iGEM 2024 — Team ucas-china

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block page\_content %}

![Image](https://static.igem.wiki/teams/5276/wiki/pages/pages-banners/wl-notebook.jpg)

## Overview

In order to record the daily wetlab progress and analyze the experimental results timely, we log the experimental
contents for every experiment, and organize them. The compiled notebook is as follows.

##### Week 4, November 2023

Initial Planning for 2024

* Deliver presentation on team introduction
* initiate student recruitment process.

##### Week 1, December 2023

Initial Planning for 2024

* Conduct recruitment interviews.
* Finalize the selection of new members to the team.

##### Week 4, January 2024

Induction and Training of New Members

* Conduct basic wetlab experiment related to synthetic biology and biochemistry.
* Provide training sessions for drylab and model group members.

##### Week 5, January 2024

Induction and Training of New Members

* Deliver theoretical knowledge on wetlab practices.
* Continue drylab and model training sessions.

##### Week 2, March 2024

* Collect brainstorming ideas during winter vacation.
* Introduce CRISPRi as a potential component of our project.

##### Months of April - May, 2024

Concept Development and keep Brainstorming

##### Week 1, June 2024

Project Finalization and Pathway Design

* Discuss the feasibility of Programmable Logic Device (PLD) as a target of our project design.
* discuss about logic gates and recombinases as potential components of the design.
* Initiate preliminary gene circuit designing around PLD, integrating CRISPRi, logic gates and recombinases.

##### Week 2, June 2024

Project Finalization

* Engage in more detailed study of parts involved in our project.
* Develop a feasible protocol and schedule upcoming wet lab experiments.

##### Week 3, June 2024

Plasmid Design

* Complete the early stage of plasmid designs.

##### Week 4, June 2024

Fragment Design and Constitution

* Source necessary genes. Design primers for plasmid fragment and recombinase genes
* Assemble DNA fragments through PCR

##### Week 1, July 2024

Plasmid Construction

* Carry out homologous recombination to construct recombinase genes and plasmids.
* Transform the constructed plasmids into *E. coli* Top10 competent cells for propagation.

##### Week 3, July 2024

Patch Development and Innovation

* Identify a flaw in the current design.
* Develop and integrate a patch to fix the mistake.

##### Week 4, July 2024

Plasmid Construction and Validation

* Utilize overlap PCR techniques to construct plasmid fragments.
* Verify PCR results via agarose gel electrophoresis.
* Recover fragments of specific length through gel extraction.

##### Week 1, August 2024

Problem Identification and Sequencing

* Analyze and reflect on issues encountered in our design.
* Fix the mistake using PCR and homologous recombination.
* Sequence the plasmids to prove the success of modification.

##### Week 2, August 2024

Finalizing Plasmid Construction and Extraction

* Re-integrate missing sections with the plasmids.
* Extract plasmids and store them for future constitution and transfers.

##### Week 3, August 2024

Continued Plasmid Construction and Validation

* Proceed with combining the Input plasmid and Register plasmids.
* Utilize agarose gel electrophoresis to corroborate the results.

##### Week 4, August 2024

Orthogonality Verification

* Pair six recombinases and three recombination-GFP sites to confirm orthogonality.
* Transfer combinations into Top10. Promote cell incubation and growth.
* Verify the accuracy of the orthogonal genes and recombinases.

##### Week 1, September 2024

Induction and Fluorescence Testing

* Add IPTG and DAPG to induce the expression of recombinases.
* Conduct fluorescence testing and agarose gel electrophoresis to certify inversion and excision.

##### Week 2, September 2024

Continued Plasmid Construction, Extraction and Transformation

* Modify various plasmids and reconstruct them.
* Extract complete plasmids for further usage.

##### Week 3, September 2024

Screening and Fluorescence Testing

* Transform and conduct monoclonal screening and extend incubation time for successfully transformed E.coli.
* Execute colony PCR. Verify the length of plasmids using agarose gel electrophoresis.
* Carry out fluorescence testing with a microplate reader. Confirm the orthogonality of recombinases.

##### Week 4, September 2024

Continued Fluorescence Testing and Data Analysis

* Continue fluorescence testing to confirm the orthogonality of recombinases and the functionality of register
  plasmids.
* Add inducers for recombinases. Plot fluorescence-time graphs to analyze the effect of recombination.
* Apply qPCR to verify the inversion and excision of the recombinases.
* Design directed evolution experiments and collect initial results.

##### Week 1, October 2024

Wiki Freeze and Presentation Video

##### Week 2, October 2024

qPCR and Directed Evolution

* Continue using qPCR to verify the modification ability of the recombinases.
* Continue to conduct directed evolution for the efficiency of recombinase A118.

##### Week 3, October 2024

Plasmid Sequencing

* Sequence the plasmid for further confirmation of the accuracy and functionality.

##### Week 4, October 2024

Grand Jamboree

##### **Full records**

For more detailed notebook description, please [download the PDF](https://static.igem.wiki/teams/5276/wetlab/2024-ucas-china-notebook.pdf) of our lab
record on benchling.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one
iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{%
endblock %}
{% block page\_content %}

![Image](https://static.igem.wiki/teams/5276/wiki/pages/pages-banners/wl-engineering.jpg)

Methodology in synthetic biology reflects basic principles in engineering, and the engineering cycle reigns amongst
one of the most significant ones. The engineering cycle offers comprehensive and systematic guidance on the design
and test of biological systems and consists of four stages: **design, build, test, and learn.**

We propose a programmable logic framework, LANTERN, capable of switching between different logic gates based on
user requirements. We are fully aware that a standardized engineering cycle is even more critical to our
cross-disciplinary work, and we followed it strictly and explored it innovatively.

We have reached decent results with the implementation of it, and a summary is as follows.

1. We designed and refined the genetic circuits utilizing CRISPRi & recombinases.
2. We conducted orthogonality verification to test the orthogonality of recombinases.
3. We implemented directed evolution to improve the efficiency of recombinases.

## Cycle 1: Genetic Circuit Completion

### Design

Our initial circuit design incorporates four recombinases and CRISPRi to implement 16 possible logic gates (Figure
1).

![initial-circuit](https://static.igem.wiki/teams/5276/wetlab/pictures/initial-circuit.png)

In our design, CRISPRi acts as a crucial NOT gate. To verify its effect, we decided to simulate the repressive
impact of CRISPRi on the target gene in Simbiology while proceeding with plasmid construction.

### Build

Based on the mechanism of CRISPR interference, we establish reaction equations to demonstrate the inhibitory effect
of dCas9 and sgRNA on the expression of *GFP*. We used Simbiology to solve ODE and simulate the production of
GFP protein.

### Test

We obtained the change curves of GFP in two conditions: with and without expression of dCas9 (Figure 2, 3). Our
results confirmed that the combination of dCas9 and sgRNA effectively inhibits GFP expression, indicating that
CRISPRi functions as anticipated in this context and implements the NOT gate function in our circuit.

![gfpnocripri.png](https://static.igem.wiki/teams/5276/model/cripri/gfpnocripri.png)

Figure 2: Productivity without CRISPRi

![gfp-3-crispri](https://static.igem.wiki/teams/5276/model/cripri/gfp-3-crispri.png)

Figure 3: Productivity with CRISPRi

However, during discussions among wet lab and model members regarding the application of CRISPRi in our genetic
circuit, a challenge emerged: if the downstream promoter is inhibited by CRISPRi, normal expression of the gene of
interest (GOI) cannot be achieved due to the steric hindrance caused by the dCas9 protein, even when the upstream
promoter is actively initiating transcription.

### Learn

While CRISPRi successfully implements the NOT gate function in our circuit, we need to solve the problem of its
unexpected repression of upstream gene expression. So we added a patch (Figure 4) utilizing another two recombinases
(Cre & fimE) to Registers, thus completing our genetic circuit design.

![orthogonality-design-1.webp](https://static.igem.wiki/teams/5276/wetlab/pictures/patch-part.png)

Figure 4: A patch system using the Cre/loxP system and fimE inversion
system

## Cycle 2: Verification of recombinase efficiency and orthogonality

### Design

Our project is based on site-specific recombination systems involving four different recombinases, making the
orthogonality between these recombinases especially crucial. To verify this, we designed two types of plasmids:
recombinase plasmids, each containing one of the four recombinases, and verification plasmids, each containing a
pair of specific recombinase recognition sites. We transformed pairs of recombinase and verification plasmids into
*BL21(DE3)* competent cells together and designed a pair of primers in the same direction, such that only the
inverted verification plasmid could be detected through colony PCR.

![orthogonality-design-1.webp](https://static.igem.wiki/teams/5276/wetlab/qzt/verify-plasmids-and-primers.webp)

Figure 5: (a) Transformation pairs of recombinase plasmids and
verification plasmids. (b) Primers used in colony PCR.

### Build

We successfully constructed the above eight plasmids by PCR and homologous recombination. Then, we transformed
pairs of recombinase and verification plasmids into the *BL21(DE3)* competent cells for inducible expression
using IPTG and verified our results using colony PCR.

### Test

After induction with IPTG, we used colony PCR to verify the effect of recombinase inversing. The colony PCR results
(Figure 6) show that these recombinases exhibit excellent orthogonality.

![orthogonality of four recombinases](https://static.igem.wiki/teams/5276/wetlab/qzt/orthogonality-direct.webp)

Figure 6: Colony PCR results of verification plasmid using corresponding
primers.

However, during the colony PCR, we found that some of the verification plasmids were still not inverted under the
action of the corresponding recombinases, which indicates that the efficiency of the recombinases requires
improvement.

### Learn

From the results, the effect of these recombinases was not ideal. So, we decided to perform directed evolution
using A118 as an example to improve the inverting efficiency of serine recombinases.

## Cycle 3: Directed evolution of A118 recombinase

### Design

An expression module and a selection module were designed. The expression module induces recombinase expression
with IPTG, while the selection module uses a phage lysis gene *φX174 E*.

### Build

We inserted the expression module into the pET-28a(+) vector and the selection module into the pET-22b(+) vector
and transformed it into *E. coli*.

### Test

We used epPCR to amplify the expression module to generate a random mutant library for the recombinases. After
constructing the *E.coli* system, we conducted the screening plans to verify its functionality and
recombination effect.

The inversion function of the recombinase was proved by the results of colony growth, indicating the reverse of
*φX174 E* gene sequence and the suppression of the phage lysis gene.

The recombination efficiency was described by GFP fluorescence, which positively correlates to the expression level
of the inverted *GFP* gene. Thus, the inversion efficiency of the recombinases can be characterized by the
fluorescence intensity of GFP.

### Learn

Though **Traditional Mutagenesis** allows for some mutations that have high efficiency, the process of
constructing recombinase mutants and transferring them into *E. coli* is too tedious and labor-intensive.
Therefore, we considered using a continuous directed evolution approach to reduce the workload.

## Cycle 4: Continuous directed evolution

### Design

Taking advantage of autonomous reproduction of bacteria, we can construct a persistent high-mutation system for
continuous mutations during DNA replication. Therefore, we designed a bistable switch that allows reporter
*GFP* inverted by the A118 recombinase under certain conditions and then inverted back by the Gp44 protein
under different conditions, enabling a continuous selection process. To achieve continuous high mutation rates
inside E. coli, we introduced eMutaT7, a fusion of cytosine deaminase (AID) and T7 RNA polymerase (T7RNAP).

### Build

We designed three plasmids, pHyo094, Target plasmid, and Selection plasmid, to provide eMutaT7 enzyme, regulate
A118 and Gp44 expression, and indicate the selection result of the continuous evolution. Each consists of different
antibiotic resistance genes to facilitate co-transformation into *E. coli*.

### Test

After transferring the three plasmids into *E.coli*, we induced the expression of eMutaT7 to mutate the A118
recombinase sequences. If it maintains its biological function, the 'Turnover' switch can execute to inverse
the *GFP* gene, expressing protein and producing fluorescence. After sampling the strain, we removed the
mutation inducers, activated the 'Turnback' switch, and turned the *GFP* gene back to its original
state. After multiple cycles, we can screen for mutant recombinases.

### Learn

It has been demonstrated that the efficiency of recombinases can be improved through directed evolution (Sclimenti
et al., 2001). We plan to apply this approach to other suboptimal recombinases. We will conduct further verification
at the total circuit level with these enhanced recombinases.

## References

Sclimenti, C. R., Thyagarajan, B., & Calos, M. P. (2001). Directed evolution of a recombinase for improved
genomic integration at a native human sequence. *Nucleic acids research*, *29*(24), 5044–5051. <https://doi.org/10.1093/nar/29.24.5044>

{% endblock %}

{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/5276/wiki/pages/pages-banners/wl-experiments.jpg)

In the experimental stage of UCAS-China, we actively employed, developed, and refined a variety of protocols.
Recognizing the value of a well-structured protocol collection to benefit all iGEM teams, we have written a total
of fourteen protocols for the iGEM community.

# Protocols

* 1
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/01.png)

  [PCR](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol1-pcr.pdf)
* 2
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/02.png)

  [Agarose Gel Electrophoresis](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol2-agarose-gel-electrophoresis-prt-ahqi03g6.pdf)
* 3
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/03.png)

  [DNA Gel Extraction](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol3-dna-gel-extraction-prt-mxfjeur2.pdf)
* 4
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/04.png)

  [Uniclone Seamless Assembly](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol4-uniclone-one-step-seamless-cloning-new.pdf)
* 5
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/05.png)

  [Chemical Transformation](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol5-chemical-transformation-heat-shock-prt-lepyhf0e.pdf)
* 6
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/06.png)

  [Colony PCR](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol6-colony-pcr-prt-erort6rz.pdf)
* 7
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/07.png)

  [Plasmid DNA Extraction and Purification](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol7-plasmid-dna-extraction-and-purification-prt-okonlof8.pdf)
* 8
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/08.png)

  [IPTG Induction](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol8-iptg-induction-expression-new.pdf)
* 9
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/09.png)

  [Overlap PCR](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol9-overlap-pcr-prt-ddvhuioa.pdf)
* 10
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/10.png)

  [Protein Expression of Register](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol10-protein-expression-of-register-prt-ipotygcc.pdf)
* 11
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/11.png)

  [QPCR](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol11-qpcr.pdf)
* 12
  ![](https://static.igem.wiki/teams/5276/wetlab/protocol/12.png)

  [Directed Evolution of A118](https://static.igem.wiki/teams/5276/wetlab/protocol/protocol12-directed-evolution-of-a118-new.pdf)

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

![Image](https://static.igem.wiki/teams/5276/wiki/pages/pages-banners/pr-results.jpg)

## Plasmid construction

The Register plasmid is constructed by ourselves. We used PCR to construct the designed primers into fragments of
about 500bp. Agarose gel electrophoresis of some fragment extensions is shown below (Fig.1, and all others).

![orthogonality-design-1.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/pcr-fragment-3.jpg)

Figure 1: Agarose gel electrophoresis of extended PCR fragment

After successfully constructing these short fragments, we assembled them using overlap PCR and subsequently
combined them with the backbone through homologous recombination. Same method is used to construct other plasmids.

## Orthogonality of recombinases

Our Register part utilizes site-specific recombination systems involving four different recombinases, making the
orthogonality between these recombinases a critical factor. To verify this, we designed two types of plasmids:
recombinase plasmids, each containing one of the four recombinases, and verification plasmids, each containing a
pair of specific recombinase recognition sites. These plasmids were transformed by pair into *E. coli*
BL21(DE3) competent cells (fig. 2a), and colony PCR was performed using a pair of primers designed in the same
orientation (fig. 2b). This setup ensured that only inverted verification plasmids were detected. The results
demonstrated excellent orthogonality between these recombinases (fig. 2c).

![orthogonality-design-1.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/20241126162109.webp)

Figure 2: (a) Recombinase plasmids and verification plasmids transformed
by pair (4 x 4). (b) Primers used in colony PCR. (c) Colony PCR results.

## qPCR

We used qPCR to verify whether the expression of these recombinases can modify the Register plasmid, which consists
of orthogonal recombinase recognition sites to evaluate their efficiency.

We inserted the Bxb1 recombinase gene in plasmid pET28a(+) and transformed the Bxb1-pET28a(+) plasmid with Register
plasmid into *E.coli* BL21 (DE3) competent cells. Single colonies were selected, cultured, and induced with
1mM IPTG at 37° for four hours. We then extracted the plasmids from both induced and original cultures, and used the
inverted and original plasmid samples for qPCR. We designed two pairs of qPCR primers(fig. 3) to respectively detect
the original and modified Register plasmids to evaluate the inversion or not change. The result showed this process
working properly (fig. 4).

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/qs-qpcr.webp)

Figure 3. Primers used in qPCR.

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/qpcr-1.webp)

Figure 4. qPCR fluorescence intensity of Register A before and after
being incubated by bxb1 recombinase.

As the initial template amount undergoes exponential amplification in relation to the Ct value, we can evaluate
recombination rates by comparing the Ct values between different pairs of primer within the same sample. By
comparing the Ct values of the inverted and original Register plasmids, we can assess the efficiency of these
recombinases using the following formula:

$$
recombination\ rate= \frac {1} {1+2^{(Ct\_{original}-Ct\_{recombined})}}\times\ 100\%
$$

We assumed that each one-cycle difference in Ct value corresponds to a doubling of the initial template amount. The
qPCR fluorescence results (table.1 &fig. 5) underscore the exceptional performance of our Register system.

|  | Orignal | RECOMBINED |
| --- | --- | --- |
| RA\_bxb1 | Undetermined | 9.329 |
| RA\_TP901 | Undetermined | 15.952 |
| RB\_A118 | 29.96 | 23.66 |
| RB\_PhiC31 | 14.72 | 15.31 |

Table 1. Ct value of original and recombined Register region induced by
different recombinases

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/qpcr-2.webp)

Figure 5. Recombinases efficiency calculate from Ct value. 'Undetermined'
indicates that the Ct value exceeds the maximum number of amplification cycles (40) and is therefore calculated as
40.

## Sequencing

We used Bxb1 as an example to verify whether recombinases function properly. Original and
Bxb1-incubated Register A plasmids were both sequenced to obtain the precise sequences(fig. 6). The results
confirmed that Bxb1 excise the target gene correctly.

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/parts-qs/sequencing-bxb1.webp)

Figure 6. Sequence of original and Bxb1 induced Register A

## Register 0 induction

To verify whether two or more promoters can function collaboratively, we first construct Register 0 plasmid as
proof-of-concept for Register part (fig. 7). Register 0 includes overlapping and orthogonal recombinase recognition
sites, four promoters(constant promoter J23111, trc promoter, and inducible promoter lac operon, Rha promoter) with
different directions.

Using Register 0, we tested two key aspects: first, whether two promoters in series could achieve the functionality
of an OR gate; and second, whether the activity of a constitutive promoter in the reverse orientation would
interfere with the expression driven by a forward-oriented promoter.

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/pictures/qs1.png)

Figure 7: Design of Register 0

The distance between the promoter and the start codon should be minimized for optimal efficiency. Therefore, we
placed the necessary elements of the inducible promoter such as LacI, rhaS and rhaR outside the recombinase sites,
ensuring that only promoters are in the register region (fig. 8).

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/register-0-plasmid.png)

Figure 8: Register 0 plasmid

After the plasmid was successfully constructed, we transferred the Register 0 plasmid into BL21 (DE3) competent
cells. Once they reached the logarithmic growth phase, induced the cells by adding 1mM IPTG and 0.3% rhamnose at a
final concentration. We recorded the time span and fluorescence intensity as output.

The results shows that both IPTG and rhamnose (rha) induce expression effectively, and simultaneous induction of
both promoters leads to enhanced expression (fig. 9). This indicates that the Register 0 system functions as an
excellent OR gate. Since the distance between the Lac operon and the start codon is farther than that of the
rhamnose promoter, the Lac operon has a smaller effect on mRNA transcription when the rhamnose promoter is active.

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/results-qs/rb-iptg-rha.jpg)

Figure 9. OR GATE test of Lac operon and rhamnose promoter.

## Directed evolution

We use directed evolution to enhance recombinases efficiency. Initially, we employed traditional directed evolution
strategies, including error-prone PCR and the eMutaT7 directed evolution system, but these approaches are proved to
be inefficient. Consequently, we shifted our focus towards enhancing recombinase efficiency through rational design.

Since the structure of the recombinase-DNA complex is unresolved, we selected the A118 recombinase as an example
and predicted its composite structure with the attP site using AlphaFold 3 (fig. 10). Molecular docking was then
performed to study their interactions (fig. 11). Moving forward, we plan to conduct directed evolution on this
region to enhance activity of A118 and identify key amino acid residues for NNK mutagenesis.

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/parts-qs/movie-1.gif)

Figure 10. molecular animations of the predicted structure of A118 and
attP.

![orthogonality-design-2.webp](https://static.igem.wiki/teams/5276/wetlab/parts-qs/a118-dsdna-complex-and-core-residue-identified.webp)

Figure 11. A118-dsDNA complex and core residue identified.

## Supplement

The following are the results of our colony PCR verification using different primers, after we transfected the
recombinase plasmid and the verification plasmid together and induced them with IPTG;
Each fragment on the gel image is marked with two letters and a number, where the first letter represents the
recombinase corresponding to the recombinase plasmid, the second letter represents the recombination site
corresponding to the verification plasmid, and the number represents the parallel repeated test. (Abbreviations of
recombinases: A-A118, B-Bxb1, C-Cre, F-Fime, T-Tp901, φ-PhiC31)

![orthogonality-abphi-12.webp](https://static.igem.wiki/teams/5276/wetlab/qzt/orthogonality-abphi-12.webp)

Figure 12: Gel image to test the recombinase plasmids. The white target fragment length varies according to
the length of different recombinases, and is approximately between 1000bp and 2000bp. The red coded fragments are
the results of PCR using Left Primer and Right Primer. The target fragment length should be 1556bp.

![new-orthogonality-cft-12.webp](https://static.igem.wiki/teams/5276/wetlab/qzt/new-orthogonality-cft-12.webp)

Figure 13.Gel image to test the verification plasmids. The white marked fragments are the results of PCR
using PET\_28\_T7\_term and PET\_28\_T7. The target fragment length varies according to the length of different
recombinases, and is approximately between 1000bp and 2000bp.
The red coded fragments are the results of PCR
using Left Primer and Right Primer. The target fragment length should be 587bp.

![new-orthogonality-34.webp](https://static.igem.wiki/teams/5276/wetlab/qzt/new-orthogonality-34.webp)

Figure 14.Gel image to test the verification plasmids. The white and red marked fragments are the results of
PCR testing original plasmids and inverted plasmids, respectively.

{% endblock %}