Notebook

The notebook recorded the whole process of our experiment.

READING TIME:

WEEK 1

This week we measured the standard curves of two particles, compared them, sent the results of the second measurement datas to the measurement committee for verification, hoping to find the problem in time for correction.
WEEK 2

The measurement committee responded to the diagnostic report of the measurement results and confirmed that our standard curve can be used to measure the next experimental data;
This week, the coding sequence of CA2(L203K)-N-LCTPSR(BBa_K2949012) and CA2(L203K)-C-LCTPSR(BBa_K2949013) were synthesized and cloned into the expression vector pET-30a(+).

7.22

The primers designed by us began to be amplified by PCR to obtain the mutant CA2 gene;
Agarose gel electrophoresis;

Diluting All Primers

Rweagent	Volume of ddH₂O
aldcF	390μLddH₂O
aldcR	185μLddH₂O
aldnF	210μLddH₂O
aldnR	285μLddH₂O

Ald(c) PCR system

Reagent	Volume
5X Primer STAR Buffer	10μL
dNTP Mixture(2.5mM each)	4μL
Primer 1[aldcF]	1μL
Primer 1[aldcR]	1μL
Template	1μL
Primer STAR	0.5μL
ddH₂O	32.5μL

Ald(n) PCR system

Reagent	Volume
5X Primer STAR Buffer	10μL
dNTP Mixture(2.5mM each)	4μL
Primer 1[aldcF]	1μL
Primer 1[aldcR]	1μL
Template	1μL
Primer STAR	0.5μL
ddH₂O	32.5μL

At the end of experiment, the product of PCR was recovered by gel recovery;

Recycling gel (30mL; 1% agarose gel).

reagent	volume
M(DL15000)	1.5μL
N1	50μL+5μL loading(10x)
N1'	50μL+5μL loading(10x)
C1	50μL+5μL loading(10x)
C1'	50μL+5μL loading(10x)

Identifiying gel (20mL;1％agarose gel)

Reagent	Volume
M(DL15000)	1.5μL
n	2μL+0.5μL loading(10x)
c	2μL+0.5μL loading(10x)

Electrophoresis results

E_W2_01
M :1.5μL DL15000 DNA marker
①: the CA2(L203K)-N-LCTPSR mutant gene
②: the CA2(L203K)-C-LCTPSR mutant gene
The experiment supplies are sterilized to ensure that the experiment is not contaminated by bacteria and the experimental results
are not affected.

7.23

Transform
pET-30a(+) (E.coli BL21); pET-28a(+) (E.coli BL21).
7.24

Pick Monoclone(4mL non-resistant LB medium and 4μL kanamycin);

7.25

The extraction and purification of recombinant plasmid;
Measuring the Absorbance of PlasmidpET300;（5μLplasmid+55μLwater）

Plasmid	Absorbance	Potency
pET-28a(+)	A260:A280=2.0978	c=40μg/mL
pET-30a(+)	A260:A280=2.1711	c=80μg/mL

The identification of empty plasmid by electrophoresis;
Sample: 2μL DL15000mark 1μL plasmid 1μL loading buffer. E_W2_01
Lane ①: pET-28a(+)
Lane ②: pET-30a(+)
Lane ③: pET-28a(+)
Lane ④: pET-30a(+)
The CA2(L203K)-C-LCTPSR and CA2(L203K)-N-LCTPSR recombinant plasmid were constructed by double enzyme digestion.

Empty Plasmid Enzyme Digestion System(30μL)

reagent	volume
NedⅠ	1.2μL
Hind Ⅲ	1.2μL
NEB Buffer 2.1	3μL
pET-30a(+)	1.5μg(10μL)
ddH₂O	14.6μL

CA2(L203K)-C-LCTPSR Gene Enzyme Digestion System(30μL)

reagent	volume
NedⅠ	1.2μL
Hind Ⅲ	1.2μL
NEB Buffer 2.1	3μL
CA2(L203K)-C-LCTPSR gene	20μL
ddH₂O	4.6μL

CA2(L203K)-N-LCTPSR Gene Enzyme Digestion System(30μL)

reagent	volume
NedⅠ	1.2μL
Hind Ⅲ	1.2μL
NEB Buffer 2.1	3μL
CA2(L203K)-N-LCTPSR gene	20μL
ddH₂O	4.6μL

7.26

Gel recovery

Recycling gel (30mL;1％agarose gel)

reagent	volume
DL15000	2μL
c	30μL
n	30μL
pET-30a(+)	30μL

Identifying gel

reagent	volume
DL15000	2μL
c	2μL
n	2μL
pET-30a(+)	2μL

Gel electrophoresis identification results：

E_W2_02
Lane ①:CA2(L203K)-C-LCTPSR target gene fragment
Lane ②:CA2(L203K)-N-LCTPSR target gene fragment
Lane ③:Empty plasmid after enzyme digestion
Construction of recombinant plasmid by double digestion ligation;

CA2(L203K)-C-LCTPSR recombinant plasmid junction system(15μL)

reagent	volume
T4 connective buffer	1.5μL
Empty load after enzyme digestion[pET-30a(+)]	6μL
Target gene after enzyme digestion(C)	2μL
T4 DNA liganse	1.5μL
ddH²O	4μL

CA2(L203K)-N-LCTPSR recombinant plasmid junction system(15μL)

reagent	volume
T4 connective buffer	1.5μL
Empty load after enzyme digestion[pET-30a(+)]	6μL
Target gene after enzyme digestion(N)	2μL
T4 DNA liganse	1.5μL
ddH²O	4μL

WEEK 3

This week, we mainly constructed and identified the recombinant plasmid by restriction enzyme digestion and sequencing.
Transformation;
pET-30a(+)-CA2(L203K)-C-LCTPSR (E.coli BL21);
pET-30a(+)-CA2(L203K)-N-LCTPSR (E.coli BL21)
Pick Monoclone(4mL non-resistant LB medium and 4μL kanamycin):

(pET-30a(+)-CA2(L203K)-C-LCTPSR E.coli BL21 colony)
(pET-30a(+)-CA2(L203K)-N-LCTPSR E.coli BL21 colony)
The extraction and purification of recombinant plasmid；
Identification of recombinant plasmids by gel electrophoresis；
E_w3_03
LaneM:2μL DL15000 DNA marker
Lane① and ②: CA2(L203K)-C-LCTPSR target gene(no results）
Lane③ and ④: CA2(L203K)-N-LCTPSR target gene(the arrow indicated)
The identification of c-terminal recombinant plasmid by restriction enzyme digestion showed no results.

The identificiation of CA2(L203K)-N-LCTPSR recombinant plasmid by restriction enzyme digestion.

Enzyme digestion system of pET-30a(+)-CA2(L203K)-N-LCTPSR

reagent	volume
pET-30a(+)- CA2(L203K)-N-LCTPSR	5μL
NedⅠ	1.2μL
Hind Ⅲ	1.2μL
NEB Buffer 2.1	3μL
ddH²O	9.6μL

Identifying gel

reagent	volume
DL15000	2μL
pET-30a(+)-CA2(L203K)-N-LCTPSR before enzyme digestion	2μL
pET-30a(+)-CA2(L203K)-N-LCTPSR after enzyme digestion	10μL

Agarose Gel Electrophoresis of CA2(L203K)-N-LCTPSR recombinant plasmid and its identification by enzyme digestion (NdeⅠand Hind Ⅲ);
E_w3_04
Lane M: DL15000 marker;
Lane 1: CA2(L203K)-N-LCTPSR recombinant plasmid;
Lane 2: Enzyme digestion band of CA2(L203K)-N-LCTPSR, the length was 834 bp (the arrow indicated)

7.29

Since the identification of CA2(L203K)-C-LCTPSR recombinant plasmid was not successful, the transformed recombinant plasmid was used to re-select monoclones.
7.30

No result was found by gel electrophoresis.
7.31

For the second time, E. coli BL21 was used for transformation, but no colonies were found.

8.1

Start the third construction of CA2(L203K)-C-LCTPSR recombinant plasmid ;
Double digestion connection and transformation.

Connecting System（15μL）

reagent	volume
T4 connective buffer	1.5μL
Empty load after enzyme digestion[pET-30a(+)]	6μL
CA2(L203K)-C-LCTPSR recombinant plasmid after enzyme digestion	2μL
T4 DNA liganse	1.5μL
ddH₂O	4μL

8.2

Pick monoclone;
Redo CA2(L203K)-C-LCTPSR gene fragments in order to avoid further failure;

pET-30a(+) Enzyme Digestion System(30μL)

reagent	volume
NedⅠ	1.2μL
Hind Ⅲ	1.2μL
NEB Buffer 2.1	3μL
pET-30a(+)	1.5μg
ddH²O	4.6μL

Gel recovery；1％agarose gel;30mL；

Identifying gel ;1％agarose gel;20mL;

reagent	volume
DL15000	2μL
CA2(L203K)-C-LCTPSR	2μL
CA2(L203K)-N-LCTPSR	2μL
pET-30a(+)	2μL

Identification results of double digestion

①:CA2(L203K)-N-LCTPSR target gene fragment
②:CA2(L203K)-C-LCTPSR target gene fragment
③:Empty plasmid after the enzyme digestion

8.3

CA2(L203K)-C-LCTPSR recombinant plasmid was identified by agarose gel electrophoresis.
E_w3_06
①②③ are CA2(L203K)-C-LCTPSR recombinant plasmid(the arrow indicated)
superhelix occurred in plasmid ②
The identificiation of CA2(L203K)-C-LCTPSR recombinant plasmid by restriction enzyme digestion.

Enzyme digestion system

Ingredient	Dosage
NedⅠ	1.2μL
Hind Ⅲ	1.2μL
NEB Buffer 2.1	3μL
C1/C2/C4	5μL
ddH²O	9.6μL

Agarose gel electrophoresis of CA2(L203K)-C-LCTPSR recombinant plasmid and its identification by enzyme digestion (NedⅠand Hind Ⅲ);

Lane M: DL15000 marker; Lane 1: CA2(L203K)-C-LCTPSR recombinant plasmid; Lane 2: Enzyme digestion band of CA2(L203K)-C-LCTPSR,the length was 834 bp (the arrow indicated).

8.4

Sequencing results：
CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR gene sequencing is correct
WEEK 4

This week,IPTG was used to include CA2(L203k)-N-LCTPSR and CA2(L203K)-C-LCTPSR expression in recombinant E.coli BL21 (DE3)and SDS-PAGE and Western Blot were used to identify the proteins. At the same time, experiments were carried out in cooperation with Ocean University of China.
8.6

Pick monoclones
[pET-30a(+)-CA2(L203K)-C-LCTPSR(E.coli BL21) and pET-30a(+)-CA2(L203K)-N-LCTPSR(E.coli BL21)];(4mL non-resistant LB medium and 4 μ L kanamycin);
Expand culture (enrich bacterial solution).

8.7

SDS-PAGE detection of CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR protein expression;

Preparation of separation gel（15% 10mL）

Reagent	Volume
distilled water	2.3mL
Acr-Bis	5mL
Tris-hcl(pH=8.8)	2.5mL
10%SDS	0.1mL
10%Ammonium persulfate	0.1mL
TEMED	0.008mL

Preparation of concentrated gel（5% 4mL）

Reagent	Volume
distilled water	2.7mL
Acr-Bis	0.67mL
Tris-hcl(pH=6.8)	0.5mL
10%SDS	0.04mL
10%Ammonium persulfate	0.04mL
TEMED	0.004mL

8.8

Electrophoretic Sample

Reagent	Volume
mark	1.5μL
1xSDS loading Buffer	20μL
n(-)	15μL
n(+)	15μL
c(-)	15μL
c(+)	15μL
1×SDS loading Buffer 15μL	15μL

Testing

Coomassie brilliant blue staining for 20 minutes
No target protein bands were found after decolorization.

First redo

The monoclones of pET-30a(+)-CA2(L203K)-C-LCTPSR(BL21) were selected, and did not grow and the colony died due to the underlined culture, so it needed to be re-transformed and cultured.

8.9

After expanding culture, IPTG induction and SDS-PAGE detection,no significant protein expression was found.

The experimental cooperation with Ocean University of China has been started.
The operation is as follows:
Adda solid puncture bacteria (chassis: Escherichia coli DH5alphaZ1) were collected and concentrated in liquid culture. Single colonies were separated by lines after 10 hours. Monoclones were selected after 20 hours. After 12 hours, different media were added to induce culture for 2 hours, 4 hours, 6 hours, 8 hours, 10 hours and 12 hours, and then the fluorescence and OD600 were measured respectively.
The measurement results are as follows:
8.10

Transform pET-30a(+)-CA2(L203K)-N-LCTPSR (BL21)(correctly sequenced plasmid)
Pick Monoclonal; pET-30a(+)-CA2(L203K)-C-LCTPSR (BL21)
8.11

The transformation of pET-30a(+)-CA2(L203K)-N-LCTPSR(BL21) was successful.
pET-30a(+)-CA2(L203K)-C-LCTPSR(BL21) single clone grows colony, continued to expand the culture for 4 hours, induced for 4 hours, SDS-PAGE detection protein, but did not express successfully.
WEEK 5

Because no results of the CA2(L203K)-C-LCTPSR(BL21) protein expression was detected, the experiment continued,we switched to E. coli TB1 for transformation,fortunately, both of CA2(L203K)-N-LCTPSR protein and CA2(L203K)-C-LCTPSR protein could be successfully expressed in TB1 strain after SDS-PAGE. And we transformed P-BAD-FGE and pET-30a(+)-CA2(L203K)-C-LCTPSR plasmid into E.coli TB1
8.12

Expanded culture of pET-30a(+)-CA2(L203K)-C-LCTPSR (BL21), IPTG induction, sample preparation;
Transform pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1) and pET-30a(+)-CA2(L203K)-N-LCTPSR (TB1);
8.13

1.Transform result:

[pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1)]
[pET-30a(+)-CA2(L203K)-N-LCTPSR(TB1)]
2. Pick monoclonal pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1)and pET-30a(+)-CA2(L203K)-N-LCTPSR(TB1)(4mL non-resistant LB medium and 4 μ L kanamycin).
8.14

Expand culture; IPTG induction; Sample preparation; Electrophoretic dyeing and decolorization;
The results are as follows:

Fig.1 SDS-PAGE analysis for CA2(L203K)-C-LCTPSR cloned in pET-30a(+) vector and expressed in E.coli TB1
Fig.2 SDS-PAGE analysis for CA2(L203K)-N-LCTPSR cloned in pET-30a(+) vector and expressed in E.coli TB1
8.15

Pick monoclonal;
8.16

All monoclonal colonies grow.
Preserve bacteria [pET-30a(+)-CA2(L203K)-C-LCTPSR(TB1), pET-30a(+)-CA2(L203K)-N-LCTPSR(TB1) and pET30a-CcaA(BL21)](-80℃)
8.17

1.Plasmid extraction of pET-30a(+)-CA2(L203K)-C-LCTPSR (TB1) 2.Plasmid co-transformation;[pBAD-FGE+pET-30a(+)-CA2(L203K)-N-LCTPSR]
8.18

Pick monoclonal(plasmid co-transformed bacteria) [LB Medium (+1mL AMP and KANA)]
WEEK 6

This week, the pET-30a(+)-CA2(L203K)-C-LCTPSR protein was purified and concentrated.
8.19

Preparation of KANA and IPTG, Arabic Sugar Solution;
Sterilization by 0.22 μm membrane;
Expand culture E.coli TB1[2mL Bacterial Solution + 200mL LB Medium (+1mL AMP, KANA)];
Measure OD600 with 200μL;
The result of measuring OD600 is 0.641;
Induced expression with Arabic Sugar Solution, 30 minutes later + IPTG
8.20

Cell lysis to obtain protein;
Centrifugal ultrasonic comminution of cells.
8.21

Preparation of 250X, 50X, 5X imidazole 1000 mL;
Adjust PBS to pH 7.4;
Purification of Nickel Column Protein.
8.23

Protein concentrate was centrifuged at 4000rpm for 20 min at 4 ℃.
WEEK 7

This week, we completed the experiments of pET-30a(+)-CA2(L203K)-N-LCTPSR co-transformation, purification and concentration of expressed proteins, and the related experiments of validating part.
8.26

The concentrated protein was identified by dyeing, and the protein was separated.
8.28

Start characterizing the existing biobrick part BBa_K2762004 (CcaA)
Pick monoclones (BL21-CcaA)
8.29

1. Make double-resistance medium of KANA resistance + AMP resistance.
2. Expand BL21-CcaA culture, adding 4ml bacterial solution to 200 ml kanamycin culture medium (100μl), putting it in shaking table, 120rpm, 37℃ for 4 hours, and adding 1 ml IPTG to induce expression.
3. Plasmid co-transformation TB1 [P-BAD-FGE+pET-30a(+)-CA2(L203K)-N-LCTPSR]
4. SDS-PAGE analysis for CcaA cloned in pET-30a(+) vector and expressed in BL21 strain
The results showed that pET-30a(+)-CcaA could be successfully expressed in BL21 strain.
8.30

1. Centrifugal bacterial fluid (BL21-CcaA)
2. Broken strain (BL21-CcaA)
3. Pick monoclones (CA2(L203K)-N-LCTPSR-CHO-FGE)
8.31

1. Expand culture of E.coli TB1 [4ml bacterium solution + 200mL LB(100μLKANA + 100μLAMP+)]
2. Protein Purification [Nickel Column Purification]
3. Protein Concentration
4. Induced expression [+1 ml arabinose ; +1 ml IPTG after 30 min]
WEEK 8

In order to get more CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR protein,we continue to do protein purification and concentration (CA2(L203K)-N-LCTPSR-FGE) this week.
Resuspension, crushing strains, protein purification and centrifugal concentration.
WEEK 9

This week, CA2(L203K)-N-LCTPSR and CA2(L203K)-C-LCTPSR protein immobilization and esterase activity assay were completed.

9.10

Immobilization;

Standard protein concentration determination.

Standard protein curve
E_w9_01_

Sample	Absorbance	Average	After deducting PBS
CA2(L203K)-C-LCTPSR immobilization (dilution 10 times)	0.401 0.379 －	0.39	0.103
CA2(L203K)-C-LCTPSR not immobilized (diluted 10 times)	0.414 0.41 －	0.412	0.125
CA2(L203K)-N-LCTPSR immobilization (dilution 10 times)	0.412 0.416 0.404	0.410667	0.123667
CA2(L203K)-N-LCTPSR not immobilized (diluted 10 times)	－ 0.436 0.43	0.433	0.146
CA2(L203K)-C-LCTPSR (diluted 10 times)	0.431 0.431 0.432	0.431333	0.144333
CA2(L203K)-N-LCTPSR (diluted 10 times)	0.456 0.442 0.456/td>	0.451333	0.164333
PBS	0.292 0.284 0.285	0.287	0

It can be obtained from standard protein curve y=1.0633x+0.0386.

CA2(L203K)-C-LCTPSR immobilization (dilution 10 times)= 0.0605662 mg/ml	39.09%
CA2(L203K)-C-LCTPSR not immobilized (diluted 10 times)= 0.0812565 mg/ml	18.28%
CA2(L203K)-C-LCTPSR (diluted 10 times)= 0.0994385 mg/ml	20.81%
CA2(L203K)-N-LCTPSR immobilization (dilution 10 times)= 0.0800000 mg/ml	32.35%
CA2(L203K)-N-LCTPSR not immobilized (diluted 10 times)= 0.1010063 mg/ml	14.58%
CA2(L203K)-N-LCTPSR (diluted 10 times)= 0.1182479 mg/ml	17.77%

9.12

Esterase assay for enzyme activity

Preparation of Standard BSA Solution
WEEK 10

In order to obtain more proteins for enzyme immobilization and multiple assay of enzyme activity,this week we successfully re-transformed pET-30a(+)-CA2(L203K)-C-LCTPSR, pET-30a(+)-CA2(L203K)-N-LCTPSR.And completed the experiments of purification and concentration of expressed proteins.
9.13

Make double-resistance medium of KANA resistance + AMP resistance.
9.14

Transformation;
pET-30a(+)-CA2(L203K)-C-LCTPSR (E.coli TB1);
pET-30a(+)-CA2(L203K)-N-LCTPSR (E.coli TB1).
9.15

1.Expanded culture of E.coli TB1 [4ml bacterium solution + 200mL LB(100μLKANA + 100μLAMP+)]
2.Induced expression[+1 ml arabinose ; +1ml IPTG after 30min];
pET-30a(+)-CA2(L203K)-C-LCTPSR (E.coli TB1)
pET-30a(+)-CA2(L203K)-N-LCTPSR (E.coli TB1)；
9.16

1.Protein Purification [Nickel Column Purification]
2.Protein Concentration
9.17

SDS-PAGE detection

SDS-PAGE analysis of purified CA2(L203K)-N-LCTPSR protein

SDS-PAGE of purified CA2(L203K)-C-LCTPSR protein
WEEK 11

This week,CA2(L203K)-C-LCTPSR protein and CA2(L203K)-N-LCTPSR immobilization and esterase assay were completed.
9.22

Immobilized CA2(L203K)-C-LCTPSR protein and CA2(L203K)-N-LCTPSR protein.
9.24

Esterase assay for enzyme activity of free CA2(L203K) protein, free and immobilized CA2(L203K)-C-LCTPSR protein, free and immobilizedCA2(L203K)-N-LCTPSR protein at 37℃ and 50℃.
The results showed as follows:

Esterase activity analysis of free CA2(L203K) and CA2(L203K)-C-LCTPSR protein and immobilized CA2(L203K)-C-LCTPSR protein at 37℃

Esterase activity analysis of free CA2(L203K) and CA2(L203K)-C-LCTPSR protein and immobilized CA2(L203K)-C-LCTPSR protein at 50℃

Esterase activity analysis of free CA2(L203K)-N-LCTPSR protein and immobilized CA2(L203K)-N-LCTPSR protein at 37℃ and 50℃
WEEK 12

This week, the reuse ability of CO₂ capture of the immobilized CA2(L203K)-C-LCTPSR was tested by our designed simulation model.
The result as follows:

CATALOGUE

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Team:AHUT China/Notebook

WEEK 1

WEEK 2

7.22

Diluting All Primers

Ald(c) PCR system

Ald(n) PCR system

Recycling gel (30mL; 1% agarose gel).

Identifiying gel (20mL;1％agarose gel)

Electrophoresis results

7.23

7.24

7.25

Empty Plasmid Enzyme Digestion System(30μL)

CA2(L203K)-C-LCTPSR Gene Enzyme Digestion System(30μL)

CA2(L203K)-N-LCTPSR Gene Enzyme Digestion System(30μL)

7.26

Gel recovery

Recycling gel (30mL;1％agarose gel)

Identifying gel

Gel electrophoresis identification results：

CA2(L203K)-C-LCTPSR recombinant plasmid junction system(15μL)

CA2(L203K)-N-LCTPSR recombinant plasmid junction system(15μL)

WEEK 3

The identificiation of CA2(L203K)-N-LCTPSR recombinant plasmid by restriction enzyme digestion.

Enzyme digestion system of pET-30a(+)-CA2(L203K)-N-LCTPSR

Identifying gel

7.29

7.30

7.31

8.1

Connecting System（15μL）

8.2

pET-30a(+) Enzyme Digestion System(30μL)

Gel recovery；1％agarose gel;30mL；

Identifying gel ;1％agarose gel;20mL;

Identification results of double digestion

8.3

Enzyme digestion system

Agarose gel electrophoresis of CA2(L203K)-C-LCTPSR recombinant plasmid and its identification by enzyme digestion (NedⅠand Hind Ⅲ);

8.4

WEEK 4

8.6

8.7

Preparation of separation gel（15% 10mL）

Preparation of concentrated gel（5% 4mL）

8.8

Electrophoretic Sample

Testing

First redo

8.9

8.10

8.11

WEEK 5

8.12

8.13

8.14

8.15

8.16

8.17

8.18

WEEK 6

8.19

8.20

8.21

8.23

WEEK 7

8.26

8.28

8.29

8.30

8.31

WEEK 8

WEEK 9

9.10

Standard protein concentration determination.

9.12

Preparation of Standard BSA Solution

WEEK 10

9.13