iGEM 2022 — Team hainanu-china

{% extends "layout.html" %}
{% block htitle %}Notebook{% endblock %}
{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each
day for your project.{% endblock %}
{% block page\_content %}

* Non-experimental
* Experimental

# Non-Experimental Part

## 2022.3.10

The team is officially established - from different majors such as biology, chemistry, agronomy and computer
science, we form a team!

![png]()

## 2022.3.19

First formal offline meeting - get to know each other - determine initial division of labor

![png]()

## 2022.3-4

Continuously communicate with experts and professors to refine experimental ideas and determine project
feasibility

## 2022.4.29

First cooperation - Establishing long-term cooperation with JLU-China

![png]()

## 2022.7

Market research - field trips to several aquaculture companies and breeding sites

![png]()

## 2022.7.16-2022.7.18

Participate in the exchange meeting held by GXU\_China and other four schools, and simulate the demonstration

![png]()

## 2022.8.6-2022.8.8

Participate in Hangzhou HZ offline meetup

![png]()

## 2022.8.14

Collaborate with other teams to hold a meetup on crispr

![png]()

## 2022.8.19-2022.8.21

Participated in CCiC - won the best hardware design award

![png]()

## 2022.9.16

Interview - Interview with Zhong Yongjie, an engineer at Microkrypton, Hainan Province

![png]()

## 2022.9.22

Brochure - Finalizing the Cooperation Brochure with JLU-China

## 2022.8-2022.9

Education - Science outreach to middle and high school students

![png]()

## 2022.7-2022.9

Create and continuously improve our posters, videos and presentations

## 2022.9

Design, populate and improve our wiki, edit review forms

## 2022.10.26-2022.10

Grand jamboree - participation in a large gathering and presentation of our project

# Experimental Part

## 6.29-7.3

### Purification of cas14 protein.

Open group meetings to discuss experimental details and familiarize with the experimental process of purified
proteins

## 7.4

### Purification of cas14 protein.

Prepare experimental materials for the purification of cas14 protein, strains, instruments, etc.

## 7.5

### Purification of cas14 protein.

1.Preparation of LB medium 500ml\*4

2. Shake the bacteria to recover.

Take LB medium 3ml\*2 (TEV,MBP)

+Bacterial night 30 ul + antibiotic (Amp) ampicillin 3 ul

Constant temperature shaker 37℃ 180rpm 12-16he

## 7.6

### Purification of cas14 protein.

1.Culture medium sterilization

2.Expansion, induction.

Take sterilized LB medium\*4(TEV\*2,MBP\*2)

+500 ul antibiotic (Amp)+ 2ml ante-night bacteria night

Shaker 37℃ 180rpm 6-8h

Shake well for TEV and MBP

TEV+IPTG 50 ul \* 237℃ 180 rpm for 12h

MBP+IPTG 100 ul \* 218℃ 180 rpm for 12h

## 7.7

### Purification of cas14 protein.

1.Bacteria-breaking lysis.

Take TEV and MBP and centrifuge at 10,000 rpm for 5 min, discard supernatant and addlysate 5 ml/tube (operate
in ice box, move fast), blow well, and combine three tubes into onetube.

Crushing bacteria with ultrasonic 25% power on 4s off 10s working time 50-60mine

2.Preparation.

Purified water Equilibrium solution Elution solution (wash once)20% ethanol sequentialextraction on the
machine, wash the machine (full water wash once) on the HS nickel column(1ml/min), wash the column, put the tube
into the corresponding reagent bottle Syringe Filtermembrane filtration sample.

3.Purified proteins.

TEV and MBP maximum speed 5ml/min

A1 pure water A2 equilibrium solution B eluent A3 20% ethanol

When the machine is turned off, the water is washed once, and the unloading column(1ml\min) is washed once with
ethano

4. Measure protein concentration (kit) Measure protein concentration (kit), 96-well plate, 100ul BCA
working solution per well, 1ul Cu reagent 10ul protein, two protein two wells, measure absorbance, about 0.4 is
normal value.

5.Mixed digestion with protein concentration 1:1 equal volume digestion (TEV slightly more) after
mixing two tubes, 4℃ overnight

## 7.8

### Purification of cas14 protein.

1.Protein purification

Ultrafiltration tube with filtered water, centrifuge 3800r 30min twice Equilibrium solution 30min twice

Top sample, centrifuge, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times on the
machine.

Wash the machine with the same as before, wash the column to wash the equilibrium solution more than once,
heparin column 1ml/min (pay special attention to sample evacuation)

## 7.9

### Purification of cas14 protein.

Purified protein experimental results: purification failure, the experimental group met to discuss the reasons,
may be operational errors, organize the material and re-purified cas14 protein.

## 7.13

### Purification of cas14 protein.

1.Preparation of LB medium 500ml\*4e'2.Shake the bacteria to recover.

Take LB medium 3ml\*2 (TEV,MBP)

+Bacterial night 30 ul + antibiotic (Amp) ampicillin 3 ul

Constant temperature shaker 37℃ 180rpm 12-16h

## 7.14

### Purification of cas14 protein.

1.Culture medium sterilization

2.Expansion, induction.

Take sterilized LB medium\*4(TEV\*2,MBP\*2)

+500 ul antibiotic (Amp)+ 2ml ante-night bacteria night

Shaker 37℃ 180 rpm 6-8h

Shake well for TEV and MBP

TEV+IPTG 50 ul \* 237℃ 180 rpm for 12h

MBP+IPTG 100 ul \* 218℃ 180 rpm for 12h

## 7.15

### Purification of cas14 protein.

1.Bacteria-breaking lysis.

Take TEV and MBP and centrifuge at 10,000 rpm for 5 min, discard supernatant and add lysis solution 5 ml/tube
(operate in ice box, move fast), blow well, three tubes in one tube

Crushing bacteria with ultrasonic 25% power on 4s off 10s working time 50-60 min

2.Preparation.

Purified water Equilibrium solution Elution solution (wash once) 20% ethanol sequential extraction on the
machine, wash the machine (full water wash once) on the HlS nickel column (1ml/min), wash the column, put the
tube into the corresponding reagent bottle Syringe Filter membrane filtration sample

3.Purified proteins.

TEV and MBP maximum speed 5ml/min

A1 pure water A2 equilibrium solution B eluent A3 20% ethanol

When the machine is turned off, the water is washed once, and the unloading column (1ml\min) is washed once
with ethanol

## 7.16

### Purification of cas14 protein.

Measure protein concentration (kit) Measure protein concentration (kit), 96-well plate, 100ul BCA working
solution per well, 1ul Cu reagent 10ul protein, two protein two wells, measure absorbance, about 0.4 is normal
value

Mixed digestion with protein concentration 1:1 equal volume digestion(TEV slightly more) after mixing two
tubes, overnight at 4℃

## 7.17

### Purification of cas14 protein.

ultrafiltration tube, with filtered water, centrifuge 3800r 30mintwice Equilibrium solution 30min twice

Top sample, centrifuge, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times

Get on the machine

Wash the machine with the same as before, wash the column to wash the equilibrium solution more than once,
heparin column 1ml/min (pay special attention to sample evacuation)

Problems: upper two tev lower two mbp: broken tube mix leads to experiment termination

![png]()

Figure 1. Computer display when performing protein purification experiments

## 7.18

### Purification of cas14 protein.

The experiment was stopped due to an error, and members of the experimental group analyzed
the experimental process to find the error

## 7.19

### Purification of cas14 protein.

The experiment was stopped due to an error, and members of the experimental group analyzed the experimental
process to find the error

## 7.20

### Purification of cas14 protein.

After discussion, it was decided to change the eluent pH/elution column to re-purify cas14 protein.

## 7.21

### Purification of cas14 protein.

1. Preparation of LB medium 500ml\*4

2. Shake the bacteria to recover.

Take LB medium 3ml\*2 (TEV,MBP)

+ bacterial night 30μl + antibiotic (Amp) ampicillin 3μl

Constant temperature shaker 37℃ 180rpm 12-16h

## 7.22

### Purification of cas14 protein.

1. culture medium sterilization

2. Expansion, induction.

Take sterilized LB medium\*4 (TEV\*2,MBP\*2)

+500μl of antibiotics (Amp) +2ml of ante-night bacteria night

Shake at 37℃ 180rpm for 6-8h

Shaken TEV and MBP

TEV+IPTG 50μl \* 237℃ 180rpm 12h

MBP+IPTG 100μl \* 218℃ 180rpm 12h

## 7.23

### Purification of cas14 protein.

1. Bacteria-breaking lysis.

Take TEV, MBP high-speed centrifugation 10000 rpm 5min, discard the supernatant add lysate 5ml / tube
(operation in the ice box, the action should be fast), blow well, three tubes in one tube

Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min

2. Preparation.

Purified water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, wash
the machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the
corresponding reagent bottle syringe filter membrane sample

3. Purification of proteins.

TEV and MBP max speed 5ml/min

A1 pure water A2 equilibrium solution B eluent A3 20% ethanol

When the machine is turned off, water wash once, unload the column (1ml/min) ethanol wash once

![png]()
![png]()

Figure 2. Computer display when performing protein purification experiments

4. measurement of protein concentration (kit) measurement of protein concentration (kit), 96-well
plate, each well, 100ul BCA working solution, 1ul Cu reagent 10ul protein, two protein two wells, measurement of
absorbance, 0.4 or so is the normal value

![png]()

Figure 3. Reagent kit operating instructions

![png]()
![png]()

Figure 4. Computer display when performing protein purification experiments

5. Mixed digestion with protein concentration 1:1 equal volume digestion (TEV slightly more) after
mixing two tubes, 4℃ overnight

## 7.24

### Purification of cas14 protein.

1. Protein purification

Ultrafilter tube with filtered water, centrifuge 3800r 30min twice Equilibrium solution 30min twice

Top sample, centrifugation, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times

Loading the machine

Wash the machine as before, wash the column to wash the equilibrium solution once more, heparin column 1ml/min
(pay special attention to sample evacuation)

![png]()

Figure 5. Purified protein successfully

## 7.25

### Purification of cas14 protein.

After discussion and testing, the strain was suspected to be improperly preserved, and the failed experience
was summarized to optimize the experimental process

## 7.26

### Purification of cas14 protein.

Repurify cas14 protein

## 7.27

### Purification of cas14 protein.

Repurify cas14 protein

## 7.28

### Purification of cas14 protein.

Repurify cas14 protein

## 7.29

### Purification of cas14 protein.

Repurify cas14 protein

## 7.30

### Purified cas14 protein.

Protein purification successfully, continue to determine mbp, tev

## 7.31

### Purified cas14 protein.

The assay failed, the experiment held a group meeting to discuss, further analyze the reasons and optimize the
experimental process

## 8.6

### Purification of cas14 protein.

Review the experimental procedure and find the possible reasons why mbp and tev could not be measured: eluent
ph is too high/bacterial problems/column problems, etc.

![png]()

Figure 6. Computer display when performing protein purification experiments

![png]()

Figure 7. Computer display when performing protein purification experiments

## 8.7

### Purified cas14 protein.

Repurification of cas14 protein

## 8.8

### Purification of cas14 protein.

Repurify cas14 protein

## 8.9

### Purified cas14 protein.

Repurify cas14 protein

## 8.10

### Purification of cas14 protein.

Repurify cas14 protein

## 8.11

### Purification of cas14 protein.

Successful cas14 protein purification

## 8.13

### Purification of csm6 protein.

## 8.14

### Purification of csm6 protein.

1. Preparation of LB medium 500ml\*2 Sterilization

2. Shake the bacteria to recover.

Take LB medium 3ml\*2

+ 30μl of bacterial night + 3μl of caramycin

Constant temperature shaker 37℃ 180rpm 12-16h

## 8.15

### Purification of csm6 protein.

1. Expansion, induction.

Take sterilized LB medium + 500μl caramycin + 3ml antecedent bacterial night

Shake 37℃ 180rpm 6-8h

Shake the good bacteria solution

Csm6+IPTG 100μl 18℃ 180rpm 12h

## 8.16

### Purification of csm6 protein.

1. Bacterial lysis by breaking.

Take the bacteriophage solution high-speed centrifugation 10000rps 5min, discard the supernatant add lysate,
blow evenly, three tubes in one tube

Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min

### 2. Preparation.

Pure water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, cleaning
machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the
corresponding reagent bottle syringe filter membrane sample

## 8.17

### Purification of csm6 protein.

Run Gel Validation

SDS-page protein gel electrophoresis

1. gel preparation

2. sample processing

3. add maker and sample

4. add running buffer and perform electrophoresis according to the procedure

5. stain the plate and decolorize

6. observation

![png]()
![png]()

Figure 8. Running glue results are displayed under the machine

## 8.18

### Purification of csm6 protein.

Summarize the experimental results csm6 protein purification successfully

![png]()

Figure 9. Running glue results are displayed under the machine

## 8.24

### Preparing for rna extraction

## 8.25

### Extraction of RNA.

21:00 200ml LB liquid medium, 1.5ml EP tube, 10ml EP tube sterilized

## 8.26

### Extraction of RNA.

9:00 Take 5ml liquid medium + 200ul bacteria and shake it.

9:20 Mix lysozyme solution with TE buffer to a concentration of 15mg/ml, mix, dispense appropriate
amount and store at -20℃.Dilute RNA wash buffer II with 100% alcohol (alcohol:buffer2=4:1, store at room
temperature)

15:00 Extraction of total RNA using E.Z.N.A. Bacterial RNA Spin kit

(Refer to the kit instructions for the experimental procedure)

The obtained RNA samples are stored at -80°C

17:40 Preparation of TAEbuffer for gum preparation

Run gum 2% TAE agarose condensate 5ul of sample

![png]()

Figure 10. Running glue results are displayed under the machine

## 8.27

### Extraction of RNA.

21:00 100ml LB liquid medium, 10ml,1.5ml EP tube sterilization

9:00 5ml LB liquid medium+200ul bacteria reagents shake bacteria

9:20 500ml LB solid medium configuration, sterilization

11:40 Add antibiotics to the solid medium, pour the plate and put it into 4℃ refrigerator for backup

15：00 Dilute the bacterial solution by the following multiples

| 10 | 10² | 10³ | 10⁴ | 10⁵ | 10⁶ | 10⁷ | 10⁸ | 10⁹ |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

Measure the OD value of undiluted bacterial solution, diluted bacterial solution; medium is also
diluted and measure the OD value

![png]()
![png]()

Figure 11. Experimental results under different conditions

16：30 Select 10⁴, 10⁶, 10⁸ dilutions of bacterial solution to coat the plate, add 100ul of bacterial
solution to each plate

19:00 Put the coated plates into 37℃ incubator and count the bacteria the next day.

## 8.29

### Extraction of RNA.

10:00 Counting bacteria

![png]()

Figure 12. Colonies under different experimental conditions

## 8.30

### Extraction of RNA.

21:00 200ml LB liquid medium, 1.5ml EP tubes, 10ml EP tubes sterilized

## 8.31

### Extraction of RNA.

Take 5ml of liquid medium + 200ul of bacterium and shake the bacterium.

Mix lysozyme solution with TE buffer to a concentration of 15mg/ml, mix in, dispense the appropriate amount and
store at -20°C

Dilute RNA wash buffer II with 100% alcohol (alcohol:buffer2=4:1, store at room temperature)
Extract total RNA using E.Z.N.A. Bacterial RNA Spin kit

The obtained RNA samples were stored at -80°C

17:40 Preparation of TAEbuffer Gum preparation

Run gum 2% TAE agarose condensate 5ul of sample

Mutual aid experiment: the virulence gene plasmid arrives, start preparing for mutual aid experiment

![png]()

Figure 13. Extraction of RNA

## 9.1

### Extraction of RNA.

Prepare 100ml LB liquid medium, 10ml,1.5ml EP tube sterilization

Mutual experiment: learn and communicate the experimental procedure Prepare experimental materials

![png]()

Figure 14. Synthetic Report Card

## 9.2

### Extraction of RNA.

LB liquid medium + 200ul bacteria Resuscitation shaking bacteria

500ml LB solid medium configuration, sterilization

Add antibiotics to the solid medium, pour the plate and put it into 4℃ refrigerator for backup

Dilute the bacterial solution by the following multiples

| 10 | 10² | 10³ | 10⁴ | 10⁵ | 10⁶ | 10⁷ | 10⁸ | 10⁹ |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

Measure the OD value of undiluted bacterial solution, diluted bacterial solution; the same dilution of the
medium and measure the OD value

OD value of 0.444 for one group and 0.241 for one group

OD value 0.444 group choose to dilute 10⁶, 10⁷, 10⁸ times to coat the plate

OD value 0.241 group choose to dilute 10, 10², 10³ times to coat the plate

![png]()
![png]()

Figure 15. Results under different experimental conditions

## 9.3

### Extraction of RNA.

Counting bacteria

![png]()

Figure 16. Number of colonies under different experimental conditions

## 9.4

### Mutual Aid Experiment.

Learning and Communicating Experimental Procedures Preparing Experimental Materials

![png]()

Figure 17. Diagram of notes taken during the exchange

## 9.5

### Mutual Aid Experiment.

![png]()

Figure 18. Synthesis Report Instructions

## 9.6

### Mutual Aid Experiment.

![png]()

Figure 19. Notes on experimental methods

## 9.8

### Reciprocal experiment.

![png]()

Figure 20. The pro strain arrived at

## 9.9

### Extraction of RNA.

200 ml LB liquid medium, 1.5 ml EP tubes, 10 ml EP tubes sterilized

## 9.10

### Extraction of RNA.

Take 5ml of liquid medium + 200ul of bacteria and shake the bacteria.

Lysozyme solution and TE buffer mixed to a concentration of 15mg/ml, mixed in, divided into appropriate
amounts, stored at -20 ℃

Dilute RNA wash buffer II with 100% alcohol (alcohol:buffer2=4:1, store at room temperature)

Extract total RNA using E.Z.N.A. Bacterial RNA Spin kit

The obtained RNA samples were stored at -80°C

17:40 Preparation of TAEbuffer Gum preparation

Run gum 2% TAE agarose condensate on sample 5ul

Reciprocal experiment.

Amplification of 4 pairs of primers for virulence genes pcr attempt with medium (solid liquid) Sterilization
Gum preparation and running gum Plate scribing and running gum

![png]()
![png]()

![png]()

Figure 21. Experiment in progress

## 9.11

### Extraction of RNA.

Prepare 100 ml LB liquid medium, 10 ml,1.5 ml EP tubes sterilized

Reciprocal experiments.

PcR amplification (same day as PCR - the previous day is pre-experiment) But x 4 total 16) Glue making and
glue running Glue sterilization for eight consecutive tubes, gun tip

![png]()
![png]()

Figure 22. Colonies of culture medium

![png]()
![png]()

Figure 23. Extraction of pro plasmids

![png]()
![png]()

Figure 24. blra rubber recycling

## 9.12

### Extraction of RNA.

LB liquid medium + 200ul bacteria Resuscitation shaking bacteria

500ml LB solid medium configuration, sterilization

Add antibiotics to the solid medium, pour the plate and put it into 4℃ refrigerator for backup

Dilute the bacterial solution by the following multiples

| 10 | 10² | 10³ | 10⁴ | 10⁵ | 10⁶ | 10⁷ | 10⁸ | 10⁹ |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |

Measure the OD value of undiluted bacterial solution, diluted bacterial solution; the same dilution of the
medium and measure the OD value

One group 0.488, one group 0.157

OD value 0.488 group choose to dilute 10⁴, 10⁵, 10⁶ times coated plate

OD value 0.157 group choose to dilute 10³, 10⁴, 10⁵ times to coat the plate

![png]()

Figure 25. Reciprocal experiments

pro plasmid try pcr amplification (pre-experiment - probe concentration + try parameters)

![png]()
![png]()
![png]()

Figure 26. Experimental notes and results of glue running

## 9.13

### Extraction of RNA.

Counting bacteria

![png]()

Figure 27. Culture medium incubated during the experiment

### Reciprocal experiments.

Results are with heterobands all selective gel recovery, no selective column recovery. -- Do more tubes of
amplification the next day.

No band OR with trailing band: fragment is too long, template concentration is too high, annealing
temperature is too low, primer specificity is not good

![png]()
![png]()

Figure 28. The instruments we use for experiments

## 9.14

### Mutual aid experiment.

Failure reason analysis.

1. added the primer of the connector may be a little less primer specificity

2. yesterday's pcr template 1 overnight degradation

3. plasmid did not mention well: too much bacteriophage, plasmid may be mixed with a lot of broken
E. coli genomic DNA

Correct the error and re-experiment

## 9.15

### Reciprocal experiment.

Extraction of plasmids with arabinose antibiotics

![png]()
![png]()
![png]()

Figure 29. The plasmids we use for our experiments

## 9.16

### Mutual aid experiment.

Pre-amplification of pcr

Results: abnormal but with bands

![png]()
![png]()

Figure 30. Display of the results of the glue run on the computer

## 9.17

### Reciprocal experiment.

Fold amplification Recovery

Result: Failure Error during glue run

![png]()
![png]()

Figure 31. Gum recovery results of mishandling

## 9.18

### Reciprocal experiment.

Repeat 9.17 + Method II Method III for double digestion

![png]()
![png]()
![png]()

Figure 32. Display of the results of the glue run on the computer

## 9.19

### Reciprocal experiment.

Transformation Homologous recombination (method 3, method 4) Double digestion (method 1)

Results: method 1 needs to be redone Adding the wrong enzyme

![png]()

Figure 33. Experimentally produced colonies

## 9.20

### Reciprocal experiment.

Observation plate: no bacteria growth Suspect transformation problem

PCR with recovery to do 1step of pro

Make solid medium + sterilize the gun tip and eight-link tube

## 9.22

### Mutual aid experiment.

Discuss and analyze the possible causes with the mutual help subjects.

1. not completely protected from light, toxic genes in the natural light leakage

2. Failure due to inaccurate heating and time

3. plasmid base number is large, self-spitting phenomenon occurs, need to keep changing the sensory state to try

4. reverse pcr, pro plasmid is too long and dragging, etc.

5. The enzyme used is not common

## 9.23

### Mutual help experiment.

Summarize the reasons for failure and then discuss the procedure of subsequent experiments

![png]()

Figure 34. Notes for learning in experiments

## 9.24

### Reciprocal experiment.

E. coli receptor state, 1 μL of virulence gene plasmid was added to 50 μL of receptor state, plates were coated after transformation according to the transformation process, 50 μL of solution was added to each plate and placed in 30°C for overnight incubation.

![png]()

Figure 35. Results of the different colonies in the experiment

## 9.25

### Extraction of RNA.

9：00 Resuscitation shake (5ml LB medium + 200ul bacteriophage solution)

14：00 Measure OD bacteriophage value, dilute the bacteriophage to about 0.157 with medium

![png]()

Figure 36. Resulting data of the experiment

14:30 Dilute the bacterial solution to 0.127 with sterile water 10⁵ times

14:40 Total RNA was extracted simultaneously from the original and 10⁵ times diluted bacterial broth using the E.Z.N.A. Bacterial RNA Spin kit.

(Refer to the kit instructions for the experimental procedure)

The obtained RNA samples were stored at -80°C

### Reciprocal experiments.

![png]()

Figure 37. Water bath for culture solution

![jpg]()

Figure 38. Dispense medium

## 9.27

### Extraction of RNA.

11:00 Run nucleic acid gel

Prepare 2% TAE agarose agglutination solution

Sample 5ul

Failure, the strips did not run, probably because the TAE solution has been prepared for too long and has deteriorated

![jpg]()

Figure 39. Experimental results

{% endblock %}

{% extends "layout.html" %}
{% block htitle %}Engineering{% endblock %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the
engineering design cycle.{% endblock %}
{% block page\_content %}

## Overview

Our ultimate goal is to build a single-base, amplification-free, portable set of detection platforms. In the
engineering success page, we first need to implement the most important and experimentally verified part, the
single-base accuracy part. We learned from our review and experiments that Cas13a and Cas14a can tolerate 1-2 base
mutations in target sequence recognition, specifically, if the target RNA or DNA has single nucleotide polymorphism
in the detection system, it will lead to unclear signal source (from target sequence or mutated sequence), and
subsequently will produce a "false positive "false positive" characterization.

To address this problem, we thought of combining single base mutation sequence with complementary paired CLAMP to
shield the mutation sequence and enable Cas protein to recognize the target sequence more accurately. At the same
time, to ensure the stability of the CLAMP-mutation sequence, we will use peptide nucleic acid (PNA) as the backbone
of CLAMP, which will reduce the intensity of the "false positive" signal and make it easier for us to identify the
source of the signal.

![png]()
![png]()

To achieve amplification-free detection, we will use the trans cleavage activity of Cas13a and Cas14a to cleave and
degrade the labeled nucleic acid by coupling with TtCsm6 and Csm6 activator (A4-U6 oligonucleotide) to generate
fluorescent signals (through the linkage between the proteins to transmit and amplify the signal in the process),
coupled with the base complementary pairing PNA "CLAMP" to shield the single base mutation sequence, this detection
system has a specificity, sensitivity and high efficiency that no single protein detection means has.

## Engineering Cycle

## CYCLE1：

### Expression of cas and csm6 proteins.

parts:[BBa\_K4223018](http://parts.igem.org/Part%3ABBa_K4223018)、BBa K4223008

## 1.1 DESIGN：

His tag-MBP-Cas14a9 ([BBa\_K4223018](http://parts.igem.org/Part%3ABBa_K4223018)) makes an important part of
our project and we use it for efficient and high quality Cas14a protein expression and purification extraction. MBP
helps to increase the yield of soluble protein in E. coli, and TEV is used as an enzymatic site to purify MBP-Cas14a
recombinant protein after His tag binding to a Ni column, which retains the pure Cas14a protein and passed through
the heparin column.

![png]()

## 1.2 BUILD

His tag-MBP-Cas14a1 purification
Day1
1.Preparation of LB medium 500ml\*4
2.Shake the bacteria to recover.
Take LB medium 3ml\*2 (TEV,MBP)
+ bacteria night 30μl + antibiotic (Amp) ampicillin 3μl
Constant temperature shaker 37℃ 180rpm 12-16h
Day2
1.Sterilization of culture medium
2. Expansion, induction.
Take sterilized LB medium\*4 (TEV\*2,MBP\*2)
+500μl of antibiotics (Amp) +2ml of the night before the bacteria
Shake at 37℃ 180rpm for 6-8h
Shaken TEV and MBP
TEV+IPTG 50μl\*2 37℃ 180rpm 12h
MBP+IPTG 100μl\*2 18℃ 180rpm 12h
Day3
1. Bacteria-breaking lysis.
Take TEV, MBP high-speed centrifugation 10000 rpm 5min, discard supernatant add lysis solution 5ml / tube (operation
in the ice box, the action should be fast), blow well, three tubes in one tube
Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min
2. Preparation.
Purified water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, wash the
machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the corresponding
reagent bottle syringe filter membrane sample
3. Purification of proteins.
TEV and MBP max speed 5ml/min
A1 pure water A2 equilibrium solution B eluent A3 20% ethanol
When the machine is turned off, water wash once, unload the column (1ml/min) ethanol wash once

![png]()
![png]()

4. Measure protein concentration (kit) Measure protein concentration (kit), 96-well plate, 100ul BCA working
solution per well, 1ul Cu reagent 10ul protein, two protein two-well, measure absorbance, about 0.4 is normal value

![png]()
![png]()

5. Mix digestion with protein concentration 1:1 equal volume digestion (TEV slightly more) two tubes mixed, 4 ° c
overnight
Day4.
1. Protein purification
Ultra-filter tube, with filtered water, centrifuge 3800r 30min twice Equilibrium solution 30min twice
Top sample, centrifugation, 30min 3800r until protein is consumed, replenish equilibrium solution 3 times
Loading the machine
Wash the machine as before, wash the column to wash the equilibrium solution once more, heparin column 1ml/min (pay
special attention to sample evacuation)

![png]()

Csm6 purification
Day1
1.Preparation of LB medium 500ml\*2 Sterilization
2.Shake the bacteria to recover.
Take LB medium 3ml\*2
+bacterial night 30μl+carboxymycin 3μl
Constant temperature shaker 37℃ 180rpm 12-16h
Day2
1. Expansion, induction.
Take sterilized LB medium + 500μl of caramycin + 3ml of ante-night bacterium night
Shaking bed 37℃ 180rpm 6-8h
Shake the good bacteria solution
Csm6+IPTG 100μl 18℃ 180rpm 12h
Day3
1. Bacteria breaking lysis.
Take the bacterium solution high-speed centrifugation 10000rps 5min, discard the supernatant add lysate, blowing
uniformly, three tubes in one tube
Use ultrasonic crushing bacteria 25% power on 4s off 10s working time 50-60min
2. Preparation.
Pure water equilibrium solution eluent (wash once) 20% ethanol sequential extraction on the machine, cleaning
machine (full water wash once) on the HIS nickel column (1ml/min), wash the column, the tube into the corresponding
reagent bottle syringe filter membrane sample
2. Collect the sample
Run gel verification
SDS-page protein gel electrophoresis
1.Gel preparation
2.Sample processing
3.Add maker and sample
4.Add running buffer to electrophoresis according to the procedure
5.Staining of gel plate Decolorization
6.Observation

![png]()
![png]()

## 1.3 TEST

**To further examine the protein activity of Cas14a and Csm6**,

we set up the system without the addition of
target DNA as the control group and the system with the addition of target DNA as the experimental group. The
fluorescence assay was performed on 4 groups of samples at 37°C using an enzymatic standard (excitation wavelength
492 nm, emission wavelength 520 nm). Three parallel samples of each group were measured and the variance and mean
were calculated and plotted with Origin 9.0. As shown in the figure, the validation of the cleavage activity of
cas14a1 and csm6 proteins was finally completed.

![png]()

Validation of the activity of Csm6 protein

![png]()

Validation of the activity of Csm14 protein

## 1.4 LEARN

Here, we successfully expressed Cas14a and csm6 proteins, which were verified to be ready for use after activity.
However, we did not get the expected results when expressing Cas13a protein, and the expressed Cas13a concentration
was very low, which was not improved after several attempts to change the conditions of different reaction systems,
so we considered asking other teams for help or purchasing Cas13a protein in future experiments.

We will then perform a false positive test in the next step

**to complete the proof of concept of the
experimental idea done in the wet experiment.**

## CYCLE2：

## False-positive tests

## 2.1 DESIGN：

**In the previous cycle, we successfully expressed the desired Cas and Csm6 proteins and presented the problem with a
corresponding solution. We will then confirm the "false positive" characterization in the next cycle to determine
the interference of the mutant sequence with the clear signal source when the target sequence coexists with the
mutant sequence in the reaction system.**

We designed two 22-base-long target ssDNA sequences and target ssRNAs, respectively, and both designed 12 sequences
with inversions at odd sites, respectively, as mutant sequences in our laboratory:

![png]()

Test sequence of Cas13a

![png]()

Test sequence of Cas14a

## 2.2 BUILD

We took the target and mutant sequences and added them to the reaction system of Cas13a and Cas14a, which was
carried out as follows.

Cas14a
Reaction Buffer (20 mM tris-hcl, 20 mM NaCl, pH 9.0)
15 uL: Cas14a （final 500nM） &sgRNA (final 500nM) in Reaction buffer ;
6 uL: Mg ion ( final 10mM) & FQ (final 400nM in reaction buffer);
9 uL: T or Mx & Clamps in reaction buffer.
(2x) buffer: 20 mM tris-hcl, KCl 100 mM, pH=8.2.
20 uL /cell：
10uL buffer(2x);
0.8uL 500nM Cas13a;
0.1uL 5uM crRNA;
0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.)
37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ ;
（12.17 uL in total ca. 12 uL)
Cas13a Continuous system
(2x) buffer: 20 mM HEPEs, pH=7.5.
20 uL /cell：
10uL buffer(2x);
0.8uL 500nM Cas13a;0.1uL 5uM crRNA;
0.67uL 150mM Mg2+(ca. 5mM Mg2+ final.)
1uL 200mM KCL;
37 ℃ Incubation 10-15min, add probe 0.6uL（10uM） FQ , 0.5uL Csm6;
（total 13.67 uL )
Ac & Target or clamps or mutants (in DEPC water) total 6.33 uL。
Target or clamps or mutants (in DEPC water) total 7.83 uL。

## 2.3 TEST

![png]()

Detection results of Cas14a protein for DNA sequences (revealed by relative fluorescence unit RFU)

![png]()

Detection results of Cas13a protein for DNA sequences (revealed by relative fluorescence unit RFU)

The recognition and cleavage of the target sequences by Cas14a and Cas13a proteins, thus exhibiting trans cleavage
activity, and consequently the fluorescence signal report, we show as Relative fluorescence unit (RFU). We can
observe that each detected sequence exhibits a different fluorescence signal intensity, and M7, M20, RM8, RM15 and
RM19 are significantly larger than the target sequence. This indicates that false positive characterization does
exist and the specificity of the detection is affected differently depending on the mutation site.

## 2.4 LEARN:

In this engineering cycle, we validated the false positive characterization of the assay using Cas13a and Cas14a
proteins from the previous engineering cycle, and after we concluded that false positives do exist and interfere
with the source of the signal, we started to design a solution to the problem - using PNA-CLAMP to mask the
mismatched sequences

## Circle3：

## Blocking of mismatched sequences using PNA

## 3.1 DESIGN

**Here we plan to provide a solution to the false positive problem that occurred in the last engineering cycle. We
will use synthetic target sequences with mutant sequences in the wet lab to simulate the processing of samples in a
real environment.**

Here we plan to provide a solution to the false positive problem that occurred in the last engineering cycle. We
will use synthetic target sequences with mutant sequences in the wet lab to simulate the processing of samples in a
real environment.

![png]()

Comparison of PNA and DNA structures

## 3.2 BUILD

During this BUILD period, we tested multiple sets of different base mismatches and the shielding effect of PNA and
DNA on the sequences at different sites and different lengths. However, since PNA is expensive, it is costly and
time-consuming to design DNA/PNA for each of the above mentioned mutant sequences for shielding, so we selected the
more representative mutant sequences for our experiments.

## 3.3 TEST

Taking Cas14a1 as an example, based on the experimental results in Cycle 2 (Confirmation of "false positive"
characterization), sequences with mutation sites at 5', -5, 7, 20-3' bases were selected from a series of sequences
with single-base mutations. -5, 7, 20-3' bases, which showed high RFU in the false positive mock assay, were
selected as typical sequences to demonstrate the role of the shackle system (clamp).
Initially, we used the designed complementary DNA as clamp as a pre-experiment to verify the preliminary role of
Clamp. As illustrated by the experimental results presented in the figure below: the Relative fluorescence unit
(RFU) of the experimental group with the addition of DNA-clamp was significantly lower than that of the control
group, and combined with the Delta-RFU analysis, Clamp did reduce the interference of mutant sequences on the assay
results.

![png]()
![png]()

But just DNA as clamp is not enough for the goal of our project. So we designed PNA as clamp and validated it in
the same way. We also compared the control group with DNA-clamp and PNA-clamp to make the data more reliable. The
above two experiments also demonstrate that our idea of designing "Clamp" to avoid the defect of misidentification
of target sequences by Cas13a and Cas14a due to similar mutated sequences can be realized.

![png]()
![png]()
![png]()

Although we have verified that PNA as clamp has better effect than DNA as clamp, however, we are not yet sure to
what extent the shielding effect of using PNA as clamp on single base mutation false sequences can be achieved, and
it is not clear what effect different concentrations of PNA have on the effect of target sequence detection. So, we
set a certain amount of PNA (100nM) and gradient concentrations of target and mutant sequences in combination, and
found that the interference of PNA on mutant sequences was significantly greater than that of target sequences, and
even after the concentration of mutant sequences was less than 100nM, its RFU dropped in a precipitous manner. When
PNA was relatively saturated in the mutant sequence, it was able to play an almost complete shielding role. In the
absence of PNA addition, the magnitude of RFU change of both was not as obvious as when PNA was added.

![png]()
![png]()

In the system of Cas13a, as shown in the figure below, the shielding effect of PNA-CLAMP on complementary sequences
gradually increased as the concentration of PNA-CLAMP increased (0-100 nM) for a certain amount of the shielded
sequence, indicating that the shielding effect increased with the amount of PNA-CLAMP within a certain concentration
range.

![png]()
![png]()

In the data in the figure below, we show that when PNA-CLAMP coexists with target and mutant sequences, only the
signal of mutant sequences is significantly shielded, while target sequences are largely unaffected. It indicates
that the application of PNA-CLAMP in single-base detection platform is feasible and has predictable good results.

![png]()

## 3.4 LEARN

### Instrument

Currently, in vitro nucleic acid assays based on CRISPR technology are in the initial development stage. Existing
PCR nucleic acid assays require special instrumentation, laboratory testing sites and specialized technical staff,
and have strict zoning requirements for laboratories. In this environment, we decided to develop a portable enzyme
marker based on our project to validate and promote our project.
For more details, please see the Hardware page (please tie the wiki hardware link to Hardware at the Expo)

## Outlook

In the era of the epidemic, we are quite sure that our project has strong applications. However, even if our
protocol is feasible, there are still many issues and challenges to really develop it to maturity. How to further
improve the sensitivity and specificity of the nucleic acid assay, how to combine and simplify the reagent
components, how to make the CRISPR-based nucleic acid assay home-based/private, how to further reduce the cost, how
to combine the CRISPR nucleic acid assay technology with the instrumentation to automate the rapid detection, these
are all things we are committed to achieve.

We will continue to innovate and breakthrough ourselves, and not end with IGEM, and finally build a perfect testing
platform to make our own contribution to improve the society!

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## WET LAB

## Ideas and design

CRISPR (Clustered Regularly interspaced short palindromic Repeats), known as Clustered regularly interspaced short palindromic repeats, is an immune system that bacteria use to protect themselves from viruses. It's not just widely used in gene editing.

In recent years，Scientists from different countries have tried to develop new tools which are based on CRISPR-Cas system to detect nucleic acids.Fortunately,they successfully expanded the areas of CRISPR-Cas system and set up some effective platforms to detect[1].

Scientist describe a tandem nuclease assay using Cas13a and Csm6 to achieve both high sensitivity and fast signal generation without requiring a preceding target amplification step. This involved the design of potent, chemically stabilized activators for Csm6 as well as use of eight different CRISPR RNA (crRNA) sequences for Cas13a detection. they also show that this assay can be implemented in a portable device, consisting of a microfluidic chip and a compact detector, to detect viral RNA extracted from human samples. This indicates that the assay is robust and simple to adapt for use in point-of-care testing workflows. This also highlights the value of combining unrelated CRISPR–Cas effectors for sensitive, one-pot detection of RNA.[2].

Based on the above previous exploration, our project has selected Cas13a and Cas14a proteins, and we hope to build a more perfect and accurate detection platform. We will artificially design Peptide nucleic acids (PNA) for base complementary pairing with single base mutations, so as to improve the accuracy of target sequence detection and improve the specificity of the detection system, so as to realize the ability of single base recognition and detection. This scheme is an improvement of the existing nucleic acid detection method of CRISPR / Cas, system (Cas13a and Cas14a), and uses the signal amplification of csm6 protein for the detection of Cas13a and Cas14a proteins.

## Validation of protein expression and activity

We successfully obtained the target protein using BL21 (DE3) expression, and successfully purified Cas13a, Cas14a and csm6 proteins using the protein purification instrument belonging to the State Key Laboratory of Marine Resources in the South China Sea. Later, we determined that the activity of the above three proteins was at high levels, and the restriction activity of both proteins was also tested, and the results also met our expected requirements for the protein.

![png]()

## Confirmation of “false positive” characterization

We validated the false-positive characterization of Cas13a1 proteins by designing the single-nucleotide polymorphism sequence of Target RNA to simulate the false-positive recognition representations generated in real situations. The recognition and cleavage of the artificially designed RNA by the Cas13a1 protein shows the trans-cleavage activity, and then the resulting fluorescence signal is reported by Relative Fluorescence unit (RFU). We can observe that each detected sequence exhibits a different fluorescence signal intensity, and that RM8, RM15 and RM19 are significantly larger than the target sequence. This indicates that false positives do exist and the specific effects on detection varies depending on the mutation sites.

## Verification of clamp effect

Clamp is a system that we envisage to assist Cas13a, Cas14a proteins to avoid causing "false positive" characterization, and the principle is to artificially design complementary sequences of single-base mutant sequences, hoping to improve the accuracy of detection. We first designed the DNA chain as clamp, and found that the Relative fluorescence unit (RFU) of the experimental group adding DNA-clamp was significantly lower than that of the control group. Combined with the Delta-RFU analysis, Clamp did reduce the interference of the single base mutation sequence on the test results, but the effect did not meet our expectations. Ultimately, we determined that PNA has a better "yoke" effect than DNA[3]. To verify the quantitative effect, we set the quantitative PNA (100nM) and the gradient concentration of the target sequence and the mutation sequence binding, and found that the PNA interfered significantly more to the mutation sequence than the target sequence, and even after the mutation sequence concentration is less than 100nM, the RFU fell off a cliff. When PNA is relatively saturated with the mutant sequence. The magnitude of RFU change without PNA was not as pronounced as when PNA was added. At a certain concentration of PNA (100nM), the shielding effect of PNA on mutant sequence is relatively obvious at a lower concentration (<100nM), and the shielding effect of PNA gradually decreases as the concentration of mutant sequence increases; the interference rate of PNA to mutant sequence gradually decreases as the latter concentration increases until it approaches 0. The experiments also illustrate that our idea of artificially designing PNA to block the signal interference caused by false identification is feasible and enables accurate detection at the single-nucleotide level.

## DRY LAB

## Analysis and structure analysis of the physicochemical properties of proteins

Before deciding to perform the experiments with the cas13a protein and the csm6 proteins, we analyzed the physicochemical properties, the hydrophilic hydrophobicity, the stability of the protein structure, etc. For the hydrohydrophobicity part, we used the open-source program of Proscale to predict the hydrophobicity of cas13, csm6 from the amino acid composition. Because the amino acid sequence of the protein determines its hydrophilic / hydrophobicity, the hydrophobicity determines its solubility in water, the hydrophobicity is conducive to the protein folding to the inside to form a secondary structure, further form a domain, tertiary structure, and the strong hydrophobicity is more conducive to the protein forming a helix, increase stability. Later, we analyzed the physicochemical properties of the two proteins. whose basic profile is as follows, including the team model section.

![png]()
![png]()

## Kinetic simulation of the PNA shielding reaction

The CRISPR / Cas system is a technique of targeting genes for modification by RNA-directed Cas proteins derived from bacteria-acquired immunity. The CRISPR / Cas group can be divided into three types, among which, the A isoform in the type III CRISPR-Cas system has an effector complex called Csm, which consists of multiple Cas proteins, together with the crRNA. The Csm complex not only cleaves the target RNA complementary to the crRNA, Moreover, the binding of the target RNA can activate the two novel enzymatic activities generated by the Csm complex, That is, the cleavage of the ssDNA during transcription and the activity of the synthetic cyclic oligo-adenylate cOA, The cOA acting as a second messenger can activate Csm6, Non-specific degradation of the RNA, Type III CRISPR can produce two cOA: cA6 and cA4, It can bind to the CARF domain on the cas protein, In turn, activating the HEPN domain for nonspecific cleavage, Cut off the predesigned fluorescence group with the quencher, Release of the fluorescent signal, Reference to the Team model section for detailed results.

## Unwinding temperature prediction model based on near-neighbor method model and DNN neural network

In the experiment, we achieved the accuracy to single base specificity by blocking PNA for the mismatch chain, but different from using DNA as a chain, PNA, as a peptide nucleic acid, has the following advantages:
When it is complementary to the nucleic acid chain, the unwinding temperature greatly increases the relative DNA / DNA binding;
When a base mismatch occurs, a single base pair mismatch can reduce the Tm value by 8 to 20℃ (an average of 15℃), much higher than the reduced temperature during DNA / DNA binding.
We propose to find a modeling method for the simulation predictions of the optimal unwinding temperature when screened with PNA.

## Portable detection hardware

In our wet experiment, our team cut a single base and generated a fluorescent signal; to transform and apply our results, we imagined building a device that can identify the fluorescent signal. Different from the traditional microplate reader, which has a large size, large weight, high cost and an inconvenient need to connect to the computer, we have successfully developed a small and convenient microplate reader through 3D printing technology after completing the circuit design. This will help to make our project results much easier to use.

## References

[1]Gootenberg JS, Abudayyeh OO, Kellner MJ, Joung J, Collins JJ, Zhang F. Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science. 2018 Apr 27;360(6387):439-444. doi: 10.1126/science.aaq0179. Epub 2018 Feb 15. PMID: 29449508; PMCID: PMC5961727.

[2] Liu, T.Y., Knott, G.J., Smock, D.C.J. et al. Accelerated RNA detection using tandem CRISPR nucleases. Nat Chem Biol 17, 982–988 (2021).

[3] Egholm M, Buchardt O, Christensen L, et al. PNA hybridizes to complementary oligonucleotides obeying the Watson–Crick hydrogen-bonding rules[J]. Nature, 365(6446): 566-568（1993）.

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