iGEM 2023 — Team whu-china

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Notebook

Notebook

Notebook

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

- [Timeline](#Timeline)

- [Experimental Records](#Experimental Records)

- [Notice](#Notice)

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To top](#first-paragraph)

WHU-China pays attention to recording the timeline of all year's work and keeping experimental records. On the
one hand, we believe that our notebook could be instructive to future team members in WHU-China and even others.
On the other hand, we take academic ethics seriously. All the row data of experimental results in "Part",
"Engineering", and "Results" can be found in the notebook. We welcome scrutiny and oversight from external
parties.

### Timeline

[Timeline(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/timeline.pdf)

### Experimental Records

[A-Genome Editing(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/a-genome-editing.pdf)

[B-Recording in-trans(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/b-recording-in-trans.pdf)

[C-Directed Evolution(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/c-directed-evolution.pdf)

[D-sgRNA Screening(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/d-sgrna-screening.pdf)

[E-pCas Optimization(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/e-pcas-optimization.pdf)

[F-Recording in-cis(PDF)](https://static.igem.wiki/teams/4630/wiki/notebook/f-recording-in-cis.pdf)

### Notice

The timeline is exported directly from Onenote. It only has a Chinese version; The experimental records are a
combination of direct exports from OneNote and manual entry into electronic copies. Part of them are
translated with the assistance of chatGPT, which may result in occasional inaccuracies. If you have any
questions or require clarification, feel free to contact us at 2021302041074@whu.edu.cn.

{% endblock %}

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Engineering

Engineering

Engineering

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

- [Part 1: Cascade recording system](#Cascade-recording-system)

- [Cycle1: CRISPReporter over plasmids in-trans](#Cycle1-CRISPReporter-over-plasmids-in-trans)

- [Cycle2: Attempt to the cassette recorder](#Cycle2-Attempt-to-the-cassette-recorder)

- [Cycle3: Integrated device test](#Cycle3-Integrated-device-test)

- [Cycle4: Promoter substitution](#Cycle4-Promoter-substitution)

- [Part 2: EL222 directed evolution](#EL222-directed-evolution)

- [Cycle1: Characterization of EL222 with eGFP Reporter](#Cycle1-Characterization-of-EL222-with-eGFP-Reporter)

- [Cycle2: Characterization of EL222 with mRFP Reporter](#Cycle2-Characterization-of-EL222-with-mRFP-Reporter)

- [Cycle3: Modification of the EL222](#Cycle3-Modification-of-the-EL222)

- [Cycle4: Directed evolution of EL222](#Cycle4-Directed-evolution-of-EL222)

- [Part 3: sgRNA screening](#sgRNA-screening)

- [Cycle1: Blue-White Plaque Screening](#Cycle1-Blue-White-Plaque-Screening)

- [Cycle2: Green fluorescence Screening](#Cycle2-Green-fluorescence-Screening)

- [Part 4 : pCas optimization](#pCas-optimization)

- [Cycle1: Characterization of pCas](#Cycle1-Characterization-of-pCas)

- [Cycle2: Typical Site directed Ligase Independent Mutagenesis (SLIM)](#Cycle2-Typical-Site-directed-Ligase-Independent-Mutagenesis-SLIM)

- [Cycle3: Three segment assembly](#Cycle3-Three-segment-assembly)

- [Cycle4: Simple removal of sgRNA](#Cycle4-Simple-removal-of-sgRNA)

- [Cycle5: Integrated recording machine](#Cycle5-Integrated-recording-machine)

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To top](#first-paragraph)

On this page, we present the detailed Design-Build-Test-Learn (DBTL) cycles for our engineering efforts,
categorized into four distinct parts.

[### Part 1: Cascade recording system

We executed four cycles to achieve multi-level editing in this part. In the first cycle, we built the
CRISPReporter over plasmids in-trans. However, recognizing the limitations of this approach after facing
construction setbacks, we transitioned to building a cassette recorder over plasmids in-cis in the second
cycle, where we achieved preliminary engineering success. In the third cycle, for better engineering, we
conducted integrated device tests and generated a considerable volume of measurement data. Finally, the
fourth cycle witnessed the realization of promoter substitution as an initial proof-of-concept for
applications.](#Cascade-recording-system)

[### Part 2: EL222 directed evolution

To realize directed evolution of the EL222 promoter, we underwent four cycles. In the first cycle, we
adopted EGFP as the marker of the evolution system. However, the fluorescence quenching effect impeded
proper system functionality. We replaced EGFP with mRFP in the second cycle and then modified it in the
third cycle, achieving engineering success in constructing the evolution system. Finally, in the fourth
cycle, we performed directed evolution on pEL222 and collected measurement data as we planned.](#EL222-directed-evolution)

[### Part 3: sgRNA screening

We executed two cycles to establish the sgRNA screening system. In the first cycle, we employed LacZ as
the marker for successful editing. However, though successful editing occurred, it cannot be identified by
the LacZ marker. Subsequently, in the second cycle, we replaced LacZ with EGFP. However, due to time
constraints, this work was not completed prior to the WiKi freeze day.](#sgRNA-screening)

[### Part 4: pCas optimization

We went through five cycles to optimize the plasmid pCas (pRed\_ cas9\_ △poxb300). The first cycle involved
characterization of the original plasmid, encompassing assessments of its editing efficiency and metabolic
burden on bacteria. Acknowledging various limitations of the plasmid identified in the first cycle, we
pursued optimization in the subsequent three cycles through diverse methods: Site-directed mutagenesis,
Ligase-Independent Mutagenesis (SLIM), Gibson Assembly of three fragments, and simple removal of sgRNA. In
the last cycle, we built an integrated recording machine which is more suitable for us to use.](#pCas-optimization)

## Cascade recording system

### Cycle 1: CRISPReporter over plasmids in-trans

#### Design

Our initial endeavour revolves around constructing a model aimed at reaching the fundamental objective of
"recording in response to a stimulus" distributed across multiple plasmids (Fig 1a). In line with our design,
the "knock-out" event has dual functions: recording this event and triggering the subsequent event (Fig
1b).
In detail, every plasmid is categorized as a "level", corresponding to the sequence of its response to
the stimulus. The knock-out of a specific level's target accomplishes two things: it leaves a mark of the
stimulation, and the product itself becomes the sgRNA for the next level. To make it easy to quantify, we
incorporated a rationally designed qPCR sequence into the region to be deleted (Fig 2).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/cascade-model-2.svg)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig2.png)

#### Build

To validate the first-level knockout, we designed a system consisting of three plasmids: pCas, level 1, and
level 2.
The level 1 plasmid is constructed by inserting the fragments of homologous arm and N20 sequence
into the designed vector and the level 2 plasmid is constructed by inserting the qPCR fragment into the designed
vector. We outsourced the synthesis of these fragments to Atentares and Tsingke, with the addition of BsaI
recognition sites at specific locations. After receiving the synthesized sequences, we performed Golden Gate
Assembly to assemble these plasmids.

#### Test

During the initial attempt to construct level 1, we faced a challenge where the N20 sequence was absent in the
plasmid obtained through the assembly of three fragments using Golden Gate Assembly. Therefore, we improved the
N20 insertion method in the second round of experiments. We synthesized two complementary primers and annealed
them to form a double-strand N20 with sticky ends, and then inserted it into the vector. In the end, we
successfully constructed the level 1 plasmid.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig3.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig4.png)

Fig 4. The sequencing result of level1 plasmid construction

Sequences 1 to 8 represent the alignment results of the sequences obtained during plasmid construction
with the
final sequence of the expected level 1 plasmid. Plasmids 3, 4, 5, and 6 successfully inserted the homologous
arm
and N20 and are the target plasmids.

In the first round of level 2 construction, we attempted to assemble these fragments using Golden Gate
Assembly. Regrettably, we were unable to obtain the recombined plasmids, possibly due to the shared antibiotic
resistance between the vector plasmid and the donor plasmid.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig5.png)

Fig 5. The sequencing result of level2 plasmid construction for round 1

a. Sequences 1 to 7 represent the alignment results of the sequences obtained
during plasmid construction with the final sequence of the expected level 2 set 1 plasmid.

b. Sequences 1 to 5 represent the alignment results of the sequences obtained
during plasmid construction with the final sequence of the expected level 2 set 2 plasmid.

In the second round of experiments, we adopted enzymatic digestion to separately cleave the inserted fragments
and the vector plasmids. After purifying the fragments through gel extraction, we performed ligation. However,
the concentration of the gel-purified target fragments was relatively low. Sequencing results indicated that
successful fragment connection had not yet been achieved.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig6.png)

Fig 6. The sequencing result of level2 plasmid construction for round 2

Sequences 1 to 10 represent the alignment results of the sequences obtained during plasmid construction
with the final sequence of the expected level 2 set 2 plasmid.

#### Learn

* In light of plasmid incompatibility and metabolic stress, the recording capacity is significantly constrained
  by the number of plasmids. To attain a higher degree of recording, it is imperative to curtail both sequence
  length and the quantity of plasmids employed.
* The vector should undergo optimization for greater compatibility.

### Cycle 2: Attempt to the cassette recorder

#### Design-CRISPReporter over plasmids in-cis

Inspired by the work of Choi et al. (Fig 7, Choi, J., Chen, W., Minkina, A. et al., Nature 608, 98-107 2022),
we attempt to set CRISPReporter as a cassette recorder *in cis*. We attempted to assemble all the
components into one sequence. In the frame of Cas9, we found that D. Perli et al.'s and Zhao, D.'s methods can
respectively achieve self-targeting and self-recombination, as we have mentioned in the [design](design#cascade).

To validate the proof-of-concept, we made two sets of in-cis designs considering the self-recombination
efficiency. In the first set, each cassette has a unique barcode to ensure proper recombination (Fig 8a). In the
second set, the cassettes share a common segment of promoter, eliminating the need for barcode accumulation and
resulting in a more compacted and extendable device (Fig 8b,c). As the previous cassettes block the expression
of later ones, the cassettes are expected to operate sequentially (Fig 8d).
As for experimental design, we intend to test the single cassette ones, to get initial data on the mechanism.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig7.png)

Fig 7. The cassette recorder *in cis*, from the literature (Choi, J., Chen,
W., Minkina, A. et al., Nature 608, 98–107 2022)

The recorder is constructed over prime editing technology. The editing event happens over an elaborate
targeted sequence. Without the preceding 5bp insertion of the 2bp barcode and 3bp key, the subsequent cassette
cannot be targeted by the prime editing guide RNA.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig8.png)

D

Stage 1: Expression of Cas9 and LambdaRed

Stage 2: 1st signal input

Stage 3: Transcription and self-targeting of stgRNA 1

Stage 4: Reparation of homologous arm using LambdaRed system

Stage 2: 2nd signal input

Stage 3: Transcription and self-targeting of stgRNA 2

Stage 4: Reparation of homologous arm using LambdaRed system

In theory, this loop can run infinitely.

Fig 8. The cassette recorder *in cis*, in the frame of Cas9 and homologous
recombination

a. The recorder design with a barcode (stgRNA-barcode-assette 1, BBa\_4630100). Once
stimulated, the barcode will function as the homologous region.

b. The recorder design without a barcode (stgRNA-cassette 1, BBa\_4630110), demonstrating
a single cassette.

c. The recorder-set design without a barcode. The homologous arm of the later
cassettes shares homology with part of the promoter.

d. The operational pattern of the cassette recorder *in cis*.

#### Build

We synthesized the required DNA fragments in collaboration with Atantares and Tsingke, two reliable biotech
companies. Then we successfully co-transformed our working plasmid with pCas, the vector for Cas9 and Lambda
Red, into *Escherichia coli* DH5α (Fig 9).

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-addition/fig9.png)

Fig 9. Construction of the working strain

a. The parts were cloned onto the pDCF-Duet1 plasmid.

b. Colony PCR results of the recorder with barcode. The white hollow arrowheads
indicate the target bands. Index 5, 6, 7, 8, 9, 10, 11, 12 were successfully co-transformed.

c. Colony PCR results of the recorder without barcode. There was a shallow band
in negative control(ddH2O), which differed from the sample ones.

#### Test for the "stgRNA-barcode-cassette 1"

After amplification culture, the bacteria solution was diluted into 3ml of LB media with appropriate
antibiotics and cultivated to reach an OD600 of 0.3~0.5. We added a variable dosage of arabinose and IPTG close
to the working concentration. After cultivating 22hrs for arabinose and 5hrs for IPTG induction, we diluted the
media and spread plate to halt induction and get single colonies (Tbl 1). Then, the relevant sequence was
amplified and sequenced (Fig 10a), confirming a successful knock-out. Large-scale screening revealed that group
2 exhibited the highest efficiency, with efficiencies of 70%, 10%, and 38.1% for groups 2, 3, and 4,
respectively (Fig 10b). Non-induction controls substantiated that induction is the prerequisite for recording
(Fig 10c).

Tbl 1. The random test over inducers

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table1.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig10.png)

Fig 10. The induction readout over the recorder with barcode.

a. Sequencing result of the picked colonies in the conditions. "R" represents
"parallel repeat". Conditions 2, 3, and 4 exhibited positive knock-out signals.

b. After induction, the target sequence is supposed to be truncated. The yellow
reference line indicates the knock-out sample.

c. Sequencing results of the non-induction controls. All of them remained intact.
N = 24.

#### Learn from the stgRNA-cassette-barcode 1

The results demonstrated that the cassette, our building block of the recorder, can function as expected under
induction. Furthermore, a relatively suitable induction condition for proof-of-concept is 2g/L for Arabinose and
3g/L for IPTG, for 22hrs and 5hrs, respectively. However, the condition data should be measured in detail for
further multi-level induction, and the knock-out events should be supported by quantifiable data.

#### Test for the "stgRNA-cassette 1"

Using the same procedure but a different induction condition (Tbl 2), we also tested the function of a single
cassette. To our disappointment, the target sequences remained all intact (Fig 11).

Tbl 2. Induction conditions in the test for stgRNA-cassette 1

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/engineering2.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig11.png)

Fig 11. Sequencing results of stgRNA-cassette 1.

All of the sequences were aligned. Given the diverse specificity-determining sequences (SDS) used in
stgRNAs, occasional gaps were observed.

#### Learn from the stgRNA-cassette 1

Comparing the two cassette designs, the homologous arm was set downstream of each cassette in
"stgRNA-cassette-barcode" ones, while the upstream of the next cassette in "stgRNA-cassette" ones (Fig 4a, b).
As a result, the single cassette we have tested has no inherent homologous arm. However, the integration of
another cassette can solve the problem. Based on the insights gained from previous experiments, we designed to
insert an additional cassette downstream of the two devices, and then apply the appropriate induction condition
to the stgRNA-cassette (1+2+3) (BBa\_4630114) (Fig 4c) and stgRNA-cassette-barcode (1+2) (BBa\_4630102).

### Cycle 3: Integrated device test

#### Design

In line with the previous experiment, we'd like to first test the inherent capability of the recorder without
barcode, and then test the cascade knock-out event of both. We set special restriction sites flanking the
cassette to make it extendable. Using the principle of BioBrick Assembly (3A Assembly) , we
introduced an XbaI site upstream of the segment to be inserted, and SpeI & EcoRI downstream of each cassette. By
cutting the backbone with SpeI and EcoRI and cutting the segment with XbaI and EcoRI, the new cassette could be
inserted downstream of the previous one(s). Besides, the remaining restriction sites make the system fully
extendable to add additional cassettes.

#### Build

The subcloning of the recorder without a barcode, stgRNA-cassette (1+2), stgRNA-cassette (1+2+3) was executed by Atantares. Meanwhile, we carried out the subcloning of the stgRNA-barcode-cassette (0+2) and stgRNA-barcode-cassette (1+2) by ourselves (Fig 12). The stgRNA-barcode-cassette (0+2) is the ligated product of the knock-out product of
stgRNA-barcode-cassette 1 and the stgRNA-barcode-cassette 2, which can be used in verification of the second editing.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig12.png)

Fig 12. The Construction of the stgRNA-cassette (1+2)

a. The electrophoresis of the ligation product. The white hollow arrowhead indicates the target band.

b. The sequencing result of the transformants.

#### Test-Attempt

We organized our experiment into groups. Employing the same induction condition (2g/L L-Arabinose and 3g/L
IPTG, for 22hrs and 5hrs respectively) with the same tester, stgRNA-cassette (1+2), stgRNA-cassette (1+2+3) have
been successfully knocked out while stgRNA-cassette 1 was intact (Fig 13). It is noted that all the parts remain
one last cassette intact, indicating the significant role played by the inherent homologous arm in the
recombination, and confirming the occurrence of multi-level knockout. Also, the induction carried out on
stgRNA-cassette-barcode (1+2) got the expected outcome (Fig 14).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig13.png)

Fig 13. Readout of the multi-level cassette recorder

a. Sequencing result of stgRNA-cassette 1. The cassette remained intact.

b. Sequencing result of stgRNA-cassette (1+2). Cassette 2 remained intact.

c. Sequencing result of stgRNA-cassette (1+2+3). Cassette 3 remained intact.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig14.png)

Fig 14. The knock-out result of the stgRNA-barcode-cassette (1+2)

Most of the samples achieved recording. Some of the sample is knocked out twice.

#### Learn

From the results, we conclude with greater confidence that the incapability of stgRNA-cassette 1 is due to the
lack of a homologous arm. Also, all the other multi-level ones were knocked out to their fullest, showing no
tiered recording results. We assume that the induction time is so long that permits the cassettes to be knocked
out twice. As a result, we intend to make a comprehensive measurement of the building block and to carry out a
time-dependency experiment of the stgRNA-cassette (1+2+3).

#### Test-Large scale

The solution to the double knock-out issue lies in better control of the induction conditions. That is, to gain
more information about the induction strength, and determine the refractory phase of the part. Maintaining other
variables at typical levels (3g/L L-Arabinose, 2g/L IPTG, inducing 22hrs and 5hrs respectively), we carried out
a concentration matrix test (Tbl 3) and two time-gradient tests.

Tbl 3. Group design for the concentration matrix

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table2-1.png)

The test was carried out on stgRNA-barcode-cassette 1.

#### Concentration Matrix

It's noted that the induction of arabinose inhibited the growth of bacteria strikingly (Fig 15), and the Lac promoter exhibited significant leakage expression (Tbl 5, Fig 16b). Despite the missing data due to failure of sequencing, E6, E5, C2, B5 performed better (Tbl 4, Fig 16a, Tbl 6). However, a quality test based on electrophoresis provided parallel data for randomly picked groups, and the two data access showed a significant correlation, with paired t-test P = 0.7602, no significant difference (Tbl 7, Tbl 8, Fig 17). Given the substantially larger amount of data from the electrophoresis test for E6 (N = 20) compared to sequencing (N = 4), we adjusted the knock-out ratio of E6 to 60% (fig S2).
Surprisingly, we notice that at a very low rate, recombination might happen between the two plasmid-inherent Lac promoter sequences, instead of the set barcode sequence. In the first matrix, the overall unexpected knock-out ratio is 2.76%, significantly lower than the normal rate of 42.5% (Tbl 9, Fig 16b).
Further, to ensure data reliability, we tried the matrix test again. Growth inhibition and leakage expression still occur (Fig 18 a, b). Also, an integrated heatmap is plotted based on the mean value of the two matrices (Fig 18 c), and the variation pattern of the two matrices showed some kind of correlation. Paired t test result of the two matrices showed a significant difference, with P = 0.0054(\*\*) and mean of differences = -0.2071(Fig 18 d). Two-way ANOVA of the second matrix and the average matrix indicate that the IPTG concentration is the main variation factor (Tbl 10).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig15-2.png)

Fig 15. Streaking plate of Group 1, after induction

a. Bacteria without induction. The bacteria had grown to Area 3.

b-d. Bacteria under 1, 4, 8 g/L L-Arabinose induction, respectively. The growth
of the bacteria was limited to Areas 1 and 2. The bacteria in C1 had been contaminated.

e. Assessment of the bacteria amount. The introduction of L-Arabinose influences
the bacteria amount strikingly.

Tbl 4. The total sample amount of each condition

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table3.png)

Tbl 5. Results of the no-IPTG-induction group

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table5.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig16.png)

Fig 16. Results of the concentration matrix

a. Heatmap of the concentration matrix based on sequencing data. The black block
indicates no sequencing data is available. The L-Arabinose 0g/L row and IPTG 0 g/L column indicate the leakage
expression of pBAD is quite low while that of pLac is quite high.

b. Comparison in knock-out ratio of IPTG 0 g/L column and the average of total.
Though there is a slight increase along with the L- Arabinose concentration, the presence of L- Arabinose
predominates, implying a high rate of pLac leakage.

c. Sequencing result of E6-1, E6-2, and E6-3 showed normal recombination while
E6-1 showed unexpected recombination. The unexpected one is because of the recombination of the two Lac
operators flanking the knock-out target site. The actual homologous arm in the situation is 21bp. However, the
pT7-Lac promoter set flanking the DSB is 44bp.

Tbl 6. The groups with high knock-out rate

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table4.png)

Tbl 7. The paired data of the electrophoresis and sequencing

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table6.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig17.png)

Fig 17. The relation between data from sequencing and electrophoresis

a. The correlation relationship of the two data. Pearson r = 0.9837, R squared =
0.9678, P = 0.0025(\*\*).

b. The normality test of the two data. Under Shapiro-Wilk test (N = 5), the P
values for sequencing and electrophoresis are 0.9500 and 0.9364, respectively.

Tbl 8. The correlation of the electrophoresis and sequencing result

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table7.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig18.png)

Fig 18. Results of the second matrix

a. Scratch plate result of the L-Arabinose / IPTG non-induction group. As the previous data, the non- L-Arabinose group grew much better than the L-Arabinose induction ones. Also, the IPTG slightly inhibits the growth of the bacteria.

b. The heatmap plotted from the second concentration matrix. The total depth of color is lower than the first matrix.

c. The heatmap plotted from the average data of the two concentration matrices. When there is no data, data from the other compensates.

d. The demonstration of the knock-out ratio. The two matrices showed a high correlation.

e. Paired t test result of the two matrices. There is a significant difference, with P = 0.0054(\*\*) and mean of differences = -0.2071.

Tbl 9. Statistical data of the unexpected recombination

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table11.png)

Tbl 10. Two-way ANOVA result of the matrices

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table8.png)

The row factor is IPTG concentration and column factor L-Arabinose in the matrix.

#### Learn from the concentration matrix

From the data we conclude that the preferred induction concentrations are 8g/L L-Arabinose and 3g/L IPTG. The unexpected recombination proves the concept that DSB is the prerequisite of the recombination, and the recombination is preferred to happen between two near homologous arms. The data from electrophoresis and sequencing can compensate for each other.
The two parallel concentration matrix tests uncover that there is a huge variation in the measurement, and more experimental data are needed. Moreover, the variation pattern of the samples showed similarity, indicating probability of logical regression. And, the statistical analysis proves that IPTG concentration is the leading factor controlling the result, fulfilling part of our proof-of-concept.
However, the leakage of pLac makes it difficult to measure the multi-level recording. We'd like to change it to other promoters- check the next cycle, the EL222 part, and the application part.

#### First time-gradient test

We did the two tests subsequently. The first test of knock-out showed a bell-type-like shape (Tbl 11, Fig 19a).
The increasing trend ends on dot 4h and the extension of IPTG induction time does not help the editing
afterwards. Consequently, we set up another time-gradient within 2 hours on stgRNA-cassette (1+2+3), and all
of them achieve full knock-out, at a ratio of 100% (Tbl 12, Fig 19b).

Tbl 11. Group design for the first time-gradient test

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table9.png)

The test was carried out on stgRNA-barcode-cassette 1.

Tbl 12. Group design for the second time-gradient test

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table10.png)

The test was carried out on stgRNA-cassette (1+2+3).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig19.png)

Fig 19. The knock-out ratio plot of the time-gradient tests

a. Result of the first time-gradient test.

b. Result of the second time-gradient test.

#### Learn from the time-gradient tests

In line with our PI's advice, long-term induction of IPTG proved harmful to the cells. However, the
time-gradient within 2 hours did not really set up because of inconsistency with the first time-gradient and the
small data amount of both. Not surprisingly, the test carried out on stgRNA-cassette (1+2+3) showed some kind of
leakage expression. It's necessary for us to substitute the Lac promoter with a more reliable one.

### Cycle 4: Promoter Substitution

#### Design

We designed many promoter substitution plans (See also [Applications](applications)). Here, we demonstrate the substitution based on the plasmid backbone. There is a T7 promoter upstream of the constructed cassettes, so by simply omitting the Lac promoter and relevant sequences, we can let the T7 promoter take control (Fig 20).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig20.png)

Fig 20. Design of the substitution of Lac promoter to T7 promoter.

The red box indicates the sequence to be omitted, and the two purple primers are used for backbone amplification for further Golden Gate Assembly.

#### Build

We successfully amplified the sequence using stgRNA-cassette (1+2+3) and underwent Golden Gate Assembly. However, due to the length preference in PCR, we only got one cassette (Fig 21). And due to time constraints, we haven't finished the co-transformation and further induction test.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/fig21.png)

Fig 21. The construction of T7-cassette

a. Three pairs of PCR primers for Golden Gate Assembly and Gibson Assembly were designed. Only the Fgolden ones were successfully amplified.

b. Sequencing results of the constructed plasmid. The target site was omitted as expected.

c. The final sequence of the cassette part. The cassette is under the control of T7 promoter.

## EL222 directed evolution

### Cycle1: Characterization of EL222 with eGFP Reporter

#### Design

In the initial phase, our objective was to evaluate the function of EL222 as a blue-light-inducible promoter. To assess this, the eGFP gene was employed as a reporter due to its widespread use as a fluorescent marker. In the design, pEL222 and EL222 were inserted upstream of the eGFP gene (Fig 1). The expression of EL222 was regulated by a constitutive promoter.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig1.svg)

Fig 1. Map of pET-28a-[EL222-eGFP]

EL222, pEL222, and eGPF were inserted into backbone pET-28a (Kan+). The expression of EL222 was regulated by a constitutive promoter.

#### Build

We synthesized and performed subcloning of EL222 and pEL222 utilizing restriction cloning into pET-28a-[eGFP]. Then the plasmid was transformed into *E. coli* DH5α for amplification and BL21(DE3) for testing.
Colony PCR was performed to make sure the plasmid was constructed just like we designed (Fig 2).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig2.png)

Fig 2. The product of Colony PCR is about 600bp.

#### Test

Bacteria were cultured, and exposed to 400 Lux blue light around 475nm for 20 hours (Fig 3). Then the dishes were observed under UV light to confirm the expression of eGFP proteins. Regrettably, no discernible green fluorescence was observed

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig3.png)

Fig 3. Colonies with plasmid pET-28a-[EL222-eGFP] on the plates were induced by blue light around 485nm (controlled by Arduinor mega).

#### Learn

A comparative analysis revealed that the excitation wavelength of eGFP is approximately 488nm, closely aligned with the blue light employed in our study. The absence of observable green fluorescence was attributed to a quenching effect induced by the blue light.

### Cycle2: Characterization of EL222 with mRFP Reporter

#### Design

In the second iteration, we replaced eGFP with mRFP1 on the pET-28a-[EL222-eGFP] plasmid (Fig 4) to mitigate the fluorescence quenching effect.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig4.svg)

Fig 4. Map of pET-28a-[el222-mRFP]

EL222, pEL222, and mRFP were inserted into backbone pET-28a by Golden Gate Assembly.

#### Build

mRFP1 sequence was obtained via PCR and subsequently cloned into the pET-28a-[EL222-eGFP] vector utilizing Golden Gate assembly, to replace the eGFP sequence. Then the plasmid was transformed into *E. coli* DH5α for amplification and BL21(DE3) for testing.
Sequencing was performed to make sure the plasmid was constructed just like we designed (Fig 5).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig5.svg)

Fig 5. Sequencing results of EL222-mRFP indicate that EL222 factor, pEL222, and mRFP1 were inserted successfully.

#### Test

Bacteria was spread on plates and first cultured under 37℃ for 10 hours, then exposed to 400 Lux blue light around 475nm for 20 hours. We then performed observation on dishes and slides we made from colonies under UV light, to confirm the expression of mRFP1 proteins. There was no visible red fluorescence observed from our dishes, while it can be observed from the slides we made (Fig 6). However, there was no difference between the samples before and after exposure to blue light (Fig 6a,b).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig6a.png)
![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig6b.png)

Fig 6.Results of blue-light-induced mRFP expression

a. After 20 hours of induction by 400 Lux blue light around 475nm, mRFP1 was activated by green light, confirming the expression of mRFP1.

b. Without induction, the negative control also showed red fluorescence activated by green light. There was no difference between the blue-light-induced group and the negative control group, indicating the expression leakage of mRFP1.

c. All the colonies are the same color after induction.

#### Learn

Examination of the EL222 protein sequence unveiled a gap of 16 amino acids at the N-terminus of EL222, along with a missing initiation codon (Fig 7). This gap was found to be the cause of the mRFP1 leakage.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig7.jpg)

Fig 7.Comparison between different EL222 factors.

There was a gap of 16 amino acids, causing the loose efficacy of the EL222 factor, therefore leading to the leakage of mRFP1 expression.

### Cycle3: Modification of the EL222

#### Design

Upon identifying the differences in EL222 proteins, we opted to modify the EL222 sequence through PCR by adding the missing sequence (Fig 8a). Furthermore, a newly synthesized EL222 sequence was introduced, and a T7 terminator was placed downstream of EL222 (Fig 8b).

![缺个图](https://gitlab.igem.org/2023/whu-china/-/raw/main/static/thaw-changes/fig8a.svg?ref_type=heads)
![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig8b.svg)

Fig 8. Map of the plasmid containing modified EL222

a. The lacking sequence is added using PCR.

b. The EL222 factor is replaced by newly synthesized EL222, BBa\_K2332004.

#### Build

The new EL222 sequence and the modified one are obtained via PCR (Fig 9) and subsequently cloned into the pET-28a-[EL222-eGFP] vector utilizing Golden Gate Assembly to replace the former eGFP sequence. Then the plasmid was transformed into *E. coli* DH5α for amplification and BL21(DE3) for testing.
Sequencing was performed to make sure the plasmid was constructed just like we designed (Fig 9).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig9.png)

Fig 9. EL222 factor was modified using two pairs of primers and the missing sequence was added.

#### Test

We conducted the induction experiment and observation with the same protocol as in cycle 2. There is no significant difference before and after exposure of the EL222-modified transformant, while the pET-28a- [EL222 new-mRFP1] transformant exhibits stronger red fluorescence compared to the negative control (Fig 10).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig10.png)

Fig 10.  Image results of induced mRFP expression in pET-28a- [EL222 new-mRFP1] transformant.

#### Learn

EL222-modified cannot work either, but EL222-new can work as expected, which may be attributed to the codon optimization of EL222-new. However, the leaky expression of pEL222 is still considerable.

### Cycle4: Directed evolution of EL222

#### Design

We decided to first perform directed evolution on EL222 to reduce leakage and increase downstream expression. All the new sequences of pEL222 will be tested on the former plasmid by measuring the fluorescence intensity of the colony before and after induction.

#### Build

We acquired different sequences of pEL222 by error-prone PCR. Then, the generated sequences were incorporated into pET-28a-[EL222-mRFP1] by Golden Gate Assembly, replacing the original sequence of pEL222. The plasmids were transformed in BL21(DE3) directly.

#### Test

Firstly, we sequenced the PCR products (Fig 12) to confirm the success of error-prone PCR. Then we use colony PCR to confirm the changes in pEL222 cloned in pET-28a plasmids. To measure the expression of mRFP1, we cultured all the bacteria in PCR tubes and measured their absorbance at 580nm and 600nm, which present for mRFP1 and bacteria concentration respectively.
The OD580/OD600 at a specific time for a sample culture was determined after subtracting from each of the technical triplicate readings of the negative control cultures (fluorescence free) at the same time. The fluorescence synthesis rate of any sample at time *t*, was calculated by taking the difference of Fluo/OD600 values from two time points and dividing the result by the time interval.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig11a.png)
![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/el/fig11b.png)

Fig 11. Sequencing results of PCR products

Colonies with different pEL222 show differencet expression of mRFP1, resulting in disparate colors. IPTG is added to ensure the expression of EL222 protein.

#### Learn

The sequence of pEL222 is related to mRFP1 expression. Using different pEL222, we can change the rate of expression.

## sgRNA screening

### Cycle1: Blue-White Plaque Screening

#### Design

In the initial phase of our study, we aimed to employ the blue-white plaque screening technique to selectively identify sgRNA sequences exhibiting high editing efficiency. To accomplish this, we developed a screening plasmid designated as pUC57-N20s-gRNA+HA (Fig 1a).
This plasmid encompasses a sgRNA scaffold, and a sgRNA targeting sequence (named N20s), which consists of 50 individual N20 sequences concatenated, each accompanied by its respective PAM sequence. This N20s sequence is inserted into the LacZ gene, unbaling to synthesize the functional β -galactosidase. The scaffold can load a specific N20 sequence (20nts specificity-determining sequence) by Golden Gate Assembly and conduct targeting fragment knockout (Fig 1b), thereby driving correct LacZ expression when induced. Functional β- Galactosidase can decompose X-gal to produce blue products, making colonies appear blue. While the colonies that didn't correctly knock out the N20s sequence were white.
We randomly loaded 50 different N20 sequences onto the plasmid and identified efficient ones by sequencing blue colonies. When the colony count is sufficiently high, our method will examine all N20 sequences with near certainty.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig1a.svg)
![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig1b.svg)

Fig 1. The design of the screening system

a. Map of pUC57-N20s-gRNA+HA

b. Map of the N20 sequence loading site

#### Build

The plasmid synthesis was executed in collaboration with Atantares. We also synthesized various N20 sequences via primer annealing (Fig 2a) and subsequently inserted each of them into the plasmid employing Golden Gate Assembly (Fig 2b). The assembled products are first transformed into *E. coli* DH5α for amplification, then co-transformed with pCas (pRed\_cas9\_△poxb300) (Fig 2c). Of note, these plasmids with different N20 sequences are co-transformed individually but not together in one tube, as we want to first determine the feasibility of the system.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig2.png)

Fig 2. Loading of N20 sequence

a. Annealing Product
For each N20 sequence, we synthesized a pair of partially complementary 24-base pair primers. These primers were further processed through annealing to yield 20-base pair N20 sequences, with 4-base pair sticky ends on both sides.

b. Golden Gate Product
Employing Golden Gate Assembly, each plasmid was endowed with a distinct N20 sequence. The assembly process was successfully executed in conformity with our design.

#### Test

Following a 24-hour induction with 2g/L arabinose, we determined the edited colonies via colony PCR. Our analyses revealed successful N20s sequence deletions for some of the tested N20 sequences, exemplified by NO. 11, 13, 14, and 15 (Fig 3), demonstrating the system's feasibility.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig3.png)

Fig 3. Knockout of N20s sequence on plasmids

Knockout of N20s sequence causes an 1150bp deletion on the plasmid, which causes the band to move down. We tested four N20 sequences. All the single colony we picked from each plate successfully executed knockout. Regrettably, the assessment of editing ratios remained elusive due to an insufficient colony count.

Sequence alignment inspections corroborated the integrity of the LacZ coding sequence in N20s-knocked-out plasmids, devoid of any mutations. However, despite employing various IPTG induction methods, these successfully edited colonies did not exhibit a blue phenotype in the substrate-supplemented medium.

#### Learn

* Golden Gate Assembly succeeds easily when the experiment is conducted with the correct protocol.
* Knockout of fragments on plasmids is feasible.
* After consultation with professors, we understood the intricate nature of the blue-white plaque screening technique's challenges. Consequently, we received advice to consider alternative markers for N20s knockout assessments.

### Cycle2: Green fluorescence Screening

#### Design

In light of the failure of blue-white plaque screening, we adopted EGFP as the reporter of successful editing in the second DBTL cycle. EGFP is also a kind of widely used fluorescent protein in *E. coli*. We positioned it under the control of a constitutive promoter to save from the trouble of IPTG induction. We designed to insert two fragments into the pUC57 vector, HA+ gRNA & EGFP-N20s, to construct the plasmid pUC57-EGFP-N20s-HA+gRNA (Fig 4). If the N20s sequence is knocked out, EGFP would be expressed properly, resulting in colonies manifesting a green phenotype.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig4.svg)

Fig 4. Map of pUC57-EGFP-N20s-HA+gRNA

#### Build

We employed Gibson assembly to insert HA+ gRNA (Fig 5) into the vector and Restriction cloning to insert EGFP-N20s (Fig 6). The construction of pUC57-EGFP-N20s-HA+gRNA went successfully as we designed.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig5-3.png)

Fig 5. Construction of pUC57-HA+gRNA by Gibson Assembly

a. Preparation of fragments. We obtained the HA fragment (100bp) from pET-28-EGFP and the gRNA fragment (337bp) from pUC57-N20s-gRNA+HA by PCR.

b. Verification of Gibson Assembly product. The colony PCR product of assembled plasmids is expected to be 452bp.

c. Comparison between assembly product and vector. The assembled plasmid is 452bp longer than the vector.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig6a-b-2.png)
![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig6c.svg)

Fig 6. Construction of pUC57-EGFP-N20s-HA+gRNA by Restriction Cloning

a. Preparation of EGFP-N20s by Gibson Assembly
We obtained the EGFP-L (596bp) & EGFP-R (343bp) fragment from pET-28-EGFP and the N20s fragment (1150bp) from pUC57-N20s-gRNA+HA. Then we connected the three fragments using Gibson Assembly.

b. Verification of Restriction cloning product by Colony PCR

c. Verification of Restriction cloning product by Sequencing

After successful construction, we loaded those N20 sequences described in the first cycle onto the plasmid in one tube and performed co-transformation with pCas into *E. coli*.

#### Test

We have run through different culture procedures to identify the optimal screening protocol (Chart 1). However, none of them achieved N20s knockout in any colony. After an extensive investigation, it was found that the preserved plasmid had undergone fragment loss. We then asked Genescript for synthesis and subcloning of the plasmid. Unfortunately, when we received it, there was no time for further testing.

Tbl 1. Culture Procedures

![缺个表](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/cascade/table.png)

#### Learn

We surmise that the concentration of Golden Gate products is too low. To solve this problem, we cultured *E. coli* in liquid LB media to expand the clone population. However, it led to false positive results: colony PCR indicates that plasmids harbored by the bacteria were origin vectors rather than Golden Gate products. Consequently, the batch random experimentation method remains to be optimized.

## pCas optimization

### Cycle1: Characterization of pRed\_cas9\_△poxb300

#### Design

As the material we got, pCas (pRed-Cas9-△poxb300) in Zhao, et al. 's work (Fig 1a, Zhao et al. Microb Cell Fact 2016, 15:205) is capable of conducting genome deletion and will be cured when cultivated at 37°C or higher. We first tested its genome editing efficiency. When induced by arabinose, the plasmid can express Cas9, Lambda-Red, and a poxb-targeting sgRNA, causing a 513bp deletion of poxb in the genome.

#### Build

We chose *Escherichia coli* DH5α as the cloning host and MG1655 to conduct editing. After transformation, we performed induction following the method offered by Zhao et al..

#### Test

We used 2g/L arabinose to induce editing. After induction, the medium was diluted 2000-fold and spread on the plate. Then, using colony PCR, we identified the editing efficiency of different induction durations by calculating the ratio of the shortened PCR fragments (Fig 1b).

We first found that 6h induction is not sufficient for 100% editing. Therefore, we conducted a gradient experiment on the arabinose induction time to figure out the optimal duration of induced editing. As it is said that the induction should last at least 6h, gradient induction was set up from 6h to 30h. Although double bents always existed (Fig 1c), which means that bacteria are not completely edited in this colony, 24h was considered as the optimal induction time with minimal double bents and almost 100% editing efficiency (Fig 1d).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig1a.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig1b.png)
![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/fig1b-overlay-3.svg)

Fig 1. Characterization of pCas

a. pCas plasmid in the literature (Zhao et al. Microb Cell Fact 2016, 15:205)

b. poxb gene knockout with 513 bp deletion.

c. Electrophoresis result of genome editing

d. Results of time gradient experiment. The modified rate is calculated by weighting single band as 1 and double band as 0.5.

#### Learn

* In the experiment, 24h induction can lead to optimal editing efficiency.
* *E. coli* grows slower after being transformed with pCas
  + The culture temperature is limited to 30 °C.
  + Expression of Cas9 and Lambda-Red increases the metabolic burden of bacteria.
  + DSB in the Genome may cause death to bacteria.

Considering these limitations of pCas, we intend to optimize it from the following aspects:

* Removal of the genome-targeting guide RNA and the homologous arm for poxb
* Removal of the temperature-sensitive site and the addition of restriction sites for Golden Gate assembly.

In this case, the pCasop (short for pCas\_optimized) plasmid suits our project better.

### Cycle2: Typical Site-directed, Ligase-Independent Mutagenesis (SLIM)

#### Design

Initially, we obtained a comprehensive set of experimental procedures from the literature. The method utilizes two long primers with overhang and two short primers in a single reaction for reverse PCR amplification of the template, meanwhile, the 5' end of each primer incorporates the desired sequences on the complementary strand at the end of the PCR product. Then, heteroduplexes are formed between mixed PCR products, resulting in the desired plasmids (Fig 2).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig2.png)

Correspondingly, we designed 4 primers to replace the original specificity-determined sequence (SDS, or N20 in the literature) with BsmBI restriction site. Then the original pCas template was digested using DpnI enzyme, to prevent false positives.

#### Build

Long fragments were successfully cloned using PCR. After denaturation and renaturation, the DNA product was obtained. Then it was transformed into *E. coli* DH5α and spread on Kanamycin plates. Positive transformants were verified and selected. To our disappointment, it turned out to be the original pCas, concluding from sequencing results (Fig 3).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig3.png)

Fig 3. The result of PCR product sequencing
We utilized a pair of primers targeting the pCas skeleton to amplify the fragment, which was subsequently sent for testing. The sequencing results were then compared to both the pCas plasmid and pCasop plasmid.

a. The sequencing result was aligned with the original pCas. The two sequences fix well.

b.The sequencing result was aligned with the designed optimized pCas. There is a gap in the expected Golden Gate site.

#### Learn

We assumed that the original pCas had not been fully digested by DpnI, and pCas has better capability to be transformed, as it was less modified. Also, the PCR product has a similar length to the original pCas, making it hard to purify pCasop segments using a DpnI-independent way. As a result, we intend to amplify shorter and more fragments for better purification, and then use Gibson Assembly to get the final plasmid.

### Cycle3: Three-segment assembly

#### Design

The whole plasmid was separated into three parts (Fig 4) and each part was amplified using well-designed primers. In addition, the Gibson primers were designed to clone fragments using pCas as the template, remove the poxb homologous arm sequence, and mutate the original 30°C sensitive sequence of pCas to enable rapid *E. coli* growth at 37°C.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig4.png)

Fig 4. The design of pCas optimization using three-fragment Gibson Assembly

#### Build

The designed three fragments were successfully PCR amplified using Gibson primers (Fig 5). These fragments were then ligated using Gibson assembly. But we found it hard to get positive transformants of *E. coli* DH5α.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig5.png)

Fig 5. Preparation of fragments of pCas
The white hollow arrowheads indicate target bands. The three fragments are exposed to be 5694bp, 5303bp, and 435bp respectively.

#### Learn

Judging from the results and advice from our PIs, we realized that simply ligating two long fragments and one short fragment using Gibson Assembly was of low efficiency. Also, we are not so familiar with the technical details of Gibson Assembly. Consequently, we intend to jump to another path.

### Cycle4: Simple removal of sgRNA

#### Design

As the length of the segments varies, we decided to remove redundant parts step by step, especially the functional sgRNA. We designed primers for the backbone to delete the sgRNA sequence using Gibson Assembly and Golden Gate Assembly (Fig 6).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig6.png)

Fig 6. The sgRNA-removal design of pCas optimization

#### Build

We successfully amplified the target sequence of pCas (Fig 7a). Subsequently, we employed Golden Gate Assembly. Then sequencing result unveiled that the sgRNA part of pCas had been removed (Fig 7c).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig7.png)

Fig 7. The result of sgRNA deletion in pCas

a. The white hollow arrowheads indicate target bands. The long fragments for Gibson Assembly and Golden Gate Assembly are expected to be 11610 and 11744 bp, respectively. The regular PCR procedure is effective.

b. The white hollow arrowheads indicate target pCasop bands. The primers were designed to amplify the sequence covering the omitted sgRNA.

c. Sequencing results were aligned with the original pCas sequence. There is a gap at sgRNA.

d. Sequencing results were aligned with the designed pCasop sequence.

#### Test

Set the original pCas as control, we transformed the pCasop into *E. coli* DH5α and induced (Fig 8a-d). The bacteria amount of pCasop-containing group is much larger than pCas group, indicating an increase in bacteria viability (Fig 8e). The sequencing results prove that there is no targeting deletion in the pCasop group (Fig 8f).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig8.png)

Fig 8. Genome-editing test for the pCasop

a-b. The induced pCas-containing bacteria were spread on plates, after 100-fold and 1,000-fold dilution, respectively.

c-d. The induced pCasop-containing bacteria were spread on plates, after 100-fold and 1,000-fold dilution, respectively.

e. The quantification of bacteria on the corresponding plates (a-d). They were evaluated by three individual testers. The bacteria amount of pCasop is strikingly larger than the original pCas.

f. The sequencing results. The genomic knock-out ratio of pCas is 70% (N = 10), while pCasop is 0. The pCas-containing strain without induction was set as the positive control.

#### Learn

The data demonstrated that we have got a capable pCasop, without knocking out the genome. Also, the prefix of this set of Golden Gate Assembly primers is quite useful, to omit a segment in a plasmid using single fragment self-assembly is feasible.

### Cycle5: Integrated recording machine

#### Design

Though we get a useful pCasop, we intend to make the entire device more compact and better contribute to synthetic biology. In detail, we design Golden Gate Assembly primers for ligation of the Cas9 coding sequence and the Lambda Red, araC sequence (Fig 9a). Considering the multi-cloning site on the pCDF-Duet1 backbone, we attempt to add XhoI and NotI prefixes on the ligated Cas-Lambda sequence (Fig 9b). Then the segment can be ligated to the plasmid by restriction cloning. We re-organize them in one integrated plasmid (Fig 9c).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig9.png)

Fig 9. Design of the integrated plasmid, using Golden Gate Assembly and restriction cloning

a. Using 5'-modified primers, we flank the target sequence with restriction site.

b. XhoI and NotI recognition sites are present in the multi-cloning site of the recorder plasmid.

c. The constructed plasmid.

#### Build

We successfully amplified the target sequence and finished the Golden Gate assembly (Fig 10). Unfortunately, due to the limitation of experimental materials, we can only get the two enzymes from different companies, causing the buffer incompatibility. We found it hard to ligate the segment and backbone by restriction cloning.

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig10.png)

Fig 10. The preparation of the target sequence

a. The white hollow arrowheads indicate target bands. The Cas9 and Lambda fragments are expected to be 4619 and 3434bp, respectively.

b. The white hollow arrowheads indicate target bands. The primers are designed to cover the sequence of the ligated site. The products are expected to be 2085 bp.

#### Learn

From the plasmid construction, we confirm that the set of Golden Gate Assembly primers is quite versatile and the risk of restriction buffer incompatibility should be noticed.

#### Design-Three-segment Golden Gate Assembly

In this turn, we decided to amplify the two segments and the backbone using Golden Gate Assembly primers (see also [Expeiments, primer list](experiments#5-Key-PCR-primers)) (Fig 11).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig11.png)

Fig 11. Design of the integrated plasmid

a. Prefix design for the three-segment Golden Gate Assembly.

b. The constructed plasmid.

#### Build

The three segments were successfully amplified, and we performed Golden Gate Assembly (Fig 12a-c). The sequencing results suggested that we have got the expected product (Fig 12d).

![缺个图](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/fig12-thaw-2.webp)

Fig 12. Integrated plasmid construction
The prepared Cas, Lambda and backbone DNA sample were used as positive control, as the paired samples differ at several 10 bp.

a. The preparation of backbone. The white hollow arrowhead indicates the target band. The segments are expected to be 4197 bp.

b. The preparation of Cas and Lambda segments. The white hollow arrowheads indicate the target band. The Cas and Lambda segments are expected to be 4593 and 3399 bp, respectively.

c. The verification of the plasmid. The primers are designed to cover the ligation site.

#### Test

Due to the limitation of time, we have not finished the induction of the integrated plasmid.

[Supplementary data for cascade system](https://static.igem.wiki/teams/4630/wiki/parts/cascade-addition/supplementary-data-copy.pdf)

{% endblock %}

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Experiments

Experiments

Experiments

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

- [Part 1: Fragments Obtaining](#Part-1-Fragments-Obtaining)

- [PCR](#PCR)

- [Annealing](#Annealing)

- [Gel Extraction](#Gel-Extraction-1)

- [Plasmid Preparation](#Plasmid-Preparation)

- [RNA Extraction](#RNA-Extraction)

- [Part 2: Subcloning](#Part-2-Subcloning)

- [Restriction-Enzyme-Cut-&-LigationCloning](#Restriction-Enzyme-Cut-LigationCloning)

- [Golden Gate Assembly](#Golden-Gate-Assembly)

- [Gibson Assembly](#Gibson-Assembly)

- [Chemically Competent Cell Transformation](#Chemically-Competent-Cell-Transformation)

- [Part 3: Key Experiments](#Part-3-Key-Experiments)

- [1.Induction of Editing on Genome](#1-Induction-of-Editing-on-Genome)

- [2.Induction of Cascade Editing on Plasmids](#2-Induction-of-Cascade-Editing-on-Plasmids)

- [3.Fluoroscopy](#3-Fluoroscopy)

- [4.Reverse Transcription](#4-Reverse-Transcription)

- [5.Key PCR primers](#5-Key-PCR-primers)

[![](https://static.igem.wiki/teams/4630/wiki/webpage/navbar/whu-gradient-arrow-down.svg)

To top](#first-paragraph)

The construction of CRISPReporter primarily involves common molecular experimental techniques, which are
detailed on this page. Additionally, we have summarized the essential experimental protocols and PCR primer
sequences used in our project, which can serve as a reference for others. If you have any inquiries or need
further clarification, feel free to contact us at 2021302041074@whu.edu.cn.

## Part 1: Fragments Obtaining

### PCR

#### Taq PCR

Reaction system

* Total
* 2X Taq Master Mix
* Primer Forward
* Primer Reverse
* Template
* ddH2O

* 20(µl)
* 10
* 1
* 1
* 50ng
* Add up to 20µl

Program:

* 1
* 2
* 3
* 4
* 5
* 6

* Pre-Denaturation
* Denaturation
* Annealing
* Extension
* Jump to step 2, repeat for 30times
* Final Extension

* 95°C
* 95°C
* Tm-5°C
* 72°C
* Jump to step 2, repeat for 30times
* 72°C

* 30s (DNA) / 2min (Bacteria)
* 30s
* 30s
* 1min/Kb
* Jump to step 2, repeat for 30times
* 5min

#### HiFi PCR

Reaction system:

* Total
* HiFi PCR Enzyme
* 2 X Buffer
* dNTPs
* Primer Forward
* Primer Reverse
* Template
* ddH2O

* 50(µl)
* 1
* 25
* 1
* 2
* 2
* 100ng
* Add up to 50µl

Program:

* 1
* 2
* 3
* 4
* 5
* 6

* Pre-Denaturation
* Denaturation
* Annealing
* Extension
* Jump to step 2, repeat for 30times
* Final Extension

* 95°C
* 95°C
* Tm-5°C
* 72°C
* Jump to step 2, repeat for 30times
* 72°C

* 30s (DNA) / 2min (Bacteria)
* 30s
* 30s
* 1min/Kb
* Jump to step 2, repeat for 30times
* 5min

#### Touch Down PCR

Reaction system:

* Total
* 2X Taq Master Mix
* Primer Forward
* Primer Reverse
* Template
* ddH2O

* 20(µl)
* 10
* 1
* 1
* 50ng
* Add up to 20µl

Program:

* 1
* 2
* 3
* 4
* 5
* 6
* 7
* 8
* 9
* 10

* Pre-Denaturation
* Denaturation
* Annealing
* Extension
* Jump to step 2, repeat for 10 times, touch down 0.7°C per cycle
* Denaturation
* Annealing
* Extension
* Jump to step 6, repeat for 22 times
* Final Extension

* 94°C
* 94°C
* Tm°C
* 72°C
* Jump to step 2, repeat for 10 times, touch down 0.7°C per cycle
* 94°C
* Tm-7°C
* 72°C
* Jump to step 6, repeat for 22 times
* 72°C

* 30s (DNA) / 3min (Bacteria)
* 30s
* 30s
* 1min/Kb
* Jump to step 2, repeat for 10 times, touch down 0.7°C per cycle
* 30s
* 30s
* 1min/Kb
* Jump to step 6, repeat for 22 times
* 5min for fragments less than 10kb, 7min for fragments in 10-15kb

#### Error-Prone PCR

Reaction system:

* Total
* 2X Taq Master Mix
* Primer Forward
* Primer Reverse
* Template
* 50mM MnCl2
* ddH2O

* 50(µl)
* 25
* 2
* 2
* 50ng
* 1
* Add up to 50µl

Program:

* 1
* 2
* 3
* 4
* 5
* 6

* Pre-Denaturation
* Denaturation
* Annealing
* Extension
* Jump to step 2, repeat for 30times
* Final Extension

* 95°C
* 95°C
* Tm-3°C
* 72°C
* Jump to step 2, repeat for 30times
* 72°C

* 30s (DNA) / 2min (Bacteria)
* 30s
* 30s
* 1min/Kb
* Jump to step 2, repeat for 30times
* 5min

### Annealing

#### Reaction system:

* Total
* Oligo I
* Oligo II
* 10X T4 Ligase buffer (NEB)
* T4 PNK (NEB)
* ddH2O

* 10 (µl)
* 100µM
* 100µM
* 1
* 1
* Add up to 10µl

Program:

* 37°C
* 95°C
* 4°C

* 30 min
* 5min and then ramp down to 25°C, at 5°C/min
* Hold until ready to proceed

### Gel Extraction

* Cut out the target band minimizing extra gel.
* Put it in an Eppendorf tube (EP tube) and add 600μL QG.
* Incubate at 50°C until the gel is fully dissolved.
* Pour into the gel extraction column, spin through and discard the waste.
* Wash with 750μL PE buffer twice.
* Centrifuge at 13000rpm for 2min to dry.
* Elute with 50μl ddH2O.

### Plasmid Preparation

* Pick a single colony, and cultivate in 5mL LB medium with antibiotic to saturation.
* Put the media into tubes repetitively.
* Centrifuge at 13000rpm for 1min.
* Discard the medium, and make the remaining pellet as dry as possible.
* Add 240μl SolutionBuffer I (mainly contains Glucose, NaCl) into the 1.5mL EP tube and resuspend the
  pellet.
* Add 240ul Buffer II (whose key component is NaOH) into the same EP tube.
* Mix it by gentle reversal 5 times until the solution gets clear.
* Add 350μl SolutionBuffer III (whose key component is acetate) into the same EP tube.
* Vortex the tube until the solution is uniform.
* Centrifuge at 12000rpm for 10min.
* Transfer the supernatant to a new mini-prep column.
* Centrifuge at 12000rpm for 1min and discard the flowthrough.
* Add 750μl SolutionBuffer PW (absolute alcohol containing) into the column.
* Centrifuge at 12000rpm for 1min and discard the flowthrough.
* Centrifuge again at 12000rpm for 2min to completely dry the column.
* Put the column into a new EP tube, and add 50μl ddH2O.
* Centrifuge at 12000rpm for 1min.

### RNA Extraction

* Pick a single colony, and cultivate in 5mL LB medium with antibiotic to saturation.
* Put 1.5mL culture into a cooled 1.5mL EP tube.
* Centrifuge at 10000rpm for 3min.
* Discard the medium, add 200μl Bacteria RNA Plus Reagent at 95°C. Mix them together.
* Incubate at 95°C for 4 min.
* Add 1ml RNA isolator Total RNA Extraction Reagent, and place the mixture on ice for 5 min.
* Add 1/5 volume of pre-cooled chloroform to the above lysate. Vortex and shake for 15 sec, form an
  emulsion and let stand on ice for 5 min. Centrifuge at 4 °C at 12,000 rpm (13,800× g) for 15 min.
* Carefully remove the centrifuge tube. At this point, the solution is divided into three layers: the
  colorless upper layer, the white middle layer, and the red lower layer. Carefully pipette the upper
  aqueous phase into a new centrifuge tube.
* Add an equal volume of pre-cooled isopropanol and mix upside down. Let stand at -20°C for 10 min.
  Centrifuge at 4 °C at 12,000 rpm (13,800 × g) for 10 min. White deposits are usually visible.
* Carefully discard the supernatant and add 1 ml of pre-cooled RNase-free ddH2O prepared 75% ethanol.
  Wash the lid and wall well, flick the bottom of the tube, let the pellet suspension, and let stand
  at 4°C or ice for 3-5 min. Centrifuge at 4 °C at 12,000 rpm (13,800 × g) for 5 min. Discard the
  supernatant.
* Dry the precipitation in a clean environment at room temperature for 2-5 min.
* Add an appropriate amount of RNase-free ddH2O. Dissolve the precipitate, gently pipette until the
  precipitate is completely dissolved. After complete dissolution, take a small amount for testing,
  and store the rest at -80 °C.

## Part 2: Subcloning

### Restriction Cloning

#### One step cut digestion

Reaction system:

* Total
* Restriction Enzyme 1&2
* DNA
* 10X NEBuffer
* ddH2O

* 50 µl
* 1 µl each
* 1 µg
* 5 µl
* Add up to 50µl

#### Two steps cutdigestion

Reaction system 1:

* Total
* Restriction Enzyme 1
* DNA
* 10X NEBuffer
* ddH2O

* 50µl
* 1µl
* 1µg
* 5µl
* Add up to 50µl

Program 1:

* Incubation Time
* Incubation Temperature

* 15min
* 37°C

Reaction system 2:

* Total
* Restriction Enzyme 2
* Reaction system 1
* 10X NEBuffer
* ddH2O

* 150µl
* 3µl
* 50 µl
* 15µl
* 82 µl

Program 2:

* Incubation Time
* Incubation Temperature

* 15min
* 37°C

#### Ligation

Reaction system:

* 10X T4 DNA Ligase Buffer
* Vector DNA (4 kb)
* Insert DNA (1 kb)
* ddH2O
* T4 DNA Ligase

* 2 μl
* 50 ng (0.020 pmol)
* 37.5 ng (0.060 pmol)
* Add up to 20 μl
* 1 μl

Program: 25°C for 10min or 16°C overnight

### Golden Gate Assembly

Reaction system:

* Vector
* Fragment
* T4 DNA ligase buffer
* T4 DNA ligase
* BsmB I / Bsa I
* H2O

* 1 μl
* 1 μl
* 1 μl
* 0.5 μl
* 0.5 μl
* 6 μl

Program

* 1
* 2
* 3
* 4
* 5
* 6

* 37°C
* 16°C
* Jump to Step1 and repeat for 30 times
* 50°C
* 80°C
* 10°C

* 3min
* 4min
* Jump to Step1 and repeat for 30 times
* 5min
* 15min
* forever

### Gibson Assembly

Reaction system:

* Total
* 2× MultiF Seamless Assembly Mix
* Total DNA
* ddH2O

* 20µl
* 10µl
* 0.5 pmol (Vector:50ng)
* Add up to 20µl

### Chemically Competent Cell Transformation

* Thaw chemically competent cells on ice.
* Add DNA (30ng), and pipette gently to mix.
* Let sit for 30 minutes on ice.
* Incubate cells for 45 seconds at 42℃.
* Chill on ice for 2 min.
* Add 1 ml LB at room temperature.
* Cultivate for 1 hour at 37℃, 150rpm.
* Centrifuge at 5000rpm for 2min, discard the supernatant to 100 μl and spread onto a plate with
  appropriate antibiotics.
* Grow overnight at proper temperature.

## Part 3: Key Experiments

### 1.Induction of Editing on Genome

Plasmid pCas\_Red\_Δpoxb is transformed into the host by chemical transformation. The transformants carrying the editing plasmid are grown in 5 mL LB cultures with 50mg/L kanamycin at 30°C for 2 hours, and 2g/L arabinose is added to the culture. Then culture is incubated for approximately 24 hours at 30°C. At last, cells were diluted 100- fold and 20μL were plated on LB dishes containing 50mg/L kanamycin and 2g/L arabinose. Obtained colonies are identified by colony PCR with or without DNA sequencing.

### 2.Induction of Cascade Editing on Plasmids

Co-transform pCasop and relevant recording plasmid into *Escherichia coli* DH5[alpha] chemical component cells. Grow the transformants carrying the plasmids in 5 ml LB media with proper antibiotics at 30°C, 250 rpm for 12 hours. Then 100 µl bacteria liquid is transferred into 3 ml new LB media with proper antibiotics and cultures to OD 0.4-0.6. Then add 2g/L arabinose and cultivate for 17 hrs. Then add 3g/L IPTG and cultivate another 5hrs. After induction, dilute the cells to 100 folds and spread them on a plate with proper antibiotics.

### 3.Fluoroscopy

Place the dish in the gel image system, and turn on the UV lights. Observe the fluorescence from the window directly, or save the image for later measurement.

### 4.Reverse Transcription

Reaction system:

A. Prepare the first strand cDNA synthesis reaction solution

Prepare the following mixture in RNase-free centrifuge tubes:

* RNAse free ddH2O
* 5×HiScript 11 Select qRT SuperMix
* Random hexamers (50 ng/μl) or
  gene specific primers (2µM)
* Template RNA

* Add up to 20μl
* 4μl
* 1μl
* Total RNA:1pg-1vg

B. No RT Control reaction

No RT Control refers to a reverse transcription negative control reaction without reverse transcriptase to test RNA templates for genomic DNA residues. Prepare the following mixture in RNase-free centrifuge tubes:

* RNAse free ddH2O
* 5×Select No RT SuperMix
* Random hexamers (50 ng/μl) or
  gene specific primers (2µM)
* Template RNA

* Add up to 20μl
* 4μl
* 1μl
* Total RNA:1pg-1vg

Program:

* 50°C
* 85°C

* 15 min
* 5 sec

### 5.Key PCR primers

* Index
* 1
* 2
* 3
* 4
* 5
* 6
* 7
* 8
* 9
* 10
* 11
* 12
* 13
* 14
* 15
* 16
* 17
* 18
* 19
* 20
* 21
* 22
* 23
* 24
* 25
* 26
* 27
* 28
* 29
* 30
* 31
* 32
* 33
* 34
* 35
* 36
* 37
* 38
* 39
* 40
* 41
* 42
* 43
* 44
* 45
* 46
* 47
* 48
* 49
* 50
* 51
* 52
* 53
* 54
* 55
* 56
* 57
* 58
* 59
* 60
* 61
* 62
* 63
* 64
* 65
* 66
* 67
* 68
* 69
* 70
* 71
* 72
* 73
* 74
* 75
* 76
* 77
* 78
* 79
* 80
* 81
* 82
* 83
* 84
* 85
* 86
* 87
* 88
* 89
* 90
* 91
* 92
* 93
* 94
* 95
* 96
* 97

* Name
* Colony\_PCR\_N20s\_For
* Colony\_PCR\_N20s\_Rev
* Colony\_KD\_For
* Colony\_KD\_Rev
* pCas-goldenGate-For
* pCas-goldenGate-Rev
* NEW\_Gib\_frag1\_assembly.FOR
* Gib\_frag1\_assembly.REV
* Gib\_frag2.FOR
* Gib\_frag2.REV
* Gib\_frag3\_assembly.FOR
* Gib\_frag3\_assembly.REV
* pCasop\_1+3\_For
* pCasop\_1+3\_Rev
* pCasop\_1-3\_For
* pCasop\_1-3\_Rev
* pET\_For(T7\_Ter)
* pET\_Rev(T7\_Pro)
* ACYCDuetUP1
* DuetDOWN1
* Gib\_Assembly\_PCR\_1+3\_For
* Gib\_Assembly\_PCR\_1+3\_Rev
* Gib\_Assembly\_PCR\_2+3\_For
* Gib\_Assembly\_PCR\_2+3\_Rev
* Gib\_Assembly\_PCR\_1+2\_For
* Gib\_Assembly\_PCR\_1+2\_Rev
* Golden\_frag1\_For
* Golden\_frag1\_Rev
* Gib\_frag3\_assembly.FOR
* Golden\_frag2+3\_Rev
* Cas\_NotI\_For
* Golden\_frag1\_Rev
* Lambda\_XhoI\_For
* Gib\_frag3\_assembly.REV
* Golden\_RemoveN20\_For
* Golden\_frag1\_Rev
* GibRemoval\_For
* GibRemoval\_Rev
* Cas\_gGate\_For
* Cas\_gGate\_Rev
* Lambda\_gGate\_For
* Lambda\_gGate\_Rev
* Duet\_gGate\_For
* Duet\_gGate\_Rev
* New\_pCasop\_For
* New\_pCasop\_Rev
* New\_pCasop\_forDirect\_Rev
* Duet\_T7\_Fgolden\_For
* Duet\_T7\_Fgolden\_Rev
* Duet\_T7\_Gib\_For
* Duet\_T7\_Gib\_Rev
* Duet\_T7\_Sgolden\_For
* Duet\_T7\_Sgolden\_Rev
* Ligation\_For
* Ligation\_Rev
* el-seq-F
* el-seq-R
* qPCR-mRFP-F
* qPCR-mRFP-R
* mdh-F
* mdh-R
* recA-F
* recA-R
* pET-28a-gol-F(RFP)
* pET-28a-gol-R(RFP)
* El222-add-1-F
* El222-add-2-F
* El222-add2-gol-F
* El222-add2-gol-R
* BamH1-Sph1-el222-F
* BamH1-Sph1-el222-R
* el222-gib-asmb-F
* el222-gib-asmb-R
* El222-fac-F-Xba1
* El222-fac-R-Sph1
* El222-add-1-F
* El222-add-2-F
* El222-add2-gol-F
* El222-add2-gol-R
* pET-28a-gol-F(RFP)
* pET-28a-gol-R(RFP)
* el-RBS-add1-R
* el-RBS-add2-R
* El222-new-add3-R
* El222-new-F
* pET-ElN-F
* pET-ElN-R
* T7-F
* T7-R
* mRFP-lac-golden-F
* mRFP-lac-golden-R
* pET-golden-F
* pET-golden-R
* pET-lac-golden-F
* pET-lac-golden-R
* el222-seq-F
* el222-seq-R

* Sequence
* CATGAGCGGATACATATTTG
* AGTGTGACTCTAGTAGAGAG
* GCTTGTCTGTAAGCGGATGC
* GCGTTATCCCCTGATTCTGTG
* ACGTTGACGAATCTTGGAGC
* TTCGTTCTCATGGCTCACGC
* GGGTCTCAACTGTATCTTAAAGCGAGACCGCTGAATGGAAGCTTGGATTCTCACC
* AAACTGAGACGCGTCTCACTAACGTAAGATCTGACTCCATAACAGAGTACTCG
* TTAGTGAGACGCGTCTCAGTTTTAGAGCTAGAAATAGCAAGTT
* GAGTGACATGCAAAGTAAGCATGATCTCAATGG
* GAACGAACCATTGAGATCATGCTTACTTTGCATGTCACT
* GCTTTAAGATACAGTTGAGACCCAGGCCCATGGATTCTTCGT
* GCATCGCCTTCTATCGCCTT
* TCCGGCGTGAATGTTACTGA
* CTATGCCAAGTTCTCAAGCG
* CCAACGGTAGCTTATTCAGTC
* GCTAGTTATTGCTCAGCGG
* TAATACGACTCACTATAGGG
* GGATCTCGACGCTCTCCCT
* GATTATGCGGCCGTGTACAA
* GAACGAACCATTGAGATCATGCT
* AAACTGAGACGCGTCTCACT
* GCTTTAAGATACAGTTGAGACCCAG
* TTAGTGAGACGCGTCTCAGTTTTA
* GAGTGACATGCAAAGTAAGCATG
* GGGTCTCAACTGTATCTTAAAGCG
* CCAGGTCTCACTAAGCTGAATGGAAGCTTGGATTCTCACC
* CCAGGTCTCAACTGCTAAGATCTGACTCCATAACAGAGTACTCG
* GAACGAACCATTGAGATCATGCTTACTTTGCATGTCACT
* CCAGGTCTCATTAGTGAGACGCGTCTCAGTTTTA
* ATAAGAATGCGGCCGCTAAACTAAGCTGGCAATTCCGACGC
* CCAGGTCTCAACTGCTAAGATCTGACTCCATAACAGAGTACTCG
* CCGCTCGAGCGGGCATCGATTTATTATGACAACTTGACGG
* GCTTTAAGATACAGTTGAGACCCAGGCCCATGGATTCTTCGT
* CCAGGTCTCACAGTCTCTCTACTAGAGTCACACTGGCTC
* CCAGGTCTCAACTGCTAAGATCTGACTCCATAACAGAGTACTCG
* TAGGCGAGTATTCACTGATAGATACAAGAGC
* TATCAGTGAATACTCGCCTATGTATAAACC
* CCAGGTCTCAAGTCAGCTGGCAATTCCGACGC
* CCAGGTCTCAACTGCTAAGATCTGACTCCATAACAGAGTACTCG
* CCAGGTCTCACAGTGCATCGATTTATTATGACAACTTGACGG
* CCAGGTCTCAGCCTCAGGCCCATGGATTCTTCGT
* CCAGGTCTCAAGGCCCGCTGAGCAATAACTAGC
* CCAGGTCTCAGACTGATTATGCGGCCGTGTAC
* GAACACATCCGGTACATGGT
* CGTTGACGAATCTTGGAGCT
* AGCACCACATAGCAGACC
* CCAGGTCTCACAGTGTGGAATTGTGAGCGGATAACAAGAT
* CCAGGTCTCAACTGCCTATAGTGAGTCGTATTAATTTCC
* TATAGGGGAATTGTGAGCGGATAACAAGATCC
* CCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCC
* CCAGGTCTCACAGTCCTCCCTACTTCTAAAGTCTGG
* CCAGGTCTCAACTGCTACAGGGGAATTGTTATCCG
* GGATCTCGACGCTCTCCC
* GCCGATATCTAAGCCTATTGAGTATTTC
* GAACCGTACTGGAACTGCGGG
* CTAGAGGAATGCTGGATATGGGC
* AGCTTTGGAACCGTACTGGAA
* TGCGTTTCAAAGTTCGTATGG
* GTCACAGGTTCCTGGCGTTA
* CGAACCAGAGACAGACCAA
* GCGCGAAGGTAAAACCTGTG
* AACGTCTACTGCGCCAGAAC
* GGCTACGGTCTCTCTCTAGTATTTCTCCTCTTTCTCTAGTAGC
* GGCTACGGTCTCCGCCAGGCATCAAATAAAAC
* CCCGATTGATGGCAGCGGCGCGCCGGGGGCAGACGACACACGCGTTG
* ATGCTGGATATGGGCCAGGATCGCCCGATTGATGGCAGCGGCGC
* GGCTACGGTCTCCTGGCTCTAGTATTAGATTCCGGCTTCGAC
* GGCTACGGTCTCTAGAGATGCTGGATATGGGCCAG
* TGCTCACCATGGTGGCGATGCTAGTATTTCTCCTCTTTCGG
* GCCGCAAGGAATGGTGCATGGAATTCGAGCTCGGTACC
* TGCTCACCATGGTGGCGATGTAGTATTTCTCCTCTTTCGG
* TGGACAGCAAATGGGTCGCGGAATTCGAGCTCGGTACC
* GCTCTAGACTAGTATTTCTCCTCTTTCGGATC
* AAGCATGCGATATCGTTGACGGCTAGCTCAG
* CCCGATTGATGGCAGCGGCGCGCCGGGGGCAGACGACACACGCGTTG
* ATGCTGGATATGGGCCAGGATCGCCCGATTGATGGCAGCGGCGC
* GGCTACGGTCTCCTGGCTCTAGTATTAGATTCCGGCTTCGAC
* GGCTACGGTCTCTAGAGATGCTGGATATGGGCCAG
* GGCTACGGTCTCTCTCTAGTATTTCTCCTCTTTCTCTAGTAGC
* GGCTACGGTCTCCGCCAGGCATCAAATAAAAC
* GAGAAAGAGGAGAAATACTAGAGGAATGCTGGATATGGGCCAGGA
* GGCGGTATCATCAATCGGAGAAAGAGGAGAAATACTAGAGGA
* GGCTACGGTCTCAACTCACTATAGGGGCGGTATCATCAATCGG
* GGCTACGGTCTCTCCGCTTATTAAATGCCCG
* GGCTACGGTCTCAGAGTCGTATTAATTTCGCG
* GGCTACGGTCTCATTTGCTGAAAGGGAGCCAGGCATCAAATAAAAC
* GGCTACGGTCTCACAAAAAACCCCTCAAGAC
* GGCTACGGTCTCTGCGGCCGCGGAAGACTACACCATCGTTG
* GGCTACGGTCTCGTGTTAAGCACCGGTGGAGTG
* GGCTACGGTCTCCTTTGATGGCTTCCTCCGAAGAC
* GGCTACGGTCTCACGAATTCCAAAATTATTTCTAGAGGGGAATTGTTATCCG
* GGCTACGGTCTCAAACACTCGAGCACCACCACC
* GGCTACGGTCTCCCAAAATTATTTCTAGAGGGGAATTGTTATCCG
* GGCTACGGTCTCGAACACTCGAGCACCACCACC
* CCGGATATAGTTCCTCCTTTCAG
* TGCGACTCCTGCATTAGGAAG

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Results

Results

Results

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

- [The Cascade Recording System](#The-cascade-recording-system)

- [Optimization of Plasmid pCas](#Optimization-of-plasmid-pCas)

- [EL222 Directed Evolution](#EL222-Directed-Evolution)

- [sgRNA Screening](#sgRNA-screening)

- [Application](#Application)

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To top](#The-cascade-recording-system)

## The Cascade Recording System

To verify the feasibility of our cascade recording system, we established two series of parts, recorder with a barcode (stgRNA-barcode-cassette series) and recorder without barcode (stgRNA-cassette series). We co-transformed the plasmids with pCas plasmid into *Escherichia coli* DH5α, and performed several validations and measurements.

### Pilot experiment: validations of the knockout effectiveness

We firstly preformed a preliminary concentration gradient experiment on stgRNA-barcode-cassette 1 (BBa\_K4630100). After cultivating 22hrs for L-Arabinose and 5hrs for IPTG induction, we diluted the media and spread plate to halt induction and get single colonies (Tbl 1).

The relevant sequence was amplified and sequenced (Fig 1a), confirming a successful knock-out. Large-scale screening revealed that Condition 2 exhibited the highest efficiency, with efficiencies of 70%, 10%, and 38.1% for Condition 2, 3, and 4, respectively (Fig 1b). Non-induction controls substantiated that induction is the prerequisite for recording (Fig 1c).

Tbl 1.The random test over inducers

![](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table1.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-1.png)

### Concentration matrix and time-gradient test

To get a comprehensive view of the stable relationship between the inducer and the functional consequence, we perform two rounds of concentration matrix (Tbl 2).

Tbl 2.Group design for the concentration matrix

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table1.png)

The results are documented from aspect of the growth of bacteria, electrophoresis result, sequencing result and the correlation between the two readouts.

#### The first concentration matrix

It's noted that the induction of arabinose inhibited the growth of bacteria strikingly (Fig 2), and the Lac promoter exhibited significant leakage expression (Tbl 3, Fig 16b). Despite the missing data due to failure of sequencing, E6, E5, C2, B5 performed better (Tbl 4, Fig 3a). However, a quality test based on electrophoresis provided parallel data for randomly picked groups, and the two data access showed a significant correlation, with paired t-test P = 0.7602, no significant difference (Tbl 5, Tbl 6, Fig 4). Given the substantially larger amount of data from the electrophoresis test for E6 (N = 20) compared to sequencing (N = 4), we adjusted the knock-out ratio of E6 to 60%.

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-2.png)

Fig 2. Streaking plate of Group 1, after induction

a. Bacteria without induction. The bacteria had grown to Area 3.

b-d. Bacteria under 1, 4, 8 g/L L-Arabinose induction, respectively. The growth of the bacteria was limited to Areas 1 and 2. The bacteria in C1 had been contaminated.

e. Assessment of the bacteria amount. The introduction of L-Arabinose influences the bacteria amount strikingly.

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-3.png)

Fig 3. Results of the concentration matrix

a. Heatmap of the concentration matrix based on sequencing data. The black block indicates no sequencing data is available. The L-Arabinose 0g/L row and IPTG 0 g/L column indicate the leakage expression of pBAD is quite low while that of pLac is quite high.

b. Comparison in knock-out ratio of IPTG 0 g/L column and the average of total. Though there is a slight increase along with the L- Arabinose concentration, the presence of L- Arabinose predominates, implying a high ratio of pLac leakage.

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-4.png)

Fig 4.The relation between data from sequencing and electrophoresis

a.The correlation relationship of the two data. Pearson r = 0.9837, R squared = 0.9678, P = 0.0025(\*\*).

b.The normality test of the two data. Under Shapiro-Wilk test (N = 5), the P values for sequencing and electrophoresis are 0.9500 and 0.9364, respectively.

Tbl 3.Results of the no-IPTG-induction group

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table5.png)

Tbl 4.The groups with high knock-out ratio

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table4.png)

Tbl 5.The paired data of the electrophoresis and sequencing

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table6.png)

Tbl 6.The correlation of the electrophoresis and sequencing result

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table7.png)

#### The second matrix and statistical analysis

In the second matrix, growth inhibition and leakage expression still occur (Fig 5a, b). Also, an integrated heatmap is plotted based on the mean value of the two matrices (Fig 5c), and the variation pattern of the two matrices showed some kind of correlation. Paired t test result of the two matrices showed a significant difference, with P = 0.0054(\*\*) and mean of differences = -0.2071(Fig 5d). Two-way ANOVA of the second matrix and the average matrix indicate that the IPTG concentration is the main variation factor (Tbl 7).

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-5.png)

Fig 5. Results of the second matrix

a. Scratch plate result of the L-Arabinose / IPTG non-induction group. As the previous data, the non- L-Arabinose group growth much better than the L-Arabinose induction ones. Also, the IPTG slightly inhibit the growth of the bacteria.

b. The normality test of the two data. Under Shapiro-Wilk test (N = 5), the P values for sequencing and electrophoresis are 0.9500 and 0.9364, respectively.

c. The heatmap plotted from the average data of the two concentration matrices. When there is no data, data from the other compensate.

d. The demonstration of the knock-out ratio. The two matrices showed high correlation.

e. The graphical result of the paired t test, of the second matrix and average matrix.

Tbl 7.  Two-way ANOVA result of the matrices

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table8.png)

The row factor is IPTG concentration and column factor L-Arabinose in the matrix.

#### Time-gradient test on stgRNA-barcode-cassette 1

We performed a time-gradient test to provide guidance for inducing multi-level knockout. The experimental results showed a bell-shaped induction time-knockout ratio curve (Tbl 8, Fig 6). The increasing trend ends on dot 4h and the extension of IPTG induction time does not help the editing afterwards.

Tbl 8.Group design for the time-gradient test

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table9.png)

The test was carried out on stgRNA-barcode-cassette 1.

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-6.png)

Fig 6.The knock-out ratio plot of the time-gradient test of stgRNA-cassette 1.

### Multiple cassette recorder

Firstly, we concluded from the previous statistic data that the multi-level recombination might occur properly in most of the cases.

Indeed, in the matrix tests, we meet several cases of unexpected knock-out (Tbl 9, Fig 7). Theoretically, the larger the homologous arm, the higher the recombination ratio. Interestingly, there are two sets of homologous arms intrinsically flanking the DSB target site. However, the preferred unexpected recombination occurred at the low ratio, 0.028, with a shorter homologous arm. The result demonstrates that the effect of distance is larger than the effect of homologous arm, and both of them are weak. We have more confidence that the change of cassette is sequential.

Tbl 9.  Statistical data of the unexpected recombination

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table11.png)

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-7.png)

Fig 7.  Sequencing result of E6-1, E6-2, and E6-3
It showed normal recombination while E6-1 showed unexpected recombination. The unexpected one is due to the recombination of the two Lac operators flanking the knock-out target site. The actual homologous arm in the situation is 21bp. However, the pT7-Lac promoter set flanking the DSB is 44bp.

Then, employing the condition with the highest editing efficiency from the previous experiment (2g/L L-Arabinose and 3g/L IPTG, for 22hrs and 5hrs respectively) with stgRNA-cassette (1+2) (Fig 8a), stgRNA-cassette (1+2+3) (Fig 8b), and stgRNA-barcode-cassette (1+2) (Fig 8c), we achieved successful self-targeted knockout in all sets.

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-8.png)

Fig 8.  Readout of the multi-level cassette recorder

a. Sequencing result of stgRNA-cassette (1+2). Cassette 2 remained intact.

b. Sequencing result of stgRNA-cassette (1+2+3). Cassette 3 remained intact.

c. Sequencing result of stgRNA-barcode-cassette (1+2).

#### Time-gradient test on stgRNA-cassette (1+2+3)

Consequently, we set up another time-gradient within 2 hours on stgRNA-cassette (1+2+3), to verify the time reliance of the multi-level knock-out (Tbl 10). All of them achieve full knock-out, at a ratio of 100% (Fig 9). However, the time-gradient within 2 hours did not really set up because of inconsistency with the first time-gradient and the small data amount of both. In addition, the Plac leakage was observed once again in the time-gradient test.

Tbl 10.Group design for the time-gradient test

![缺个表](https://static.igem.wiki/teams/4630/wiki/parts/bba-k4630100-stgrna-barcode-cassette-1/table10.png)

The test was carried out on stgRNA-cassette (1+2+3).

![缺个图](https://static.igem.wiki/teams/4630/wiki/parts/cascade-result/asset-9.png)

Fig 9.The knock-out ratio plot of the time-gradient test of stgRNA-cassette (1+2+3)

## Optimization of plasmid pCas

### 1. Elimination of the sgRNA on pCas

In order to prevent the reduction of activity due to genome knockout in *Escherichia coli* where the pCas expression system was transformed, we made optimizations to pCas by eliminating the sgRNA segment on the plasmid.

We eliminated the essential sgRNA segment responsible for genome knockout, and subsequently performed PCR amplification of the long sgRNA fragment using specific primers (Fig 1a). Our DNA product was then circularized through Golden Gate Assembly and transformed into competent *E. coli* DH5α cells. Subsequently, we conducted verification by selecting multiple positive transformants, and both electrophoresis and sequencing analysis confirmed the successful removal of sgRNA (Fig 1b-d).

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig7.png)

Fig 1.  The result of sgRNA deletion in pCas

a. The white hollow arrowheads indicate target bands. The long fragments for Gibson Assembly and Golden Gate Assembly are expected to be 11610 and 11744 bp, respectively. The regular PCR procedure is effective.

b. The white hollow arrowheads indicate target pCasop bands. The primers were designed to amplify sequence covering the omitted sgRNA.

c. Sequencing results were aligned with the original pCas sequence. There is a gap at sgRNA.

d. Sequencing results were aligned with the designed pCasop sequence.

### 2. pCasop exhibited superior growth

Then we proceeded to prove the genome knockout capability. Upon induction, we observed that the bacteria transformed with pCasop exhibited superior growth compared to those transformed with pCas (Fig 2a-e). Through specific primers, we successfully verified that the poxb gene's genome did not knockout in the case of pCasop (Fig 2f).

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/oppcas/fig8.png)

Fig 2.  Genome-editing test for the pCasop

a-b. The induced pCas-containing bacteria were spread on plate, after 100-fold and 1,000-fold dilution, respectively.

c-d. The induced pCasop-containing bacteria were spread on plate, after 100-fold and 1,000-fold dilution, respectively.

e. The quantification of bacteria on the corresponding plates. They were evaluated by three individual testers. The bacteria amount of pCasop is strikingly larger than original pCas.

f. The sequencing results. The genomic knock-out ratio of pCas is 70% (N = 10), while pCasop is 0. The pCas-containing strain without induction was set as positive control.

### 3. Integrated the functional parts of pCas into the recorder

Finally, to make more contribution to synthetic biology, we integrated the functional parts of pCas, the Cas9 and Lambda system, into the plasmid of the recorder device (fig 3).

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/fig12-thaw-2.webp)

Fig 3.  Integrated plasmid construction

a. The preparation of backbone. The white hollow arrowhead indicates the target band. The segments are expected to be 4197 bp.

b. The preparation of Cas and Lambda segments. The white hollow arrowheads indicate the target band. The Cas and Lambda segments are expected to be 4593 and 3399 bp, respectively.

c. The verification of the plasmid. The primers are designed to cover the ligation site.

The prepared Cas, Lambda and backbone DNA sample were used as positive control, as the paired samples differ at several 10 bp.

## EL222 Directed Evolution

### 1. BL21 with pET-28a-[EL222 new-mRFP1] can express mRFP1 when induced by blue light.

#### Part 1: gene synthesis

We synthesized two gene sequences, blue light sensitive protein EL222(BBa\_K2332004), and EL222 controlled promoter pEL222(BBa\_K2332002). Terminators and mRFP1 are acquired using PCR from pET-28a plasmid and iGEM 2019 contribute plate3 5G BBa\_J04450, respectively (Fig 1). Subsequently, they are cloned in pET-28a vector(Fig 2) and expressed in *E. coli* BL21(DE3).

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig1.png)

Fig 1.  Design of EL222-mRFP1. EL222 sequence is inserted between T7 promoter and terminator, located at upstream of pEL222 controlled mRFP1.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig2.png)

Fig 2.  Design of pET-28a-[EL222-mRFP1] transformed into *E. coli* BL21(DE3).

#### Part 2: Blue light induction pre-experiment

Bacteria are spread on LB plates with kanamycin and first cultured under 37℃ for 10 hours, then exposed to 400 Lux blue light around 475nm for 20 and 22 hours with half of the plates covered. Blue lights are controlled using Arduino Mega 2560. On the slides we made, the pET-EIN-mRFP-Golden plasmid transformant exhibits stronger red fluorescence compared to the negative control after 20 hours induction (Fig 3).

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig3.png)

Fig 3.  Image results of induced mRFP1 expression in pET-28a- [EL222 new-mRFP1] transformant, shot at 1/15S, ISO 2000.

#### Part 3: measurement of mRFP1 expression level

We use spectrophotometry to measure the expression level of mRFP1 at protein level (Fig 4). We measured their absorbance at 540nm and 600nm, which present for mRFP1 and bacteria concentration respectively. The OD540/OD600 at a specific time for a sample culture was determined after subtracting from each of the technical triplicate readings of the negative control cultures (fluorescence free) at the same time.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig4.png)

Fig 4.  Absorbance of *E. coli* BL21(DE3) in LB media.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig-5.png)

Fig 5.  Relative express level of *E. coli* BL21(DE3) carrying pET-EIN-mRFP-Golden plasmid after exposing to blue light.

#### Part 4:Bacteria with different pEL222 expressed different amounts of mRFP after induction

We acquired different sequence of pEL222 by error-prone PCR and incorporated the generated sequences into pET-28a-[EL222-mRFP1] to replace the original sequence of pEL222. The plasmid was transformed in BL21(DE3) directly. We sequenced the PCR products (Fig 6) to confirm the success of error-prone PCR.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig6-1.png)
![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig6-2.png)

Fig 6.  Sequencing results of PCR products and transformed plasmids confirm the success in ep-PCR, the sequence of pEL222 has been changed.

To ensure the expression of EL222 protein, We performed the pre-experiment first. We used IPTG to induce the expression of mRFP in pET-EIN-mRFP-Golden plasmid instead of the blue light induction. After 22 hours induction of IPTG, Colonies with different pEL222 show differencet expression of mRFP1, resulting in disparate colors (Fig 7). This work demonstrates that different pEL222 has different sensitivity to the inducer.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig7.png)

Fig 7.  The results of the expression of mRFP1 after induction,The induction conditions are 1.0mM IPTG for 22 hours.

Moreover, we measured the sensitivity and leakage of different pEL222 by debugging different blue light induction times (Fig 8). We cultured all the bacteria in PCR tubes and measure the expression level of mRFP following the same steps in part 3.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/el222-result/fig8.png)

Fig 8.  Relative expression level of different pEL222 sequences after induced by blue light.

## sgRNA Screening

### 1. Construcrion of pUC57-N20s-gRNA+HA

N20 sequence can be loaded onto pUC57-N20s-gRNA+HA. The plasmid synthesis was executed in collaboration with Atantares. Employing Golden Gate Assembly, each plasmid can be endowed with a distinct N20 sequence. The assembly process was successfully executed in conformity with our design, demonstrating the feasibility of the concept of BREM.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig2.png)

Fig 1.  Golden Gate Product

### 2. Knockout for N20s sequence

pUC57-N20s-gRNA+HA with pRed\_cas9\_△poxb300 in *E.coli* DH5α can achieve N20s fragment knockout.Following a 24-hour induction with 2g/L arabinose, we determined the edited colonies via colony PCR. Our analyses revealed successful N20s sequence deletions for some of the tested N20 sequences, exemplified by NO. 11, 13, 14, and 15 (Fig 2), demonstrating the feasibility of the knock out system.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig3.png)

Fig 2.  Knockout of N20s sequence on plasmids

### 3. Construction of pUC57-EGFP-N20s-HA+gRNA

pUC57-EGFP-N20s-HA+gRNA is constructed by Gibson Assembly and Restriction Cloning.

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig6a-b-2.png)

![](https://static.igem.wiki/teams/4630/wiki/webpage/page-content/engineering/sgrna/fig6c.svg)

Fig 3.

a. Preparation of EGFP-N20s by Gibson Assembly

b. Verification of Gibson Assembly product by Colony PCR

c. Verification of Restriction cloning product by Sequencing

## Application

In order to validate the functionality of our recorder in various scenarios, we designed to replace the Pel222 in the stgRNA-cassette system with Pphsa, PluxR, and PcaU activated promoter, which are responsive to biomarker of IBD, biomarker of PA infection, and product of polyethylene terephthalate degradation, respectively. Therefore editing can be initiated by the promoters mentioned above. We synthesized the required DNA fragments in collaboration with biotech companies but received only the sequence for the PluxR system before wiki freeze. We conducted induction experiments with a concentration gradient and time gradient for the PluxR system, however, we were unable to obtain the necessary experimental data due to lack of time.

{% endblock %}