iGEM 2020 — Team SJTU-BioX-Shanghai

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# Team:SJTU-BioX-Shanghai/Notebook

home

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* [Home](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai)
* Project

  + [Description](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Description)
  + [Design](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Design)
  + [Results](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Results)
  + [LUCAS and the world](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Implementation)
  + [Safety](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Safety)
  + [Proof of concept](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Proof_Of_Concept)
* Wet-lab

  + [Parts](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Parts)
  + [Part Collection](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Part_Collection)
  + [Measurement](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Measurement)
  + [Contribution](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Contribution)
  + [Engineering](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Engineering)
  + [Notebook](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Notebook)
  + [Protocol](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Protocol)
* Model

  + [Overview](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Model)
  + [Prediction Model](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Prediction_Model)
  + [Kinetics Model](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Kinetics_Model)
  + [Molecular Dynamics](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Molecular_Dynamics)
  + [Rational Design](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Rational_Design)
  + [Graphic Interpretation](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Graphic_Interpretation)
  + [Software tools](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Software)
* H.P.

  + [Overview](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/HP_overview)
  + [Integrated Human Practices](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practices)
  + [Public Education](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Education)
  + [Protein Atlas](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Protein_Atlas)
  + [Supplements](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Supplements)
* Team

  + [Members](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Team)
  + [Collaborations](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Collaborations)
  + [Attributions](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Attributions)
  + [Partnership](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Partnership)
  + [Inclusion](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Inclusion)
  + [Awards](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Awards)

overview

![](https://static.igem.org/mediawiki/2020/7/78/T--SJTU-BioX-Shanghai--notebook.png)

* [PWJ66 plasmid construction/
  mutant library construction](#section0)
* [Knock out ω subunit](#section1)
* [CRISPRa](#section2)
* [CRISPRi](#section3)

# Notebook

## PWJ66 plasmid construction/mutant library construction

## Click here for more

## Knock out ω subunit

## Click here for more

## CRISPRa

## Click here for more

## CRISPRi

## Click here for more

[![](https://static.igem.org/mediawiki/2020/c/c4/T--SJTU-BioX-Shanghai--totop3.png)](#section0)

Acknowledgements

[![](https://static.igem.org/mediawiki/2020/8/85/T--SJTU-BioX-Shanghai--SJTU.png)](https://www.sjtu.edu.cn/)

[![](https://static.igem.org/mediawiki/2020/5/50/T--SJTU-BioX-Shanghai--bioxlogo.png)](http://www.bio-x.cn/)

[![](https://static.igem.org/mediawiki/2020/8/83/T--SJTU-BioX-Shanghai--lifeshcool.png)](http://life.sjtu.edu.cn/)

# SJTU-BioX-Shanghai

## contact us

Contact us: sjtuigem@gmail.com or sjtuigem@gmail.com

Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800

---

Copyrights © 2020 SJTU-BioX-Shanghai

![](https://static.igem.org/mediawiki/2020/5/54/T--SJTU-BioX-Shanghai--logo3.png)

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# Team:SJTU-BioX-Shanghai/Engineering

home

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* [Home](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai)
* Project

  + [Description](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Description)
  + [Design](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Design)
  + [Results](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Results)
  + [LUCAS and the world](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Implementation)
  + [Safety](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Safety)
  + [Proof of concept](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Proof_Of_Concept)
* Wet-lab

  + [Parts](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Parts)
  + [Part Collection](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Part_Collection)
  + [Measurement](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Measurement)
  + [Contribution](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Contribution)
  + [Engineering](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Engineering)
  + [Notebook](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Notebook)
  + [Protocol](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Protocol)
* Model

  + [Overview](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Model)
  + [Prediction Model](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Prediction_Model)
  + [Kinetics Model](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Kinetics_Model)
  + [Molecular Dynamics](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Molecular_Dynamics)
  + [Rational Design](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Rational_Design)
  + [Graphic Interpretation](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Graphic_Interpretation)
  + [Software tools](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Software)
* H.P.

  + [Overview](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/HP_overview)
  + [Integrated Human Practices](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practices)
  + [Public Education](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Education)
  + [Protein Atlas](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Protein_Atlas)
  + [Supplements](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Supplements)
* Team

  + [Members](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Team)
  + [Collaborations](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Collaborations)
  + [Attributions](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Attributions)
  + [Partnership](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Partnership)
  + [Inclusion](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Inclusion)
  + [Awards](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Awards)

overview

![](https://static.igem.org/mediawiki/2020/4/4e/T--SJTU-BioX-Shanghai--engineering.png)

* [Overview](#section0)
* [Verification-Construction](#section1)
* [Verification-Expression](#section2)

# Engineering

# Part construction and expression verification

## Overview

To facilitate the evaluation of the on/off-target level of the dCas9, one part, which is the pBAD/araC upstream of RFP with inhibition unit containing target
[(BBa\_K3365014)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3365014)
is constructed and the RFP is
used as
one of the reporter proteins.

The RFP gene sequence [(BBa\_K3365052)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3365052) is located directly downstream of one target sequence [(BBa\_K3365006)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3365006)
followed by rrnBT1-T7TE [(BBa\_B0015)](http://parts.igem.org/wiki/index.php?title=Part:BBa_B0015). The transcription of the RFP
gene
is under the control of the inducible pBAD/araC promoter designed to carry PAM and target sequence
downstream of pBAD. In our part, the “target” is the target sequence for *PDCD1* CRISPR gene
editing in one clinical trial, which can be identified and bound by the complex of dCas9 and
corresponding sgRNA[1].

![](https://static.igem.org/mediawiki/2020/4/4a/T--SJTU-BioX-Shanghai--engineering1.png)

Figure1. Gene circuit of BBa\_K3365014

This part is used to express RFP protein regulated by the signal of arabinose and dCas9.
The pBAD is regulated by the AraC protein, which is both a positive and a
negative regulator[2]. The binding of dCas9 to any position within the region between the promotor and
RBS
might prevent transcription[3][4]. Therefore, the uninduced transcriptional level of RFP is very low. In
the
presence of arabinose, transcription from the pBAD promoter is turned on and there will be a
relatively
strong fluorescence expression. In the presence of both arabinose and the complex of dCas9 and
sgRNA,
the complex might bind to the target sequence and the transcription is partially inhibited because
of
the block of RNAP. So, a relatively weak fluorescence expression of bacteria indicates a dCas9 with
higher on-target rate that effectively inhibits the expression of reporter gene.

## Verification of the successful construction of this part

The inducible pBAD/araC promoter [(BBa\_K3365013)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3365013) is amplified with the primer F1/R1 to add
restriction cutting sites, target sequence and part of the RBS sequence [(BBa\_K3365002)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3365002). The RFP with
double terminator is amplified with the primer F2/R2 to add restriction cutting sites and part of
the RBS sequence [(BBa\_K3365002)](http://parts.igem.org/wiki/index.php?title=Part:BBa_K3365002). The above PCR products are ligated through overlap PCR with the
primer F1/R2 to get the fragment with suitable restriction sites and the product can be ligated into
its backbone pUC57 by enzymic digestion and connection.

The electrophoretic profile of the overlap PCR (F1/R2) and colony PCR (M13 fwd/ M13 rev)
product and the sequencing result reveal the successful construction of the fragment and the
plasmid, named pIn-RTr.

F1: 5’-CGAGCTCTTATGACAACTTGACGGCTACATCATTCAC-3’
R1: 5’-TTCTTAAAGGCAGTTGTGTGACACGGAAGCGGATGGAGAAACAGTAGAGAGTTGCG-3’
F2: 5’-TTCCGTGTCACACAACTGCCTTTAAGAAGGAGATATACATATGGCGAGTAGCGAAGACG-3’
R2: 5’-GGAATTCCCGCCCTAGGTATAAACGCAGAAAG-3’
M13 fwd: 5’-GTAAAACGACGGCCAGT-3’
M13 rev: 5’-GTCATAGCTGTTTCCTG-3’

![](https://static.igem.org/mediawiki/2020/2/2c/T--SJTU-BioX-Shanghai--engineering2.png)

Figure2. Electrophoretic profile of the overlap PCR product
Lane 1: 5000 bp marker; Lane 2-13: overlap PCR product

![](https://static.igem.org/mediawiki/2020/2/27/T--SJTU-BioX-Shanghai--engineering3.png)

Figure3. Electrophoretic profile of Colony PCR
product
Lane 13-15 show positive results.

## Verification of the RFP protein expression

To verify the inhibition effect of pdCas9 on pIn-RTr, we set up six culturing system (as shown in the table below) and used microplate spectrophotometer to examine fluorescence intensity. L-arabinose was added to induced the expression of RFP.

|  | pIn-RTr | pdCas9 | L-arabinose |
| --- | --- | --- | --- |
| MG1655 | + | + | + |
| MG1655 | + | + | - |
| MG1655 | + | - | + |
| MG1655 | + | - | - |
| MG1655 | - | - | + |
| MG1655 | - | - | - |

The results are shown below, from which we could see 64.39 times of fluorescence/OD600 difference between transform group (without the expression of pdCas9) and co-transform group (with the expression of pdCas9), indicating our transcription inhibition system did work.

![](https://static.igem.org/mediawiki/2020/8/85/T--SJTU-BioX-Shanghai--plot-1.png)

Figure4. The inhibition effect of dCas9 on pIn-RTr.

To know about the inhibition of the expression of RFP by dCas9 in a single cell as well as verify our mutant library, the obtained dCas9 mutant library and the wild-type dCas9 are transformed into MG1655 carrying pIN-RTr. Then, we analyze the red fluorescence by flow cytometry.

The results are shown below. 77.12% cells have positive red fluorescence in the absence of dCas9, while the proportion of positive cells decreases to 0.08% in the presence of dCas9, showing a quite efficient inhibition of wild-type dCas9. And the positive rate rises to 78.37% when the cells are transformed into dCas9 mutant library, which means the lack of function in most of mutant dCas9 and the successful construction of our mutant library.

![](https://static.igem.org/mediawiki/2020/a/a6/T--SJTU-BioX-Shanghai--Engr_temp1.png)

Figure5. The result of flow cytometry analysis

## References

[1] Lu Y, Xue J, Deng T, Zhou X, Yu K, Deng L, Huang M, Yi X, Liang M, Wang Y, Shen H, Tong R, Wang W, Li L, Song J, Li J, Su X, Ding Z, Gong Y, Zhu J, Wang Y, Zou B, Zhang Y, Li Y, Zhou L, Liu Y, Yu M, Wang Y, Zhang X, Yin L, Xia X, Zeng Y, Zhou Q, Ying B, Chen C, Wei Y, Li W, Mok T. Safety and feasibility of CRISPR-edited T cells in patients with refractory non-small-cell lung cancer. *Nat Med*. 2020 May;26(5):732-740.

[2] Guzman LM, Belin D, Carson MJ, Beckwith J. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. *J Bacteriol*. 1995 Jul;177(14):4121-30.

[3] Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, Lim WA. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. *Cell*. 2013 Feb 28;152(5):1173-83.

[4]Vigouroux A, Bikard D. CRISPR Tools To Control Gene Expression in Bacteria. *Microbiol Mol Biol Rev*. 2020 Apr 1;84(2):e00077-19.

[![](https://static.igem.org/mediawiki/2020/c/c4/T--SJTU-BioX-Shanghai--totop3.png)](#section0)

Acknowledgements

[![](https://static.igem.org/mediawiki/2020/8/85/T--SJTU-BioX-Shanghai--SJTU.png)](https://www.sjtu.edu.cn/)

[![](https://static.igem.org/mediawiki/2020/5/50/T--SJTU-BioX-Shanghai--bioxlogo.png)](http://www.bio-x.cn/)

[![](https://static.igem.org/mediawiki/2020/8/83/T--SJTU-BioX-Shanghai--lifeshcool.png)](http://life.sjtu.edu.cn/)

# SJTU-BioX-Shanghai

## contact us

Contact us: sjtuigem@gmail.com or sjtuigem@gmail.com

Bio-X Institute, Shanghai Jiao Tong University, Dongchuan Rd. 800

---

Copyrights © 2020 SJTU-BioX-Shanghai

![](https://static.igem.org/mediawiki/2020/5/54/T--SJTU-BioX-Shanghai--logo3.png)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:SJTU-BioX-Shanghai/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

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:SJTU-BioX-Shanghai/Results

home

[![](https://static.igem.org/mediawiki/2020/b/be/T--SJTU-BioX-Shanghai--logo.png)](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai)

![](https://static.igem.org/mediawiki/2020/0/0b/T--SJTU-BioX-Shanghai--more.png)

* [Home](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai)
* Project

  + [Description](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Description)
  + [Design](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Design)
  + [Results](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Results)
  + [LUCAS and the world](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Implementation)
  + [Safety](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Safety)
  + [Proof of concept](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Proof_Of_Concept)
* Wet-lab

  + [Parts](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Parts)
  + [Part Collection](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Part_Collection)
  + [Measurement](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Measurement)
  + [Contribution](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Contribution)
  + [Engineering](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Engineering)
  + [Notebook](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Notebook)
  + [Protocol](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Protocol)
* Model

  + [Overview](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Model)
  + [Prediction Model](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Prediction_Model)
  + [Kinetics Model](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Kinetics_Model)
  + [Molecular Dynamics](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Molecular_Dynamics)
  + [Rational Design](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Rational_Design)
  + [Graphic Interpretation](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Graphic_Interpretation)
  + [Software tools](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Software)
* H.P.

  + [Overview](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/HP_overview)
  + [Integrated Human Practices](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Human_Practices)
  + [Public Education](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Education)
  + [Protein Atlas](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Protein_Atlas)
  + [Supplements](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Supplements)
* Team

  + [Members](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Team)
  + [Collaborations](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Collaborations)
  + [Attributions](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Attributions)
  + [Partnership](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Partnership)
  + [Inclusion](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Inclusion)
  + [Awards](https://2020.igem.org/Team%3ASJTU-BioX-Shanghai/Awards)

overview

![](https://static.igem.org/mediawiki/2020/b/b8/T--SJTU-BioX-Shanghai--Result_MAIN.png)

* [Transcription activation](#section1)
* [Transcription inhibition](#section2)
* [Targeting progression](#section3)
* [Mutagenesis
  library construction](#section4)
* [Mutagenesis
   library screening](#section5)

# Result

In order to select dCas9 protein with higher on-target and lower off-target rate, we designed a
screening system and two different judging methods by monitoring alteration of two kinds of
fluorescence
level.

## Transcription activation

In this circuit, when dCas9 binds with target or lure sequence, the $\omega$ subunit will
recruit RNA polymerase, initiating transcription of downstream reporter gene – RFP and eGFP,
respectively.
The design of transcription activation system is inspired by a published paper, with target or
lure
sequence located 59bp upstream of the -35 element.
Target and potential off-target sequence (lure) of *PDCD1* are taken from previous paper as well
as
predicted by our own software.

### Construct plasmid for verifying transcription activation

## Click here for more

To verify the feasibility of our system, we first constructed a
module
with only target
sequence and each reporter gene.
J23117 promotor and 35bp spacer sequence were obtained through PCR, which were
then
ligated to target
sequence by designing target sequence on the primer.
We used overlap PCR to ligate the target+spacer+J23117 with RFP and eGFP
separately.

![](https://static.igem.org/mediawiki/2020/2/26/T--SJTU-BioX-Shanghai--design2wyt.png)

Distance between the protospacer and promoter of reporter gene.

RFP fragment with terminator was obtained from pE1a-RFP kindly offered by
**Tongji-China**.
Since our team did not have available eGFP gene with terminator, we conducted
the
procedure below to
add a double terminator to eGFP.
Due to complex secondary structure of double terminator, we gave up the idea to
perform PCR twice to
add terminator to eGFP fragment.
Rather, we first obtained terminator by performing PCR on pE1a-RFP and then link
target+spacer+eGFP
and terminator through overlap PCR.
Success was proven by both gel electrophoresis of PCR product and DNA
sequencing.

![](https://static.igem.org/mediawiki/2020/5/53/T--SJTU-BioX-Shanghai--Result_Fig.2.png)

PCR product of
target+spacer+eGFP+terminator .
(Lane 1: 2000 bp marker;Lane 2-9: overlap PCR product)

![](https://static.igem.org/mediawiki/2020/a/a1/T--SJTU-BioX-Shanghai--result_temp.png)

Circuit of target+spacer+J23117+RFP/eGFP+terminator.

Gibson homologous regions were added to this fragment through another PCR process.

We then linearized the backbone of pUC57 for Gibson assembly.

Sequentially, we assembled pUC57 backbone and the PCR product with Gibson assembly. The assembled product was transformed into DH5α. Colony PCR was conducted for positive colony selection and verification. And we named this plasmid for verifying transcription activation effect as pScreen-RTr/g(r for RFP while g for eGFP).

![](https://static.igem.org/mediawiki/2020/4/4f/T--SJTU-BioX-Shanghai--Result_Fig.3.png)

Verification of pScreen-RTg
assembly through
colony PCR. (Lane 1-8 and Lane 10-17: result of PCR product; Lane 9: 2000bpmarker. Lane 16
shows positive result.)

![](https://static.igem.org/mediawiki/2020/f/f8/T--SJTU-BioX-Shanghai--pScreen-RTg.png)

Circuit of pScreen-RTg.

![](https://static.igem.org/mediawiki/2020/8/85/T--SJTU-BioX-Shanghai--Result_Fig.4.png)

Verification of pScreen-RTr
assembly through colony PCR. (Lane 2-25: result of PCR product; Lane 1: 2000bp marker. Lane 10 shows positive result.)

![](https://static.igem.org/mediawiki/2020/3/30/T--SJTU-BioX-Shanghai--pScreen-RTr.png)

Circuit of pScreen-RTr.

### Construct plasmid for mutant libraries screening

## Click here for more

After successfully constructed plasmid with only target and eGFP/RFP,
we
continued to add
lures to this screening module.
We first chose four lure sequences with highest off-target rate, two from
literature
and two from our
software prediction (named lure 1/2/3/4).

We first tried Gibson assembly.
Lure+eGFP fragment was obtained by conducting PCR and replacing target sequence
with
lure on the
primer.
Homologous fragments were added to lure+eGFP fragments.
![](https://static.igem.org/mediawiki/2020/b/b2/T--SJTU-BioX-Shanghai--Result_temp2.png)

Circuit of curcuit of Lure1/2/3/4+spacer+J23117+eGFP+terminator

Sadly, positive clone were only found for lure2. Due to its low successful rate,
we
turned to
restriction enzyme digestion and ligation.
SacI and EcoRI cutting sites were added to lure+eGFP fragment ends.
pScreen-RTr vector and lure fragment were both digested by the two restriction
enzyme mentioned
above, and were then ligated using T4 DNA ligase and transformed into DH5α. And We named this plasmid for screening mutant libraries as pScreen-RTL1/2/3/4.

Success was proven by PCR.

![](https://static.igem.org/mediawiki/2020/c/c0/T--SJTU-BioX-Shanghai--RTL1.png)

Verification of pScreen-RTL1/3/4 assembly through colony PCR. (Lane 1-8: result of PCR product of pScreen-RTL1,Lane 2、3、7 show positive result; Lane 9-16: result of PCR product of pScreen-RTL3，Lane 11、14、15 show positive result; Lane 17-24: result of PCR product of pScreen-RTL4，Lane 17 shows positive result; Lane 25: 2000bp marker. )

![](https://static.igem.org/mediawiki/2020/e/e3/T--SJTU-BioX-Shanghai--RTL2.png)

Verification of pScreen-RTL2 assembly through colony PCR. (Lane 1-12、14-25: result of PCR product; Lane 13: 5000bp marker. Lane 1 shows positive result.)

![](https://static.igem.org/mediawiki/2020/3/30/T--SJTU-BioX-Shanghai--pScreen-RTL.png)

Circuit of pScreen-RTL1/2/3/4.

### Preparing $\Delta$rpoZ MG1655 strain

## Click here for more

Since our team want to recruit RNA polymerase and initiate
transcription
of reporter gene
through $\omega$ subunit-fusion dCas9
binding with target or lure sequence, innate $\omega$ subunit of *E.coli* must be removed
to
avoid
disturbance.
So we decided to knock out *rpoZ* - $\omega$ subunit encoding gene in *E.coli*, by
homologous
recombination of
Lambda Red recombineering system.

pKD46 plasmid was transformed into *E.coli* strain MG1655 by preparing MG1655
electro-competent cells.
Kanamycin resistant gene with homologous arm of *rpoZ* on MG1655 genome
was obtained through PCR using pET28a plasmid as template.

L-arabinose was added to induce pKD46 gene expression of Exo, Beta, Gam protein, and Kana-homo fragment was transformed in to knock out *rpoZ* gene.

![](https://static.igem.org/mediawiki/2020/0/00/T--SJTU-BioX-Shanghai--knockout_rpoZ.png)

Knock out *rpoZ* gene

Knockout of *rpoZ* was selected and verified by using kanamycin selective medium
and
verifying the
length of PCR product. We named this strain $\Delta$MG1655.

![](https://static.igem.org/mediawiki/2020/5/5b/T--SJTU-BioX-Shanghai--Result_KanaPart.png)

PCR product of Kanamycin resistant gene with homologous arm of *rpoZ* on MG1655 genome.(Lane 1, 3：result of PCR product. Lane 2：2000 Marker）

![](https://static.igem.org/mediawiki/2020/8/81/T--SJTU-BioX-Shanghai--Result_Fig.1.png)

Verification of *rpoZ*
deletion through colony PCR. (Lane 1: 5000 bp marker; Lane 2-25: result of PCR product. The length of PCR product of *rpoZ* is 464bp, while cells successfully knocking out *rpoZ* will have PCR product of 1155bp- the length of kanamycin resistant gene. Lane 3, 4, 10 and 21: positive colonies.)

### System verification

After we finished the construction of pScreen-RTg and pScreen-RTr, we immediately
verified the feasibility of our activation system.
We prepared 5 kinds of strains carrying different plasmids and test their fluorescence intensity
using flow cytometer for general screening and flowcytometry for single-cell
screening.

| Strain | pdCas9\* | pScreen-RTg | pScreen-RTr |
| --- | --- | --- | --- |
| $\Delta$MG1655 | - | + | - |
| $\Delta$MG1655 | - | - | + |
| $\Delta$MG1655 | + | + | - |
| $\Delta$MG1655 | + | - | + |
| $\Delta$MG1655 | - | - | - |

\*pdCas9 is generated by adding the dCas9-ω with a constitutive promoter to the plasmid vector pACYC184.

![](https://static.igem.org/mediawiki/2020/3/39/T--SJTU-BioX-Shanghai--Result_temp3.png)

The results are shown below. Unfortunately, it seemed that dCas9-ω didn’t bring about transcription-activation effect for certain reason. We hypothesized that the force of ω-subunit recruiting RNP is too weak to activate transcription. Meanwhile, dCas9-ω binding to target may instead interfere with transcription as it acts as a “roadblock”.

![](https://static.igem.org/mediawiki/2020/3/32/T--SJTU-BioX-Shanghai--plot-3.png)

![](https://static.igem.org/mediawiki/2020/2/20/T--SJTU-BioX-Shanghai--flow_1.png)

Raw data: [CRISPRa data](https://static.igem.org/mediawiki/2020/0/02/T--SJTU-BioX-Shanghai--CRISPRa.pdf)

## Transcription inhibition

In this circuit, target and lure sequence are designed right after promoters of
reporter
genes, RFP and eGFP, respectively.
Binding and staying still of dCas9 prevents RNA polymerase from sliding by, thus inhibit
transcription of downstream gene.

### System verification

To validate the inhibition system can work, plasmids pE1a-RFP-sgRNA and pS8K-dCas9
kindly
offered by Prof.
Yi Xiao were transformed into MG1655 and underwent microplate photometer to see fluorescence
difference.

MG1655 co-transformed with pE1a-RFP-sgRNA and pS8K-dCas9 were divided into four
groups
with three repeats in each group.
The culture condition of each group is mentioned below.
IPTG was added in the liquid culture to induce the expression of RFP, and L-arabinose was added
to
induce the expression of dCas9.
Glucose was used to inhibit leaky expression of dCas9.

|  | 1M arabinose stock solution | 1M IPTG stock solution | 0.5g/ml glucose stock solution |
| --- | --- | --- | --- |
| 1 | Added to liquid culture in a ratio of 1:200 at the start | Added to liquid culture in a ratio of 1:1000 when OD600 reaches 0.6 | No |
| 2 | Added to liquid culture in a ratio of 1:200 when OD600 reaches 0.4 | Added to liquid culture in a ratio of 1:1000 when OD600 reaches 0.6 | No |
| 3 | No | Added to liquid culture in a ratio of 1:1000 when OD600 reaches 0.6 | Added to liquid culture in a ratio of 1:250 when OD600 reaches 0.4 |
| 4 | No | Added to liquid culture in a ratio of 1:1000 when OD600 reaches 0.6 | No |

The results are shown below, from which we could see 9.44 times of fluorescence/OD600
difference between control group
(without any operation, only express RFP) and dCas9 expression group, indicating dCas9 do
inhibit
the expression of reporter gene.

![](https://static.igem.org/mediawiki/2020/9/9b/T--SJTU-BioX-Shanghai--Result_temp5.png)

Raw data: [Transcription inhibition system verification](https://static.igem.org/mediawiki/2020/5/59/T--SJTU-BioX-Shanghai--Inhibition.pdf)

### Construct plasmid for verifying transcription inhibition

## Click here for more

Seeing the feasibility of the inhibition system, we started to
construct
our own
plasmids.

Inducible araC and pBAD promoter
was amplified from pS8k-dCas9 given by Prof.
Yi Xiao. Our target sequence and restriction cutting sites were added to the
fragment during PCR.
In the meantime, target+RBS+RFP+term with restriction enzyme cutting site was
amplified from
pScreen-RTr.

The two fragments were ligated through overlap PCR, and the product
was
ligated into
pUC57 using SacⅠ and EcoRⅠ.
We named this plasmid for verifying transcription inhibition as pIn-RTr.

![](https://static.igem.org/mediawiki/2020/e/e6/T--SJTU-BioX-Shanghai--Result_pln-RTr_PCR.png)

PCR product of araC+pBAD+target+RBS+RFP+terminator. (Lane 1: 5000 bp marker; Lane 2-13: overlap PCR product)

![](https://static.igem.org/mediawiki/2020/c/c2/T--SJTU-BioX-Shanghai--Result_temp7.png)

Circuit of araC+pBAD+target+RBS+RFP+terminator.

![](https://static.igem.org/mediawiki/2020/9/95/T--SJTU-BioX-Shanghai--Result_batP.png)

Verification of pIn-RTr assembly through colony PCR. (Lane 1-8、11-18: result of PCR product; Lane 9: 2000bp marker；Lane 10:5000bp marker. Lane 13-15 show positive result.)

![](https://static.igem.org/mediawiki/2020/5/5a/T--SJTU-BioX-Shanghai--pIn-RTr.png)

Circuit of pIn-RTr.

### Verifying the inhibition effect of dCas9 on pIn-RTr

We set up six culturing system (as shown in the table below) and used microplate
spectrophotometer to examine fluorescence intensity.
L-arabinose was added to induce the expression of RFP.

|  | pln-RTr | pdCas9 | L-arabinose |
| --- | --- | --- | --- |
| MG1655 | + | + | + |
| MG1655 | + | + | - |
| MG1655 | + | - | + |
| MG1655 | + | - | - |
| MG1655 | - | - | + |
| MG1655 | - | - | - |

![](https://static.igem.org/mediawiki/2020/f/f7/T--SJTU-BioX-Shanghai--Result_temp6.png)

The results are shown below, from which we could see 64.39 times of fluorescence/OD600 difference between transform group (without the expression of dCas9) and co-transform group(with the expression of dCas9), indicating our transcription inhibition system did work.

![](https://static.igem.org/mediawiki/2020/8/85/T--SJTU-BioX-Shanghai--plot-1.png)

Raw data: [Transcription inhibition system verification(pIn-RTr)](https://static.igem.org/mediawiki/2020/a/af/T--SJTU-BioX-Shanghai--CRISPRi-pIn-RTr-data.pdf)

### Construct plasmid for mutant libraries screening

## Click here for more

Then we continued the construction of screening plasmid containing
both
target and lure
with their reporter gene.

Inducible pBAD promoter
was amplified from pS8k-dCas9 given
by Prof. Yi Xiao. Our lure1/2/3 sequence and restriction cutting sites were
added to the fragment during PCR. In the meantime, lure1/2/3+RBS+eGFP+term with
restriction enzyme cutting site was amplified from pScreen-RTr.

The two fragments were ligated through overlap PCR, and the product
was ligated into pUC57 using Pst1. We named this
plasmid for screening transcription inhibition as pIn-RTL1/2/3. However, only lure3's overlap product is successfully connected. Due to the limited time, we decide to carry out screening experiment with lure3 first, and the rest will be completed later.

![](https://static.igem.org/mediawiki/2020/8/8f/T--SJTU-BioX-Shanghai--jiaotu-1.png)

PCR product of pBAD+lure1+RBS+eGFP+term. (Lane 1: 2000 bp marker; Lane 2-10: overlap PCR product)

![](https://static.igem.org/mediawiki/2020/b/b0/T--SJTU-BioX-Shanghai--wyt2.png)

PCR product of pBAD+lure3+RBS+eGFP+term. (Lane 1: 2000 bp marker; Lane 2-11: overlap PCR product)

![](https://static.igem.org/mediawiki/2020/0/0c/T--SJTU-BioX-Shanghai--Result_temp8.png)

Circuit of pBAD+Lure3+RBS+eGFP+terminator

![](https://static.igem.org/mediawiki/2020/d/d7/T--SJTU-BioX-Shanghai--Result_pln-RTL_2.png)

PCR product of pBAD+lure2+RBS+eGFP+term. (Lane 1: 2000 bp marker; Lane 2-10: overlap PCR product)

![](https://static.igem.org/mediawiki/2020/5/59/T--SJTU-BioX-Shanghai--result11.png)

Verification of pIn-RTL1/2/3 assembly through colony PCR. (Lane 1:5000bp marker；Lane 2-9: result of PCR product of pIn-RTL1; Lane 10-17: result of PCR product of pIn-RTL2;Lane 18-25: result of PCR product of pIn-RTL3. Only Lane 19 show positive result.)

![](https://static.igem.org/mediawiki/2020/6/64/T--SJTU-BioX-Shanghai--pIn-RTL3.png)

circuit of pIn-RTL3

As an alternative，we also inserted our Lure1/3 sequence into an unfunctional site from pIn-RTr through site-directed mutagenesis PCR. The sequencing results show that we successfully inserted the target sequence, but due to the limited time, we have not yet conducted subsequent screening. We will continue to do this afterwards.

### Verifying the inhibition effect of dCas9 on pIn-RTL3

We set up six culturing system (as shown in the table below) and used microplate spectrophotometer to examine fluorescence intensity.L-arabinose was added to induce the expression of RFP and eGFP.

|  | pln-RTL3 | pdCas9 | L-arabinose |
| --- | --- | --- | --- |
| MG1655 | + | + | + |
| MG1655 | + | + | - |
| MG1655 | + | - | + |
| MG1655 | + | - | - |
| MG1655 | - | - | + |
| MG1655 | - | - | - |

![](https://static.igem.org/mediawiki/2020/6/65/T--SJTU-BioX-Shanghai--Result_temp9.png)

The results are shown below, from which we could see 38.5 times of fluorescence/OD600 difference between transform group (without the expression of dCas9) and co-transform group(with the expression of dCas9) in RFP and 3.96 times in eGFP, indicating our transcription inhibition system has better effect on target site than lure sites, which matches our prediction.

![](https://static.igem.org/mediawiki/2020/a/ab/T--SJTU-BioX-Shanghai--plot-2.png)

Raw data: [Transcription inhibition system verification(pIn-RTL3)](https://static.igem.org/mediawiki/2020/f/f2/T--SJTU-BioX-Shanghai--CRISPRi-pIn-RTL3.pdf)

## Targeting progression

We use our kinetic model to infer the difference between the off-target binding
process of SpCas9 and xCas9. The improvement of on-target rate by directed evolution is derived
from a more stable system after PAM recognition. Meanwhile, the energy penalty for incorrect
matching obviously increases due to the variation of the interaction between DNA and Cas9-sgRNA
complex. The free energy of the entire system is generally in a higher state during the
targeting progression so that Cas9 is less likely to cleave the off-target site. This kinetics
interpretation for xCas9 proves that directed evolution method can help reduce the off-target
effects.

![](https://static.igem.org/mediawiki/2020/9/90/T--SJTU-BioX-Shanghai--targeting_progression.png)

## Mutagenesis library construction

The dCas9 mutant libraries are constructed using error-prone PCR.

To restrict the mutagenesis region to dCas9, we first amplified the dCas9 fragment
and pdCas9 backbone through high-fidelity PCR. Homologous arms for Gibson assembly were added to
both dCas9 and the backbone during PCR process. Then this dCas9 fragment was used as a template
for error-prone PCR using error-prone PCR kit to obtain dCas9 randomly mutagenized library.

Then the randomly mutagenized library and the backbone are Gibson assembled. And
theassembled library was transformed into DH5α for later plasimid library extraction, which was
then transformed into MG1655 carrying pIN-RTr to verify the dCas9 mutant
library.

According to our design, we do not need to sequence the plasmids immediately after acquiring the mutant library, but only to verify the success of our mutant library by flow cytometry analysis.

As is shown in the figure below, after mutating dCas9, we can see a significant change in its inhibition effect of gene circuits. Before mutation, the bacteria which was transformed initial pdCas9 plasmid showed almost no red fluorescent characterization. But after the mutation, red fluorescence characterization is very obvious, which means dCas9 with inhibiting effect of circuit has a significant change, and the dCas9 proteins are improved a lot. This also illustrates that the error - prone PCR is effective, and the our mutant library is built successfully.

![](https://static.igem.org/mediawiki/2020/e/e2/T--SJTU-BioX-Shanghai--flow_2.png)

## Mutagenesis library screening

To select dCas9 mutants with higher on-target level and lower off-target level, the obtained dCas9 mutant library and the wild-type dCas9 were transformed by electroporation into MG1655 carrying pIN-RTL3, respectively.

We used flow cytometer to examine fluorescence intensity. L-arabinose was added to induce the expression of RFP and eGFP .

The results are shown below, from which we can see few of the proteins were optimal before mutation (i.e. on-target and not off-target), but the proportion of target proteins in the dCas9 protein library after mutation increased significantly, from 0.066% to 0.46%.
At the same time, the proportion of proteins in the mutant dCas9 library that were neither on target nor off target also increased significantly, which may be caused by the inactivation of some mutant proteins.
So this suggests that we have constructed a relatively ideal mutant library. And there were both positive and negative mutants, which also indicated that our previous work was effective and the design was reliable.
Due to the limited time, the subsequent rounds of sorting have not been done yet, and we will continue to do it..

![](https://static.igem.org/mediawiki/2020/0/09/T--SJTU-BioX-Shanghai--flow_3.png)

[![](https://static.igem.org/mediawiki/2020/c/c4/T--SJTU-BioX-Shanghai--totop3.png)](#section0)