iGEM 2021 — Team ZJUT-China

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# Team:ZJUT-China/Notebook

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* [Awards](https://2021.igem.org/Team%3AZJUT-China/Awards)

## Notebook

### ZJUT-China

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# Team:ZJUT-China/Engineering

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Engineering

## We demonstrate engineering success in the **gene circuit** of our project on this page. By going through iterations of the engineering design cycle, we proved the success of our gene circuits.

![slidedown](https://static.igem.org/mediawiki/2021/3/38/T--ZJUT-China--slidedown.svg)

![figure-1](https://static.igem.org/mediawiki/2021/4/42/T--ZJUT-China--engineering_1.png "Click to see more clearly")

This year, we focused on using Cell-Free system to detect RNA. As
a biosensor, our project consists of three units, including **the recognition
element**, **the
transducer** and **the detection system**.

In transducer unit, we designed a gene circuit specifically to
convert molecular signals into fluorescence signals. The whole network topology comprises
four DNA constructs: P70a-σ28, P28-tetR (P28-tetR-ssrA), P28-tetO-deGFP, P70a-ClpXP. The
Cell-Free system contains σ70, which binds to the P70 promoter and initiates the expression
of σ28 and ClpXP. σ28 then binds to P28 promoter and initiates the expression of tetR (tetR-ssrA). TetR exclusively binds to the tetO and represses the production of deGFP.
Additionally, the repressor protein encoded by tetR contains a C-terminal ssrA tag for
targeted protein degradation by ClpXP proteases[1].

The gene circuit has many parts, the typical ones are **P28-tetR [(BBa\_K3885211)](http://parts.igem.org/Part%3ABBa_K3885211)** ,**P28-tetR-ssrA [(BBa\_K3885212)](http://parts.igem.org/Part%3ABBa_K3885212)** ,and **P70-σ28 [(BBa\_K3885202)](http://parts.igem.org/Part%3ABBa_K3885202)** which achieved what we expected. Additionally, P28-tetR-ssrA is one of the **new parts** that we registered this year. By adding ssrA tag to the **tet Repressor part [(BBa\_K337028)](http://parts.igem.org/Part%3ABBa_K337028)** registered by iGEM 10\_Heidelberg, we endowed novel function to the original biobrick.

![figure-1-2](https://static.igem.org/mediawiki/2021/e/e2/T--ZJUT-China--engineering_3.png "Click to see more clearly")
**Figure 1.** Whole gene
circuit.

P28-tetR (repressor protein)

Design

Gene tetR can express **repressor protein**
while the repressor
protein inhibits the expression of deGFP. Once the Cas9 protein is activated to cut gene
tetR, the inhibition effect will be eliminated and reach the purpose of signal
conversion. When combined with promoter P28, tetR can achieve better expression effect,
thus we plan to construct plasmid P28-tetR.

Test

Agarose Gel Electrophoresis

P28-tetO-deGFP was used as the vector plasmid and the
fragment comes from puc57-tetR.

After recombination, a recombinant plasmid P28-tetR was
successfully constructed confirmed by sequencing results.

The sequencing results were consistent with expectations,
plasmid P28-tetR constructed successfully.

![figure-2](https://static.igem.org/mediawiki/2021/4/44/T--ZJUT-China--engineering_5.png "Click to see more clearly")
**Figure 2.**
Schematic of the plasmid P28-tetR construction.
(a) On the left is 5k marker, in the middle is tetR segment, on the right is the
vector from plasmid P28-tetO-deGFP-ssrA reverse PCR;
(b) On the left is 5k marker, on the right is colony PCR verification strip.

Verification of repressor protein about inhibitory effect

**Experiment group**: P70a-σ28, P28-tetR, P28-tetO-deGFP
**Control group**: P70a-σ28, P28-tetO-deGFP

![figure-2](https://static.igem.org/mediawiki/2021/5/51/T--ZJUT-China--engineering_6.png "Click to see more clearly")
**Figure 3.**
The gene circuit for verification of repressor protein functioned.

If the fluorescence intensity of control group is stronger than the experiment
group, the inhibitory effect of plasmid P28-tetR can be proved.

**Figure 4.** Green fluorescence of the experimental group and the control group.

**The effect of plasmid P28-tetR is proved.**

P28-tetR-ssrA

Design and improvement

Next, we added CRISPR system to our gene circuit and
investigated its function into Cell-Free system by adding Cas9 and gRNA.

**Experiment group**: P70a-σ28，P28-tetR, P28-tetO-deGFP
**Control group**: P70a-σ28, P28-tetO-deGFP

![figure-2](https://static.igem.org/mediawiki/2021/6/60/T--ZJUT-China--engineering_7.png "Click to see more clearly")
**Figure 5.**
The gene circuit used to detect CXCL9 mRNA with CRISPR system.

We found that even after the Cas9 protein exercises cleavage activity to disable tetR,
repressor protein remain in the system still prevent fluorescent protein expression and
have an impact on the signaling output of the system [2]. After reviewing the article,
we found **ssrA tags**. SsrA tags are encoded by amino acids and can be
added to the
C-terminal of proteins to degrade abnormal proteins during translation. Therefore by
adding ssrA tag to the end of tetR gene, we can solve the problem.

Test

Agarose Gel Electrophoresis

The vector comes from P28-tetO-deGFP-ssrA, the fragment
comes from puc57-tetR.

After recombination, we got recombinant plasmids
P28-tetR-ssrA about 3.2k bp.

![figure-6](https://static.igem.org/mediawiki/2021/4/42/T--ZJUT-China--engineering_9.png "Click to see more clearly")
**Figure 6.**
Schematic of the plasmid P28-tetR-ssrA construction.
(a) On the left is 5k marker, in the middle is tetR segment, on the right is the
vector from plasmid P28-tetO-deGFP-ssrA reverse PCR;
(b) On the left is 5k marker, on the right is colony PCR verification strip.

The sequencing results were consistent with expectations,
plasmid P28-tetR constructed successfully.

Verification of repressor protein about inhibitory effect

**Experiment group**: P70a-σ28，P28-tetR-ssrA，P28-tetO-deGFP
**Control group**: P70a-σ28，P28-tetR, P28-tetO-deGFP

![figure-2](https://static.igem.org/mediawiki/2021/3/30/T--ZJUT-China--engineering_8.png "Click to see more clearly")
**Figure 7.**
The gene circuit used to verify ssrA tag.

If the fluorescence intensity of experiment group is stronger than the control
group, then the degradation effect of ssrA tag can be proved.

**Figure 8.** Green fluorescence of the experimental group and the control group.

**As shown by the data, the fluorescence difference between the experimental and
control groups was not significant. This indicates that the degradation effect
of ssrA label did not reach our expectation.**

P70a-ClpXP

Design and learn

With the experience of constructing P28-tetR-ssrA, we
followed steps in 2.1, combined it to CRISPR system and verified it in Cell-Free system.
But it failed to achieve the ideal effect.

By viewing literature, we found that **ClpXP can improve the degrading effect of
ssrA tag**.
Thus, we verify the combination effect of P28-tetR-ssrA and P28-tetO-deGFP while adding
P70a-ClpXP to the system. Hoping to eliminate the effects of residual repressor protein
in the system and provide data for modeling.

Verification of the combination degradation effct of ssrA tag and ClpXP

**Experiment group**:P70a-σ28+P28-tetR-ssrA+P28-tetO-deGFP+P70a-ClpXP
**Control group**: P70a-σ28+P28-tetR+P28-tetO-deGFP+P70a-ClpXP

![figure-2](https://static.igem.org/mediawiki/2021/3/32/T--ZJUT-China--engineering_10.png "Click to see more clearly")
**Figure 9.**
If the fluorescence intensity of experiment group is stronger than the control
group, then the combination degradation effct of ssrA tag and ClpXP can be
proved.

If the fluorescence intensity of experiment group is stronger than the control group,
then the combination degradation effct of ssrA tag and ClpXP can be proved.

**Figure 10.** Green fluorescence of the experimental group and the control group.

Reference

[1]吕璐. mf-ssrA标签介导的大肠杆菌的正交性降解研究[D].天津大学,2019.
[Link](https://kns.cnki.net/kcms/detail/detail.aspx?dbcode=CMFD&dbname=CMFD202101&filename=1020434042.nh&uniplatform=NZKPT&v=GBs24zXntLm%mmd2B9h80xHoxkxQDHQnmAwHMBwaC3Olv%mmd2FCVGbw6hjGtqX9LmBjT7Znjv "Click to read")

[2]Structural basis of ClpXP recognition and unfolding of ssrA-tagged substrates.[J]. eLife,2020,9:
[Link](https://pubmed.ncbi.nlm.nih.gov/33089779/ "Click to read")

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# Team:ZJUT-China/Experiments

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Experiments

## This page provides all standardized protocols used in our experiments, including Plasmid Construction, Transformation, Cas9&gRNA *in vitro* Experiment, Cell-Free system and Fluorescence-based Analysis.

![slidedown](https://static.igem.org/mediawiki/2021/3/38/T--ZJUT-China--slidedown.svg)

* [Experiments](#experiments)
* [Protocol](#protocol)

RNA-Biomarker

Construction of CXCL9 mRNA

We constructed a new plasmid that can transcribe CXCL9 mRNA by using two existing plasmids (igRNA-CXCL9 and pTargetF). The primers were designed for PCR, and the PCR products were obtained after template elimination and purification. Then the constructed plasmid was transformed the plasmid into *E.coli* DH5αcompetent cells to make it self-replicating. Then we did colony PCR using colonies grown on resistant plates. We got the right plasmid after agarose gel electrophoresis and further sequencing.

Recognition Element

Design and screening of gRNA

CGG and AGG were selected as the protospacer adjacent motif (PAM) to alter the original N20 sequence in plasmid pTargetF. Primers gRNA-R and gRNA-F (Figure 1) were designed for PCR, and the PCR products should be dealt with template elimination and purification, and then transferred the fragments into  *E. coli* DH5α to make them self-ligating. On this basis, we obtained five different gRNAs and measured their cleavage activity and electroporated gRNAs into cells with spCas9 and tetR plasmids and obtained highly active gRNAs by resistance selection in *E. coli*. Meanwhile, we transcribed gRNAs *in vitro*, cleaved the target segments with Cas9 protein, and obtained highly active gRNAs by agarose gel electrophoresis.

## Note

---

spCas9(kanamycin
resistence), gRNA(spectinomycin resistence), tetR(Ampicillin)

These plasmids have different replicons.

![figure-1](https://static.igem.org/mediawiki/2021/2/25/T--ZJUT-China--experiment_1.png "Click to see more clearly")
**Figure 1.** The map of gRNA from
SnapGene

|  |  |  |
| --- | --- | --- |
| electroporate | gRNA+spCas9+tetR | resistance screening：Ampicillin |
| electroporate | spCas9+tetR | resistance screening：Ampicillin |

Design and *in vitro* transcription of igRNA

On the basis of screening gRNA1 with high cleavage activity, we added the sequence at the 3 'end to make it complementary with the RNA biomarker, and sent the sequence to Tsingke Biotechnology for synthesis. Primers with T7 promoters were designed, and igRNA transcription templates with T7 promoters were obtained through igRNA plasmids. *In vitro* transcription experiments were conducted according to the kit, followed by purification with chloroform or RNA purification kit, and the integrity of the bands was verified by agarose gel electrophoresis. After confirming the integrity of the strips (no degradation), the next step *in vitro* verification experiment can be carried out and The igRNA was stored at -80℃.

For details, please refer to Protocol:
IVT,
RNA
purification.

*In vitro* validation of igRNA-CXCL9 mRNA Complex (EMSA)

In order to verify whether igRNA and
CXCL9 mRNA can have base complementary pairing and bind to Cas9, an electrophoretic mobility shift assay(EMSA)
experiment was carried out. First, the RNA in the -80℃ refrigerator was
taken out and added into the annealing buffer for annealing, so as to form
the correct secondary structure. Then，the experimental group and the control
group were set according to the table. After Cas9 protein was mixed on ice for 30 min, samples were loaded into two pieces of 6% non-denatured polyacrylamide gel, and 120 V electrophoresis was performed in 4℃ for 1 hour. Finally, one was stained with GelRed for visualizing nucleic acid and the other was stained with Coomassie blue to check bright blue, and the binding was observed according to the GelRed-gel in the UV light.

For details, please refer to Protocol:
EMSA.

|  |  |  |  |  |
| --- | --- | --- | --- | --- |
|  | igRNA | CXCL9+igRNA | CXCL9 | Cas9 |
| Test(CXCL9 mRNA-igRNA-Cas9) | - | + | - | + |
| Control 1(igRNA-Cas9) | + | - | - | + |
| Control 2(igRNA) | + | - | - | - |
| Control 3(CXCL9 mRNA) | - | - | + | - |
| Control 4(Cas9) | - | - | - | + |

Plasmid construction of a simplified version of spCas9

To make the spCas9 plasmid better for
expression in the Cell-Free system, we used one-step cloning technique to
remove unnecessary genes and constructed a simplified
version of the spCas9 plasmid.

Considering the difficulty of plasmid
ligation due to excessive plasmid assembly and low transformation
efficiency, we chose to synthesize a new plasmid P15A ori-CmR. Then we used spCas9 plasmid from laboratory and P15A ori-CmR to construct plasmid spCas9.

We first designed primers ori-F, ori-R, spCas9-F, and pCas-R for the two plasmids to conduct PCR. After clean-up and eliminate templates experiments, we measured the concentration and converted it, using the enzymes cloned in one-step for ligation of the fragments. Finally the plasmid was transformed into *E. coli*  DH5α to culture overnight.

After the colonies grew, we picked out the colonies for colony PCR, and finally we got the right length and sequencing results.

For details, please refer to Protocol:
Plasmid construction.

Electroporation

In order to verify whether our components can operate normally, we first perform in-bacteria verification, and mainly rely on electro- transformation for verification. First, we need to prepare BW25113 competent cells with deionized water and store them at -80°C for later use. Then prepare the P70a-σ28-P28-deGFP, P70a-σ28-P28-tetR and P28-tetO-deGFP required for our verification.

Design four sets of
experiments, as follows.

|  |  |
| --- | --- |
| Group | design |
| Control group | BW25113 |
| test group 1 | BW25113+P70a-σ28-P28-deGFP |
| test group 2 | BW25113+P28-tetO-deGFP |
| test group 3 | BW25113+P28-tetO-deGFP+P70a-σ28-P28-tetR |

Conduct the electroporation experiment
with an electroporator. After the electroporation is successful, add 970μL of
LB liquid medium to the electroporation cup, pipette it evenly, transfer it
to the EP tube, and culture with shaking for 1 hour. Centrifuge, discard 900
μL of supernatant, resuspend the cells, spread and culture. For details of
the results, please view the Result：Cas9 group.

For details, please refer to Protocol:
Electroporation-Transformation
*E. coli* BW25113 cells.

Transducer

Gene circuits validation

All of the plasmids were validated in
the Cell-Free system.

Each Cell-Free reaction system was
12 μL, including 9 μL σ70 reagent and 3 μL plasmid template. All experimental
groups were set up as follows:

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
|  | P70a-σ28 | P28-tetR | P28-tetR-ssrA | P28-tetO-dGFP | P70a-ClpXP | Purpose |
| Test 1 | 2nM | - | - | 10nM | - | Verify the effect of σ28 |
| Test 2 | 2nM | 5nM | - | 10nM | - | Verify the effect of tetR |
| Test 3 | 2nM | - | 5nM | 10nM | 1nM | Verify the effect of ClpXP |
| Test 4 | 2nM | - | 5nM | 10nM | - | Control group |

Since the total volume of plasmid template was only 3 μL, the more kinds of plasmids were added, the higher the concentration of plasmids we needed to prepare. Through plasmid extraction, we obtained plasmids with a concentration of about 500 ng/μL, which was concentrated enough for us to carry on.

Then we calculated the concentration,
diluted and mixed each plasmid in equal volume, vortex gently, and add 3μL
template mixture to the Cell-Free system. After vortex again, we transfer
5μL to the two wells of the 96-well V plate preheated at 29℃ for 30 minutes
in advance. After sealing the film, put it into a microplate for
fluorescence dynamic detection.

Detailed analysis of the results
obtained from the experiment is on the page of Engineering Success.

For details, please refer to Protocol:
Cell-Free
system.

Plasmid
Construction

* LB
  Media
* Polymerase
  Chain Reaction（PCR）&Colony
  PCR
* Elimination
  template and PCR
  Clean-up
* DNA
  Gel Extraction
* Agrose
  Gel Electrophoresis
* One-Step
  Cloning
* Preparation
  of chemo-competent *E. coli*
  KL740 cells
* Transformation
  of
  chemo-competent
  *E. coli* KL740 cells
* Transformation
  of
  chemo-competent
  *E. coli* DH5α cells
* Plasmid
  microextraction
* Mass
  plasmid DNA
  extraction

Transformation

* Preparation
  of chemo-competent
  *E. coli* BW25113 cells
* Electroporation-Transformation
  *E. coli* BW25113 cells

Cas9&gRNA
*in vitro* experiment

* *In
  vitro* transcription
* RNA
  purification
* *In
  vitro* DNA digestion with
  Cas9 Nuclease
* EMSA(Gel-shift
  Assay)

Cell-Free
system

Fluorescence-based
Analysis

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# Team:ZJUT-China/Results

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* Lab
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* [Awards](https://2021.igem.org/Team%3AZJUT-China/Awards)

Results

## The results page involved almost all of achievements in our experiments. There are many flow diagrams before the detailed procedure. We hope it will improve the feeling of reading and makes the results more visual.

![slidedown](https://static.igem.org/mediawiki/2021/3/38/T--ZJUT-China--slidedown.svg)

What did we achieve?

1. We constructed **a simplified plasmid of spCas9**
and a plasmid
that can transcribe **CXCL9 mRNA**.

2. We designed an **igRNA** based on a gRNA with
high cutting
activity, and carried out an *in vitro* verification experiment for igRNA and RNA
biomarker.

3. We constructed **P70a-σ28-P28-deGFP and
P70a-σ28-P28-tetR**
plasmids to verify the intracellular function of transcription factor **σ28**
and inhibitor
protein **tetR** in gene circuits.

4. We constructed plasmids with fluorescent genes that remove
ssrA degradation tag to explore **the effect of degradation tag ssrA** on
deGFP.

5. We successfully constructed the plasmid in the gene circuits
and completed the function verification in **Cell-Free system**.

Plasmid spCas9 construction

![figure-1](https://static.igem.org/mediawiki/2021/5/55/T--ZJUT-China--results_1.svg "Click to see more clearly")
**Figure 1.** Schematic of the procedure of
plasmids construction. After colony Polymerase Chain Reaction (PCR), the monoclonal
colonies were cultured and sent to the company for sequencing.

Plasmid spCas9 (Figure 1) was constructed from the original
plasmid spCas9 and the P15A Ori-CmR gene fragment of the synthetic plasmid puc57, which
for **expressing Cas9 protein and complex the corresponding CXCL9 mRNA-igRNA for
cleavage
of target genes in Cell-Free system**. The building process is shown in the
figure above.

![figure-2](https://static.igem.org/mediawiki/2021/1/1c/T--ZJUT-China--results_2.png "Click to see more clearly")
**Figure 2.** Schematic of the plasmid spCas9
construction.
(a) The map of spCas9 from SnapGene.
(b) Gel electrophoresis analysis of PCR products. The marker is 5k (lane 1), the
fragment is spCas9 (lane 3&4), the vector is P15A Ori-CmR (lane 5&6).
(c) Gel electrophoresis analysis of cloning PCR products. The marker is 5k (lane 1)
, the colony PCR fragment is a part of the goal plasmid (lane 2&3).

Plasmid CXCL9 mRNA construction

![figure-3](https://static.igem.org/mediawiki/2021/b/b9/T--ZJUT-China--results_3.png "Click to see more clearly")
**Figure 3.** Schematic of the procedure of
plasmid CXCL9 mRNA construction. After cloning PCR, the monoclonal colonies were
cultured and sent to the company for sequencing.

**We constructed a new plasmid which can transcribe
CXCL9 mRNA
by using two existing plasmids (igRNA-CXCL9 and pTargetF, Figure 4a).** Then
designed
primers CXCL9-F and CXCL9-R to conduct PCR, clean-up, and eliminate templates
experiments (Figure 4b). Transformed the fragment andvector with one-step cloning
enzyme into *E. coli* DH5α to make it self-replication. Colony PCR using colonies
grown on
resistant plates. We had fundamental success after agarose gel electrophoresis and a
final success after sequencing.

![figure-4](https://static.igem.org/mediawiki/2021/0/06/T--ZJUT-China--results_4.png "Click to see more clearly")
**Figure 4.** Schematic of the plasmid
construction.
(a) Maps of plasmid pTargetF-gRNA and CXCL9-igRNA from SnapGene;
(b) On the left is
5k marker, in the middle is CXCL9 segment, on the right is the vector from plasmid
pTargetF reverse PCR;
(c) On the left is 5k marker, on the right is colony PCR verification strip.

Construction of plasmid gRNA and screening of cleavage activity

![figure-5](https://static.igem.org/mediawiki/2021/a/a8/T--ZJUT-China--results_5.png "Click to see more clearly")
**Figure 5.** Schematic of the design procedure of
the gRNA construction and screening. There are five 20nt sequences to change into five
different gRNAs.

In the CRISPR/Cas9 system, **gRNA** can direct Cas9
protein to cut
dsDNA at the sequence target site. Simultaneously, **a hybridization region of
igRNA** is adding
additional sequences in the **5'** of sgRNA. Therefore, designing an active
gRNA with high
cleavage ability is important.

Based on the target gene (tetR), we chose **CGG and AGG as
PAM**, and
designed 5 gRNAs in the plasmid pTargetF (Figure 5).

*In vitro* cleavage validation

![figure-6](https://static.igem.org/mediawiki/2021/c/c0/T--ZJUT-China--results_6.png "Click to see more clearly")
**Figure 6.** Gel electrophoresis analysis of *in
vitro* cleavage products with different gRNA.Target dsDNA was incubated with the
following gRNA for 1 h at 37 °C under the condition of *in vitro* Cas9
cleavage assay:
marker (lane 1); gRNA1 (lane 2); gRNA2 (lane 3); gRNA3 (lane 4); gRNA5 (lane
5).

After the plasmid of gRNA was constructed, 5 corresponding
RNAs were transcribed by *in vitro* transcription. The **gRNA, Cas9 and the
target
gene
fragment were mixed together** for reaction according to the kit handbook.
Then carry out
the agarose gel electrophoresis (Figure 6).

Subsequently, we analyzed why **the cutting ability of
different gRNAs** varies greatly. According to some research, we explored
this question
with the professionals from **Genscript** and found an answer from their
official CRISPR
handbook: "The **10 to 12 bp** base of gRNA near PAM, also known as
**the seed sequence**,
determines the specificity of gRNA and target recognition. The results showed that
controlling **GC content** in seed sequence at **40%~60%**
during gRNA sequence design could
significantly improve the specificity of gRNA and target recognition."

CG content of 5 gRNA sequences designed is:

| gRNA | sequence | CG (%) |
| --- | --- | --- |
| gRNA1 | ttagagctgcttaatgaggt | 30-42 |
| gRNA2 | attggcatgtaaaaaataag | 10-17 |
| gRNA3 | caaaagtacatttaggtaca | 30-33 |
| gRNA4 | tagccattgagatgttagat | 30-33 |

Obviously, the CG content of **gRNA1** seed
region with obvious
cutting ability obtained from *in vitro* validation experiments is
**between
40%-60%**, but
gRNA4 does not meet this requirement. More strangely, gRNA3 and gRNA4 have the same CG
content, but gRNA3 does not show obvious cutting ability. Therefore, we speculated that
gRNA3 could not guide Cas9 protein to perform the cleavage function because of the
sequence of bases or the easy degradation of RNA transcribed *in vitro*.

After comprehensive consideration, **we chose gRNA1
with higher
CG content as the basis of igRNA design**.

*In vitro* validation of RNA biomarkers and igRNA

![figure-7](https://static.igem.org/mediawiki/2021/1/1a/T--ZJUT-China--results_7.png "Click to see more clearly")
**Figure 7.** Polyacrylamide gel-shift assay
analysis of the
igRNA hybridizating with CXCL9 mRNA.

We selected the **CXCL9 mRNA**, a biomarker
related to rejection
**reaction after kidney transplantation**, and designed the sequence of
igRNA. Please refer
to Enginering Success for the specific design scheme. In order to verify that igRNA can
complement CXCL9 mRNA, we conducted **EMSA experiment** *in vitro*
(Figure
7).

In column A, non-igRNA and CXCL9 mRNA could
**not complement
each other**, and the bands were separated after binding Cas9, and the
migration rate of
the complex was slower as the number of Cas9-binding RNA bases increased. The column D
was CXCL9 mRNA-igRNA-Cas9 complex, and the migration rate was **slower**
than the column A.
**The results indicated that igRNA was successfully combined with CXCL9 mRNA
*in vitro*.**

How to verify that every part is functional in gene circuits?

In Cell-Free systems, validation of each component is also
important to ensure proper use of the whole function. Therefore, the **σ28, tetR
repressor** in
the gene circuit was validated in bacteria **prior** to the
**validation** of the Cell-Free system.

Construction of P70a-σ28-P28-deGFP plasmid

![figure-8](https://static.igem.org/mediawiki/2021/3/30/T--ZJUT-China--results_8.svg "Click to see more clearly")
**Figure 8.** Schematic of the procedure of
plasmid P70a-σ28-P28-deGFP construction. After colony PCR, the monoclonal colonies
were cultured and sent to the company for sequencing.

The plasmid P70a-σ28-P28-deGFP was constructed from the gene
fragment of the original plasmid P70a-deGFP and the synthetic fragment UTR1-σ28-P28
(Figure 9a) to verify the normal expression of σ28 and provide the original plasmid for
the synthesis of P70a-σ28-P28-tetR. After PCR, the results of agarose gel eletrophoresis
were as follows, **with correct band sizes** (Figure 9b).

![figure-9](https://static.igem.org/mediawiki/2021/3/39/T--ZJUT-China--results_9.png "Click to see more clearly")
**Figure 9.** Schematic of the plasmid
construction.
(a) The map of plasmid P70a-σ28-P28-deGFP from SnapGene;
(b) On the left is the 5k marker, in the middle is UTR1-σ28-P28 segment, on the
right is the vector from plasmid P70a-deGFP reverse PCR;
(c) On the left is the 5k marker, and on the right is colony PCR verification band.

![figure-10](https://static.igem.org/mediawiki/2021/6/6a/T--ZJUT-China--results_10.png "Click to see more clearly")
**Figure 10.** Photos of cultured plates. There
are some colonies with plasmid P70a-σ28-P28-deGFP theroratically on the left plate,
and the negative control is on the right plate.

After **recombination and transformation**,
overnight culture,
bacterial colonies grew on ampicillin plates (Figure 10).

Through the **colony PCR** verification band,
our agarose gel
eletrophoresis results were consistent with expectations (Figure 9c), and the plasmid
was **successfully constructed** after sequencing.

Construction of P70a-σ28-P28-tetR plasmid

![figure-11](https://static.igem.org/mediawiki/2021/4/45/T--ZJUT-China--results_11.svg "Click to see more clearly")
**Figure 11.** Schematic of the procedure of
plasmid P70a-σ28-P28-tetR construction. After colony PCR, the monoclonal colonies
were cultured and sent to the company for sequencing.

Plasmids P70a-σ28-P28-tetR were constructed from the gene
fragment of original plasmid puc57 and the gene fragment of P70a-σ28-P28-tetR. After
PCR, the results of agarose gel electrophoresis were as follows, **with correct
band sizes**
(Figure 12b).

![figure-12](https://static.igem.org/mediawiki/2021/f/f1/T--ZJUT-China--results_12.png "Click to see more clearly")
**Figure 12.** Schematic of the plasmid
P70a-σ28-P28-tetR construction.
(a) The map of plasmid P70a-σ28-P28-tetR from SnapGene;
(b) On the left is 5k marker, in the middle is P70a-σ28-P28-tetR segment, on the
right is the vector from plasmid P70a-σ28-P28-tetR;
(c) on the left is 5k marker, on the right is colony PCR verification strip.

After **recombination and transformation**,
overnight culture,
bacterial colonies grew on chloramphenicol plates. Through the **colony
PCR** verification
band, our gel results were consistent with expectations (Figure 12c), and the plasmid
was **successfully constructed** after sequencing.

Construction of P28-tetO-deGFP plasmid

![figure-13](https://static.igem.org/mediawiki/2021/7/75/T--ZJUT-China--results_13.svg "Click to see more clearly")
**Figure 13.** Schematic of the procedure of
plasmid P28-tetO-deGFP-ssrA construction. After colony PCR, the monoclonal colonies
were cultured and sent to the company for sequencing.

The original plasmid P28-tetO-deGFP-ssrA was used to
construct a plasmid with ssrA degradation tag removed (Figure 14a) to **explore
the
effect of ssrA degradation tag on deGFP protein**.

![figure-14](https://static.igem.org/mediawiki/2021/f/fc/T--ZJUT-China--results_14.png "Click to see more clearly")
**Figure 14.** Schematic of the plasmid
construction.
(a) The map of plasmid P28-tetO-deGFP-ssrA from SnapGene;
(b) On the left is 5k marker, in the middle is deGFP fragment, on the right is the
vector from plasmid P28-tetO-deGFP;
(c) On the left is 5k marker, on the right is colony PCR verification band.

PCR and Agarose Gel Electrophoresis (AGE) results were shown
in Figure 14b, and the band was correct.

After template removal and purification, the plasmid was
cloned and transformed into *E. coli* trans1-T1 competent cells for culture.

Colonies on resistant plates were selected for colony PCR,
and the results were shown in Figure 14c with correct bands.

Validation of σ28

![figure-15](https://static.igem.org/mediawiki/2021/f/ff/T--ZJUT-China--results_15.png "Click to see more clearly")
**Figure 15.** Schematic of the procedure of
Electroporation-Transformation *E. coli* BW25113 cells.

To **enhance** the expression intensity of each
component, we use
σ28 as the promoter factor. **σ28** is a special transcriptional factor
concerning the FliA,
which is an alternate sigma factor for the class 3 flagella operons, and it **does
not
exist in Cell-free system**. To verify the functional availability of σ28, we
build
P70a-σ28-P28-deGFP plasmid. If the expression of σ28 is normal, it shows fluorescence.
Conversely, there is no fluorescence.

To verify the normal expression of σ28, we performed
electroporation experiments with *E. coli* BW25113 (Table 1).

| Group | set |
| --- | --- |
| Control Group | BW25113 |
| Experiment Group | BW25113+P70a-σ28-P28-deGFP |

**Table 1.**

![figure-16](https://static.igem.org/mediawiki/2021/d/d1/T--ZJUT-China--results_17.jpg "Click to see more clearly")

No colony in the control group fluorescenced (Figure 16a)
,
while the single colony in the experimental group fluorescenced (Figure 16b) ,
indicating that P70a-σ28-P28-deGFP was successfully transferred, all gene elements
on
the plasmid were functional, and **σ28 was normally expressed**.

Validation of tetR

Tet Repressor (tetR) is a **repressor protein**
that inhibits
fluorescence and indirectly feedback the sensity of the entire gene circuits. TetR
repressoracts as a guard on gene circuits. Normally, tetR **inhibits the
expression of
deGFP**, but when it is cut by Cas9 protein, it will lose its inhibition
effect. And it
sends us a message by means of fluorescence.

TetR binds to the tetO site and thus blocks deGFP expression.
Therefore, if P70a-σ28-P28-tetR is normally constructed, fluorescence is present in the
control group and inhibited in the experimental group.

To verify the normal expression of tetR, we carried out an
electroporation experiment with *E. coli* BW25113 (Table 2).

| Group | set |
| --- | --- |
| Control Group | BW25113+P28-tetO-deGFP |
| Experiment Group | BW25113+P28-tetO-deGFP+P70a-σ28-P28-tetR |

**Table 2.**

The P28-tetO-deGFP plasmid was present in the control group
(BW25113 + P28-tetO-deGFP), the fluorescence was normal (Figure 16c). The fluorescence
in the experimental group (BW25113+ P28-tetO-deGFP +P70a-σ28-P28-tetR) should not exist
theoretically. DeGFP is theoretically suppressed. However, in fact, there were three
colonies with strong green fluorescence, as shown in Figure 16d, which may be because
of the failure of transforming P70a-σ28-P28-tetR plasmid during electroporation,
resulting in some colonies with **strong fluorescence**.

Some single colonies were selected for culture in the above
three groups and their fluorescence intensity was measured, as shown in Figure 17 and
Table 3.

**Figure 17.** Average ratio-standard deviation
of fluorescence intensity from *E. coli* BW25113 cells containing the
plasmids
carrying P70a-σ28-P28-deGFP, P28-tetO-deGFP, P70a-σ28-P28-tetR with P28-tetO-deGFP.

| Group | 1 | 2 | 3 | 4 | 5 | 6 | Average |
| --- | --- | --- | --- | --- | --- | --- | --- |
| BW25113+P70a-σ28-P28-deGFP | 33456 | 29526 | 33285 | 30745 | 28582 | 35611 | 31868 |
| BW25113+P28-tetO-deGFP | 13225 | 13112 | 14765 | 15351 | 14796 | 14305 | 14259 |
| BW25113+P28-tetO-deGFP+P70a-σ28-P28-tetR | 4125 | 5736 | 5031 | 4098 | 5421 | 4579 | 4632 |
| BW25113 | 2467 | 3308 | 3428 | 1980 | 2340 | 2766 | 2715 |

**Table 3.**

After transforming plasmids P28-tetO-deGFP and
P70a-σ28-P28-tetR into *E. coli* BW25113, the fluorescence of BW25113 was
significantly
decreased compared with that of BW25113 and P28-tetO-deGFP, indicating that **tetR
reprssor could bind tetO site and thus reduce the intensity of deGFP**.

Gene circuits

We aim to use Cell-Free system to detect RNA with CRISPR/Cas9
system. Based on the cleavage of Cas9 protein, we designed special gene circuits.

Plasmid P70a-σ28 Construction

Vector: P70a-deGFP (Figure 18a) segment: puc57-σ28

The vector and segment have been amplified by PCR. We
verified all our PCR fragments, the fragment length of σ28 is about 720~750 bp by
agarose gel electrophoresis, the fragment length of vector is about 2500~2550bp by
agarose gel electrophoresis as we expected (Figure 18b). Then we conducted plasmid
recombination.

We verified the recombination plasmid by colony PCR, the
fragment length is about 1000bp by agarose gel electrophoresis as we expected (Figure 18c).

**Plasmid p70a-σ28 was proved to be constructed
successfully after sequencing.**

![figure-18](https://static.igem.org/mediawiki/2021/2/20/T--ZJUT-China--results_18.png "Click to see more clearly")
**Figure 18.**Schematic of the plasmid P70a-σ28
construction.
(a) The map of plasmid P70a-σ28 from SnapGene;
(b) On the left is 5k marker, in the middle is σ28 segment, on the right is the
vector from plasmid P70a-deGFP reverse PCR;
(C) On the left is 5k marker, on the right is colony PCR verification strip.

Plasmid P28-tetR Construction

Vector: P28-tetO-deGFP-ssrA (Figure 19a) segment: puc57-tetR

The vector and segment have been amplified by PCR. We
verified all our PCR fragments. The fragment length of tetR is about 720~750 bp by
agarose gel electrophoresis. The fragment length of vector is about 2500~2550bp by
agarose gel electrophoresis as we expected (Figure 19b). Then we conducted plasmid
recombination.

We verified the recombination plasmid by colony PCR, the
fragment length is about 1000bp by agarose gel electrophoresis as we expected (Figure 19c).

**Plasmid P28-tetR was proved to be constructed
successfully
after sequencing.**

![figure-19](https://static.igem.org/mediawiki/2021/7/77/T--ZJUT-China--results_19.png "Click to see more clearly")
**Figure 19.**Schematic of the plasmid P28-tetR
construction.
(a) The map of plasmid P28-tetR from SnapGene;
(b) On the left is 5k marker, in the middle is tetR segment, on the right is the
vector from plasmid P28-tetO-deGFP-ssrA reverse PCR;
(c) On the left is 5k marker, on the right is colony PCR verification strip.

Plasmid P28-tetR-ssrA Construction

Vector: P28-tetO-deGFP-ssrA (Figure 20a) segment: puc57-tetR

The vector and segment have been amplified by PCR. We verified all our PCR fragments,
the fragment length of tetR is about 720~750 bp by agarose gel electrophoresis, the fragment length of the vector is about 2500~2550bp by agarose gel electrophoresis as we expected (Figure 20b). Then we conducted plasmid recombination.

We verified the recombination plasmid by colony PCR, the fragment length is about 1000bp
by agarose gel electrophoresis as we expected (Figure 20c).

**Plasmid P28-tetR-ssrA was proved to be constructed successfully after
sequencing.**

![figure-20](https://static.igem.org/mediawiki/2021/5/58/T--ZJUT-China--results_20.png "Click to see more clearly")
**Figure 20.**Schematic of the plasmid
P28-tetR-ssrA construction.
(a) The map of plasmid P28-tetR-ssrA from SnapGene;
(b) On the left is 5k marker, in the middle is tetR segment, on the right is the
vector from plasmid P28-tetO-deGFP-ssrA reverse PCR;
(c) On the left is 5k marker, on the right is colony PCR verification strip.

For more details about the verification results of "Gene Circuits", please refer to
[Engineering success](https://2021.igem.org/Team%3AZJUT-China/Engineering).