere with the protein
folding and function.

As an output response to test the CAI-1 promotor strength, we cloned the mRFP downstream of the
*CqsS*
gene to monitor the cellular response to CAI-1.

### Test Part：

To evaluate the *E. coli* response to the CAI-1 quorum sensing molecules, we monitored the RFP
expression via the fluorescence output when tested under different *V. cholerae* cellular
densities.
We tested the different cellular densities to evaluate the CAI-1 production in *V. cholerae*
under the
different stages of cellular growth. In the article published by MB Holowko[2](#re_10), they discovered that an overnight culture of *V.
cholerae* can have an accumulated concentration of CAI-1 as high as 1.25μM. Thus, through
testing the
supernatant using different dilution factors, we can establish a linear correlation between CAI-1
concentration and the gene expressions.

### Learn Part：

In the initial design of our work, we used the same promoter sequence to regulate the expression of
various proteins in the *E. coli* host. However, we observed gene truncation or gene loss in
the
extracted plasmids, indicating that similar sequences resulted in gene recombination.

Additionally, in adapting exogenous gene expression in our *E. coli* chassis, such as the CqsS
system
in *Vibrio cholerae*, all regulatory proteins need to be codon optimised to ensure a rapid
response
rate. The codon optimization is vital to prevent translational slowdown resulting from the
rate-limiting presence of tRNA carrying the desired amino acids.

### **Reference**

[1]Holowko, M. B.; Wang, H.; Jayaraman, P.; Poh, C. L., Biosensing Vibrio cholerae with
Genetically Engineered Escherichia coli. ACS Synth Biol 2016, 5 (11), 1275-1283.

[2]Higgins, D. A.; Pomianek, M. E.; Kraml, C. M.; Taylor, R. K.; Semmelhack, M. F.;
Bassler, B. L., The major Vibrio cholerae autoinducer and its role in virulence factor
production. Nature 2007, 450 (7171), 883-6.

## **Part3: Engineering dehydration detection component and cellular-cellular signaling in *Lactobacillus reuteri***

### Design part:

This experiment aims to create an upstream diarrhea prevention sensor and signal production tool,
which is used to sense the environmental changes caused by diarrhea and convey the signal to the
downstream *E. coli* engineering bacteria. A good portion of diarrhea results in an
excessive
increase in osmotic pressure (mainly due to chloride ion concentration) within the intestine,
causing water sequestration, resulting in watery stools[11](#re_11).
Therefore, in the presence of high salt concentrations, our *L. reuteri* produces the
gram-negative bacterial quorum sensing molecule, acylated homoserine lactones (AHL) and
subsequently activating autolysis. The autolysis releases the AHL into the intestinal tract that
will activate our engineered probiotic *E. coli* in the large intestine to facilitate water
retention.

### Build Part:

We achieved this cellular behaviour using two independent cellular functions. The first function
produces AHL, and the second function facilitates cellular autolysis in the presence of elevated
salt concentrations.

The first route uses the *luxI* gene regulated by the ermB promoter in the lactic acid
bacteria[3](#re_12)to produce AHL used in E.
coli quorum sensing[2](#re_13).
The second route uses chlorine ion regulatory promoters rrnhB and gadR[4](#re_14), and downstream-regulated genes
are the automatic gene acmA of Gram-positive bacteria[5](#re_15) from the previous iGEM design (Part: BBa\_K3142013
from team iGEM19\_SZPT-CHINA). We selected *L. reuteri* as our desired chassis due to its
GRAS
status and its ability to colonize the small intestine upstream of the large intestine,
essential for delivering the AHL to the localized engineered probiotic *E. coli*. As the
backbone
of our genetic engineering component, we decided on the shuttle vector pTRKH3 that can replicate
in both gram-negative and gram-positive bacteria.

![image](https://static.igem.org/mediawiki/2021/0/07/T--SUSTech_Shenzhen--engineering-picture-8.png)

**Figure 1|** The shuttle plasmid is pTRKH3-ermGFP from from Michela
Lizier[16](#re_16)

### Test Part：

Due to limited wet laboratory time during the COVID-19 pandemic and various gene synthesis issues
encountered in our work, we failed to complete the testing component on time. However, based on the
experimental design, we intend to use different concentrations of NaCl solution and IPTG, then test
its OD600 in 60 hours to compare its gene expression rate. By combining the AHL synthase
and
cellular autolysis function in the plasmid, we intend to determine the concentration of AHL released
in the supernatant through the use of UHPLC by comparing it to commercially available standards.

### Learn Part：

The gene synthesis encountered some issues due to the gadR terminator that forms a stable hairpin
during gene synthesis. Therefore, we replaced the gadR terminator with a double-series
terminator, which has successfully prevented the formation of a stable hairpin structure.

### **Reference**

[1]Berkes, J.; Viswanathan, V. K.; Savkovic, S. D.; Hecht, G., Intestinal
epithelial responses to enteric pathogens: effects on the tight junction barrier, ion
transport, and inflammation. Gut 2003, 52 (3), 439.

[2]Lizier, M.; Sarra, P. G.; Cauda, R.; Lucchini, F., Comparison of expression
vectors in Lactobacillus reuteri strains. FEMS microbiology letters 2010, 308 (1), 8-15.

[3]Prindle, A.; Samayoa, P.; Razinkov, I.; Danino, T.; Tsimring, L. S.; Hasty, J.,
A sensing array of radically coupled genetic 'biopixels'. Nature 2011, 481 (7379), 39-44.

[4]Sanders, J. W.; Leenhouts, K.; Burghoorn, J.; Brands, J. R.; Venema, G.; Kok,
J., A chloride-inducible acid resistance mechanism in Lactococcus lactis and its regulation.
Molecular Microbiology 1998, 27 (2), 299-310.

[5]Steen, A.; Buist, G.; Horsburgh, G. J.; Venema, G.; Kuipers, O. P.; Foster, S.
J.; Kok, J., AcmA of Lactococcus lactis is an N-acetylglucosaminidase with an optimal number
of LysM domains for proper functioning. The FEBS Journal 2005, 272 (11), 2854-2868.

## **Part4: Programming E. coli antimicrobial response to combat *Vibrio cholera***

### Design Part:

On top of preventing dehydration to treat diarrhea, a more direct approach to eliminating the
diarrhea-causing pathogen can treat the symptoms. Thus, in this section, we aimed to target
diarrhea-causing pathogen, V. cholerae, using antimicrobial proteins.

Human lactoferrin[1](#re_16) (flHLF) has
antibacterial properties that are regarded as safe for host consumption. The antimicrobial
properties are often attributed to the antagonism of flHLF to iron ions that are essential
for bacterial growth1. We intend to use flHLF as our output to target the V. cholerae
pathogenic infection, where the protein is expressed and secreted in the presence of CAI-1.
We are also extending the study to use an improved variant of flHLF, rtHLF4, which is more
effective and stable than conventional flHLF[1](#re_16).

### Build Part:

We cloned the *flHLF* and *rtHLF4* genes into a pET28 vector for expression in
*E. coli*. The
recombinantly expressed protein was purified via metal ion affinity chromatography and size
exclusion chromatography before evaluating the anti-*V. cholerae* properties. In the
antimicrobial screening, we tested the purified proteins against the attenuated *Vibrio
cholera* strain, BNCC 232030[2](#re_17) ( BeNa Culture Collection).

![image](https://static.igem.org/mediawiki/2021/4/47/T--SUSTech_Shenzhen--engineering-picture-13.png)

**Figure 1|** Size exclusion chromatography of rtHLF4

### Test Part：

In testing for the antimicrobial activity of the flHLF and rtHLF4 proteins, *V.
cholerae*
cultures were inoculated in fresh media containing an increasing concentration of the
proteins. The changes in the growth trend observed over absorbance at 600nm help in
determining the bactericidal or bacteriostatic properties of these proteins. Based on our
assay, we discovered that flHLF and rtHLF4 are bacteriostatic and bactericidal, respectively
(Fig. 2). Our results showed a prominent decrease in *V. cholerae* growth with the
increasing
concentrations of both flHLF and rtHLF4. flHLF showed a delay in growth, indicating the
flHLF interferes with the pathogen’s growth cycle but is insufficient to repress the
pathogenic proliferation over a duration of time.

![image](https://static.igem.org/mediawiki/2021/2/26/T--SUSTech_Shenzhen--engineering-1-1.png)

![image](https://static.igem.org/mediawiki/2021/0/0f/T--SUSTech_Shenzhen--engineering-part4-2.png)

**Figure 2|** Antibacterial test of the HLF

### Learn Part：

The antimicrobial properties of flHLF require higher concentrations of the purified
proteins
to exert the antimicrobial activities and can only slow down the *V. cholerae*
growth
rate.
Further, flHLF requires over 7 hours to have observable antimicrobial properties,
rendering
the protein unsuited for treating *V. cholerae* infections. Clinical data reported
that
severe
cases of *V. cholerae* infections could be lethal within 24 hours of infection,
where
the
flHLF’s antimicrobial activity is ill-suited to combat the pathogen (Fig. 2).

In order to resolve this issue, we substituted the lactoferrin protein with an
alternative
antibacterial peptide found from the iGEM database. A potential antibacterial candidate
is
the Eosinophil cationic protein that has antibacterial properties suited for use in the
distal region of the human large intestine[3-4](#re_18). Further studies on the antimicrobial
properties of the cationic protein will be tested in the nematode Caenorhabditis elegans
to
observe the antibacterial properties in a gut-like environment.

### **Reference**

[1]Pan, Y.; Chua, N.; Lim, K.; Ho, C. L., Engineering of Human Lactoferrin for
Improved Anticancer Activity. ACS Pharmacology & Translational Science 2021.

[2]Azman, A. S.; Rudolph, K. E.; Cummings, D. A.; Lessler, J., The incubation
period of cholera: a systematic review. J Infect 2013, 66 (5), 432-8.

[3]Wagner, M.; Peterson, C. G. B.; Stolt, I.; Sangfelt, P.; Agnarsdottir, M.;
Lampinen, M.; Carlson, M., Fecal eosinophil cationic protein as a marker of active
disease and treatment outcome in collagenous colitis: A pilot study. Scandinavian
Journal of Gastroenterology 2011, 46 (7-8), 849-854.

[4]Rivera, F. P.; Medina, A. M.; Bezada, S.; Valencia, R.; Bernal, M.; Meza,
R.; Maves, R. C.; Ochoa, T. J., Bovine lactoferrin decreases cholera-toxin-induced
intestinal fluid accumulation in mice by ganglioside interaction. PLoS One 2013, 8 (4),
e59253.

## **Part5：Gene circuit design to regulate antimicrobial properties of *E. coli* to target *V. cholerae* infection**

### Design Part:

We regulate the expression of the antimicrobial protein using an AND gate to have a
precise release of the antimicrobial agents using the two inputs are the
**transcriptional repressor** protein PhlF—its promoter (*Pphlf*)[1](#re_20), and transcriptional
activator
protein EsaR—*Esa*
box[2](#re_21). The EsaR-*Esa* box
is
regulated using the AHL produced by our engineered *L. reuteri* upon sensing
changes in
the salt concentrations in the small intestine, whereas the PhlF-*pPhlf* is
regulated by
sensing the *V. cholerae* quorum sensing molecule CAI-1. The programmed AND gate is
used
to produce the antimicrobial agents to kill *V. cholerae* in the presence of
dehydrating
conditions and CAI-1, ensuring that the produced antimicrobial agent does not cause
unnecessary perturbation to the host microbiome.

EsaR protein is constitutively expressed, functioning as a transcription repressor to the
Esa box to inhibit downstream expression[2](#re_21). The EsaR protein interacts with the AHL
quorum-sensing molecules, causing it to be released from the *Esa* box and
restoring the
expression of genes regulating colanic acid biosynthesis and the conditional expression
of the antimicrobial agent.

Many studies have demonstrated the use of the PhlF inverter system in E. coli[1](#re_20). PhlF
functions as a transcription repressor, inhibiting the downstream gene expression
regulated by the *pPhlF* promoter. The repressed levels of PhlF release the imposed
inhibition on *pPhlF*, leading to the expression of the protein sequences regulated
by
*pPhlF*.

In total, the EsaR system responds to the AHL concentration, and PhlF system respond to
CAI-1 concentrations. Thus, combining the two systems using an AND gate would promote
the downstream expression only in the presence of CAI-1 and AHL.

### Build Part：

The PhlF expression and the *pPhlF* + Esa box-regulated GFP expression were
synthesized on
opposite strands of the double-stranded DNA (fig. 1). This design was intended to
prevent transcriptional spillover resulting from two adjacent genes being close to each
other. Thus, arranging the genes on the opposing strands can increase the output
stability of the experimental system.

![image](https://static.igem.org/mediawiki/2021/4/4b/T--SUSTech_Shenzhen--egineering-picture-10.png)

**Figure 1|** PhlF + GFP + PphlF sequence design

We similarly applied the same approach to studying the EsaR-*Esa* box (fig. 2).

![image](https://static.igem.org/mediawiki/2021/5/57/T--SUSTech_Shenzhen--engineering-1-2.png)

**Figure 2|** EsaR controlling part

We integrated the EsaR and PhlF regulatory systems on a single plasmid to ensure no loss
of gene function due to the plasmid loss (fig. 3). We included RFP and GFP as our output
for both the gene circuit response, where individual regulatory proteins were tagged
with His-tag and Strep-tag to monitor its expression rate via western blot (fig. 1 &
fig. 3). We selected different terminators and RBSs to prevent gene recombination (fig.
3).

![image](https://static.igem.org/mediawiki/2021/4/40/T--SUSTech_Shenzhen--engineering-4-1.png)

**Figure 3|** Total plasmid design

In the specific selection of transformation, electrical stimulation rather than thermal
transformation was used in the experiment to *E.coil* Nissle 1917. According to
existing
experimental data, thermal transformation can lead to irreversible denature of
*E.coil*
Nissle 1917 protein and death of strain, so the required transformation cannot be
completed[3](#re_22)
.To simplify the experiment's procedure, *E.coli* Acella was used to test some of
the
parts by the transformation method of heat shock.

### Test Part：

The plasmid was initially transformed into *E.coli* Acella (fig. 4) and selected for
Kanamycin resistance. We found that the design plasmid can be transformed into
the *E.
coli* host while persevering the antibiotic resistance, as shown in figure
4. The
negative control bacteria without plasmid transformation on the left and
the right are
heat-shock bacteria.

![image](https://static.igem.org/mediawiki/2021/9/95/T--SUSTech_Shenzhen--engineering-1.png)

**Figure 4|** Transformation of the plasmid to *E.coil* Acella

Western blot was used to detect the normal expression of PhlF (fig. 5). Using anti-His
tag antibodies conjugated with Horseradish peroxidase, we found that the induced
expression of PhlF accumulates at the inclusion body, whereas uninduced cells showed no
expression of PhlF. This result suggests a tightly regulated expression of PhlF,
facilitating the following step of the study in identifying the strength of PhlF in
inhibiting *pPhlF*. A purified His tagged-GFP was used as a positive control in the
study.

![image](https://static.igem.org/mediawiki/2021/a/a8/T--SUSTech_Shenzhen--engineering-16.png)

**Figure 5|** Western blot of PhlF (E is experiment; C is control; S
is
supernatant;
P
is
precipitate; PC is positive control)

We used fluorescence tests to verify the entire pathway. AHL and IPTG with different
concentrations were added to the *E. coli* chassis harbouring the
lac+ESAR+phlf+RFPv1.1
plasmid shown in fig. 3. The RFP fluorescence should be inversely proportional to the
concentration of AHL, whereas GFP fluorescence should be inversely proportional to the
AHL and IPTG concentration. However, our results so far do not support our hypothesis
(fig. 6).

![image](https://static.igem.org/mediawiki/2021/b/b9/T--SUSTech_Shenzhen--engineering-13-1.png)

**Figure 7|** Fluorescence test of GFP with different concentrations
of IPTG and same concentration of AHL. We have conducted this experiment at a series
of AHL concentrations: 0nM, 10nM, 100nM, 1000nM. But the result is similar and do
not show obvious inhibitory effects, thus we does not show all of them here.

### Learn Part：

Due to the time limitation and travel restrictions, we could not complete the validation
of our designed circuit. Despite repeating the experiment numerous times using different
inducer concentrations and reaction conditions, the change in fluorescence level over
time does not match the profile we generated *in silico*. We hypothesize that this
might
be due to various factors, including imposed metabolic stress from the protein
expression in the host chassis. Thus, we intend to regulate the expression of
these key
proteins using RBS with differing strengths in order to limit the stress imposed
on the
host cell.

### **Reference**

[1]Moon, T. S.; Lou, C.; Tamsir, A.; Stanton, B. C.; Voigt, C. A., Genetic
programs constructed from layered logic gates in single cells. Nature 2012, 491
(7423), 249-53.

[2]Shong, J.; Collins, C. H., Quorum sensing-modulated AND-gate promoters
control gene expression in response to a combination of endogenous and exogenous
signals. ACS Synth Biol 2014, 3 (4), 238-46.

[3]Hancock, V.; Vejborg, R. M.; Klemm, P., Functional genomics of probiotic
Escherichia coli Nissle 1917 and 83972, and UPEC strain CFT073: comparison of
transcriptomes, growth and biofilm formation. Mol Genet Genomics 2010, 284 (6),
437-54.

## Quick access

[#### Project

Introduction and description of the team project](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Description)

[#### Human Practice

Introduction and description of HP's objective, process and achievements](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Human_Practices)

[#### Team

Introduction of the team, team members and their attributions](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Team)

[#### Experiment

Introduction and description of the experiment ideas, process and results](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Experiments)

[#### Model

Explain how we make predictions mathematically](https://2021.igem.org/Team%3ASUSTech_Shenzhen/Model)

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# Team:SUSTech Shenzhen/Experiments

# Experiments

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

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

### What should this page contain?

* Protocols
* Experiments

### Inspiration

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

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

# Team:SUSTech Shenzhen/Results

# Results

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

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)