iGEM 2023 — Team shanghai-city

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{% block title %}Notebook{% endblock %}
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![](https://static.igem.wiki/teams/4873/wiki/notebook.png)

**Date:** 2023.07.21

**Event:** 1st meeting at lab

**Agenda:**

1.
LB medium preparation

2.
PCR amplification ( zliE-zliS, SacB)

3.
Electrophoresis verification: agarose
gel

4.
Gel recycling

5.
Reverse PCR

**Date:** 2023.07.22

**Event:** 2nd meeting at lab

**Agenda:**

1.
Continue to complete the remaining reverse PCR on
the 21st

2.
Complete the unsuccessful process on the
21stPCR: primmer: zliE F1 + zliS R1

3.
Restriction digestion of empty plasmid and target
fragment

4.
Enzyme digestion to verify EV ( empty vector
)

5.
Recycle DNA

6.
Enzyme-linked plasmid fragments and target
fragment

**Date:** 2023.07.23

**Event:** 3rd meeting at lab

**Agenda:**

1.
PCR amplify RFP ( outline: prepare for RFP; plasmid
2--->plasmid 3 )

2. Homologous recombination ( homology arm
)

3. Transform into *CH5α E.coli* and grow overnight.

**Date:** 2023.07.24

**Event:** 4th meeting at lab

**Agenda:**

1.
PCR ( E.coli with plasmid 3/plasmid 1 ( RFP without its own promoter ) )

2.
AMP solid medium

3.
Gel electrophoresis

4.
PCR ( to amplify the promoter of RFP ( PRFP ) )

5.
PCR ( Because the DNA electrophoresis of plasmid 1 failed in the previous test, plasmid 1
should be reconstructed )

**Date:** 2023.07.25

**Event:** 5th meeting at lab

**Agenda:**

1.
Gel electrophoresis

2.
Extract plasmid 3

3.
DNA extraction

4.
Digestion

5.
Enzyme link

6.
Convert

7.
Gel electrophoresis ( only for plasmid 1, since the
previous ones failed )

8.
Culture *E.coli* with three different plasmids

**Date:** 2023.07.26

**Event:** 6th meeting at lab

**Agenda:**

1.
Colony PCR ( *E.coli* with plasmid 1/plasmid 2/plasmid 4 )

2.
AMP ( *E.coli* with plasmid 4; prepare for the next section:
IPTG )

3.
Gel electrophoresis ( PCR product with plasmid 1&2&4
)

4.
Inverse PCR ( prepare for building up plasmid 3,
amplifying plasmid 2 )

5.
Ampicillin configuration

**Date:** 2023.07.27

**Event:** 7th meeting at lab

**Agenda:**

1.
IPTG & Sucrose control variable

2.
Gel electrophoresis

3.
RM media test

4.
Gel electrophoresis ( Because the IPCR of plasmid 2
failed in the previous section )

**Date:** 2023.07.28

**Event:** 8th meeting at lab

**Agenda:**

1.
Extraction of sample fructan

2.
Testing the effect of sucrose and IPTG

3.
Prepare the solution for the standard curve of
fructan

4.
Optical density value ( OD 540nm )

**Date:** 2023.0
9
.
01-09.08

**Event:**
Repeat some experiments under the guidance of mentor

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

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering.png)

**Engineering Success**

**Overview**

Our team embarked on a rigorous scientific journey to enhance the
visibility and yield of levan. Preliminary research indicates the potential of levan as an additive
in pharmaceuticals, health supplements, and dermatological products due to its anti-aging and
potential anti-carcinogenic properties. The prevailing challenge is the prohibitive cost of levan
production and testing. Through a biosynthetic pathway, we have made contributions by altering the
promoter sequences in the levan expression pathway to further enhance levan production. We look
forward to inspiring and serving as a reference for future large-scale industrial production of
levan.

**Our D****-****B****-****T****-****L cycle**

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-1.png)

Fig.1 Overview of Designs

**Cycle 1---****Enhancing Levan Visibility**

**Design:** we planned to build 4 plasmids. At first, we only have a EV
(empty vector) pUC19, so in order to construct a plasmid that can express itself by having fluorescence,
we will have to add regulators and others. As a result, we planned that the first plasmid should be
pUC-zliES, and then plasmid 2 pUC-zli-SacB
(Part:BBa\_K4873052)
, plasmid 3 pUC-zli-SacB-RFP
((Part:BBa\_K4873054))
, and finally plasmid 4 pUCzli-SacB-PRFPT
(
Part:BBa\_K4873055
)
.
And we expected to see the fluorescence of the red color from plasmid
4.

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-2.png)

Fig.2
 The graph that illustrates the process of construction plasmids
and
transform
 the plasmids into *E.coli*.

**Build:**
I
n the lab
, we conducted a substantial amount of experimental work.
Finally
,
 we generally used PCR, IPCR, enzyme digestion, enzyme ligation,
transformation, sequencing identification, and homologous recombination to build up different
plasmids. In the end, we successfully constructed a
plasmid 4 capable of expressing RFP.

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-3.png) |

Fig.
3
 The
sequence
map
 of plasmid 4
(pUCzli-SacB-PRFPT)

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-4.png)

Fig.4 Identification of the promoter fragment after enzyme digestion
through DNA gel electrophoresis, with a fragment size of approximately 300 bp.

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-5.png)

Fig.5 Sequencing results indicate the successful
construction of pUC-Zli-SacB-
P
RFPT.

**Test:**
To validate the successful expression of our RFP reporter gene in the
system, we transformed Plasmid 4 into Escherichia coli for expression. After fluorescent imaging, we
observed a significant red fluorescence in the RFP.

![](https://static.igem.wiki/teams/4873/wiki/engineering/10-engineering-success-upaded-chirs2265.png)

Fig.6
 The recombinant plasmid containing RFP successfully expressed in
Escherichia coli, exhibiting red color.

**Cycle 2---****Evaluating the Efficiency of Various Promoters**

**Design:** we planne
d
 to compare the effects of promoter plac and
p
uv on levan production. So, based on the plasmid 4 we had constructed,
we will just have to use PCR and enzyme digestion and ligation to construct **plasmid 5****（****pUC-zli-plac-SacB-PRFPT****, BBa\_K4873056****）**
.

**Build
：**
I
n the lab
, we conducted a substantial amount of experimental work.
Finally
,
 we generally used PCR, IPCR, enzyme digestion, enzyme ligation,
transformation, sequencing identification, and homologous recombination to build up different
plasmids. In the end, we successfully constructed a
plasmid 5 capable of expressing RFP.

![](https://static.igem.wiki/teams/4873/wiki/engineering/10-engineering-success-upaded-chirs3035.png)

Fig
.7
Monoclonal culture containing plasmid 5

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-8.png)

Fig
.8
 Sequencing results indicate the successful construction of
pUC-zli-plac-SacB-PRFP

**Test
：**

In order to investigate the expression levels of SacB in different promoter
systems (plasmid 4/5), we first compared the fluorescence intensity of RFP. We observed that in the
Plasmid 5 system, SacB exhibited the highest red fluorescence intensity. Subsequently, we measured
the fluorescence intensity values of RFP, as shown , which also confirmed that Plasmid 5 had higher
values, indicating that SacB has a higher expression level in the Plasmid 5 system.

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4873/wiki/engineering/10-engineering-success-upaded-chirs3641.png) |

Fig.9
In the Plasmid 4/5 expression systems, when comparing the brightness of
RFP, Plasmid 5 exhibits stronger RFP expression.

**Cycle** **3****---****Evaluating the Efficiency of Various Promoters****(II)**

**Design**
: To further investigate the impact of changes in promoter
sequences on SacB expression, we replaced plac with the puv promoter, constructing Plasmid 6
(pUC-zli-puv-SacB-PRFPT, BBa\_K4873057), using a series of genetic engineering methods.

**Build**
：
Plasmid 6 has been successfully constructed. We validated our
experiments through DNA gel electrophoresis and sequence comparison analysis.

![](https://static.igem.wiki/teams/4873/wiki/engineering/10-engineering-success-upaded-chirs4228.png)

Fig.10
Monoclonal culture containing plasmid 6

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-11.png)

Fig.11
The sequencing results confirm the successful construction of
pUC-zli-puv-SacB-PRFP

**Test:**
In order to investigate the expression levels of SacB in different promoter
systems (plasmid 4/5/6), we first compared the fluorescence intensity of RFP. We observed that in
the Plasmid 5 system, SacB exhibited the highest red fluorescence intensity. Subsequently, we
measured the fluorescence intensity values of RFP, as shown , which also confirmed that Plasmid 5
had higher values, indicating that SacB has a higher expression level in the Plasmid 5 system.

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-12.png) |

Fig.12 In the Plasmid 4/5/6 expression systems, when comparing the
brightness of RFP, Plasmid 5 exhibits stronger RFP expression.

We conducted qualitative (RFP color brightness) and quantitative analyses
of RFP expression levels in different plasmid systems. Figure 13 demonstrates that in the Plasmid 5
system, there is also a higher level of RFP expression.

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-13.png)

Fig.13. Quantitative analysis of RFP expression levels in three different
systems, with the vertical

In order to validate which biosynthetic pathway results in higher levan
production, we compared the levan yields in the plasmid 4/5/6 systems. The average levan production
was higher in the plasmid 5/6 systems, as shown in Figure 14-B/C/D. Additionally, we also tested
levan production at different temperatures. In all three systems, levan production was higher at
around 30
°
C.

![](https://static.igem.wiki/teams/4873/wiki/engineering/engineering-14.png)

Fig14. pUCzli-SacB-PRFP, pUCzli-PlacsacBRFP, pUCzli-PUVsac-BRFP plasmids
’
expression and functional identification.

**Learn**: In the construction of Parts, every modification to
each component can have an impact on gene expression. In this design, we anticipated that the expression
levels of Levan and RFP should be the highest in the Plasmid 6 system. However, experimental results
showed higher expression in the Plasmid 5 system. This presents us with new challenges, which we will
investigate further in future experiments
.

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{% block title %}Experiments{% endblock %}
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![](https://static.igem.wiki/teams/4873/wiki/experiments.png)

1)
Experimental notes and process

l
**LB media**

- **Objective:** Culture and Amplify *E. coli*

- **Concepts:**

1.
tryptophan and yeast extract provide carbon and nitrogen needed by
*E. coli*.

2.
NaCl helps maintain the osmotic pressure.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| Tryptone | 10 g/L |
| Yeast extract | 5 g/L |
| NaCl | 10 g/L |

  Note: solid: +3 g agar powder

- **Methods:**

1.
Use an electronic balance to weigh the required reagents
separately.

2.
Mix tryptone, yeast extract, NaCl powder, agar powder, and water in a
conical
.
 Shake the container until the solute dissolves. Adjust pH to 7.0
with 5mol/LNaOH. Adjust volume to 1L with deionized water.

3.
Use millipore filter membrane to seal the conical flask.

4.
Place a piece of autoclave indicator tape on the conical flask
.

5.
Steam sterilization was performed at 121 ° C for
21min.

- **Results:**

1.
Autoclave indicator tape turns black

2.
The solution shows a yellow color

l
**PCR**

- **Objective:** Amplify a specific DNA fragment

- **Concepts:** At 95
℃
, the DNA helix unwinds transforme
d
 from double to single. When held at 72
℃
 for 10 min, all the single chains will become double. During the
cycle, the structure of DNA transformed repeatedly between single and double strands.

- **Materials:**

Mix: Mg2+, DNA polymerase, dNTP, ddH2O

Amplifying the target gene of plasmid 1 (zliE-zliS )

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: zliE-F1 | 1 |
| Primer: zliS-R1 | 1 |
| DNA template: *zm4* | 1 |
| dd H2O | 7 |
| Total | 20 |

Amplifying the target gene of plasmid 2 (SacB)

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: SacB-F1 | 1 |
| Primer: SacB-R1 | 1 |
| DNA template: *zm4* | 1 |
| ddH2O | 7 |
| Total | 20 |

- **Methods:**

1.
Mix some of the reagents mentioned above in a centrifuge tube
separately (group 1: zliE F1 + zliS R1 + ddH2O + mix + template zm4; group 2: SacB F1 + SacB R1 + ddH2O + mix + template zm4 ), except zm4, ( all tubes should be placed
in crushed ice, otherwise most enzyme will become inactive, such as DNA polymerase )

2.
Vortex and centrifuge (to avoid the occurrence of solution hanging on
the wall of the tube and Mix well )

3.
Place in PCR machine

4.
Turn on the machine

5.
Click “file” on the screen

6.
Click “program
”

7.
Click “primmer start”

PCR machine program settings:

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 sec |
| 3 | 55 | 30 sec |
| 4 | 72 | 1min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 cycles

- **Result:** Achieving DNA amplification

l
**Gel electrophoresis**

- **Objective:** To check whether the DNA sample contains the correct DNA
segments/fragments we need

- **Concepts:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the
correct DNA fragments we need according to the marker.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils dissolve ( to ensure uniform
mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL )

4.
Pour the agarose gel slowly into the mold, taking care not to create
bubbles Wait until the agarose gel setsRemove the comb

5.
Pour TAE buffer solution into the tank

6.
Use the pipette to add the four DNA samples made in the step “PCR” and
marker to the corresponding grooves respectively.

7.
Put the lid on and power on (180V, 20 min)

8.
(After 20 min) put the gel under a gel imager to document
bands

9.
Software: Genosens

10.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Result:** group 2 SacB DNA successfully separated

l
**DNA extraction**

- **Objective:** separate the target DNA fragments from the gel and detect the
concentration of DNA

- **Concepts:** binding, washing and eluting from a silica column

- **Materials:**

 Silica column

|  |  |
| --- | --- |
| Name | Dosage ( μL) |
| Buffer 2 | 300 |
| Wash solution | 500 |
| Elution buffer | 40 |

- **Methods:** (note: protein: 280 nm; DNA: 260 nm )

![](https://static.igem.wiki/teams/4873/wiki/experiment4247.png)

1.
Cut out the gel with target DNA

2.
Mix the gel that contains DNA with 300µL Buffer 2 in a
tube

3.
Place the tube in a water bath at 65˚C for 5-10 min

4.
Centrifuge (8,000
×
g ) for 30 s and pour off the underlying liquid ( liquid in the
transparent tube )

5.
Add 500 µL wash solution

6.
Centrifuge (9,000
×
g ) for 30 s and pour off the underlying liquid ( liquid in the
transparent tube )

7.

R
epeat 5 and 6

8.
Centrifuge (9,000
×
g ) for 30 s and pour off the underlying liquid ( liquid in the
transparent tube )

9.
Centrifuge (9,000
×
g ) for 1 min

10.
Place the solution (with the internal yellow tube ) into two new 1.5 mL
tubes

11.
Add 40 µL elution buffer into tube

12.
Wait for 1 min

13.
Centrifuge (9,000
×
g ) for 1 min

14.
Detect DNA concentration

15.
Click “nucleic acid” on the screen

16.
Add 1 µL elution buffer into the “small hole” on the
instrument

17.
Click “empty test” + “zero”

18.
Dropping 1 µL DNA sample to detector

19.
Click “sample testing”

(note: normally: A260/A280: 1.8-2.1 )

- **Results:**

1.
Concentration: 0.9ng/µL

2.
A260/A280: 2.25

 (reasons: RNA/DNA remains a lot of fragments; if the figure for
A280 is higher than the normal range ( 1.8-2.1 ): some proteins have not been cleaned up )

l
**Inverse PCR**

- **Objective**: amplify a specific DNA fragment

- **Concepts**: Because there is no suitable enzyme cutting site on this structure,
or there is a terminator blocking the normal direction, it is necessary to change the direction for PCR.
The purpose of inverse PCR is to amplify DNA flanking a known sequence, that is to say, this reaction
system synthesizes DNA not between a pair of primers but outside the primers.

- **Materials**:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Template: plasmid 4 | 1 |
| Primer: zliE-F1 | 1 |
| Primer: SacB-F4-avr | 1 |
| mix | 20 |
| ddH2O | 17 |
| Total | 40 |

Mix: Mg2+, DNA polymerase, dNTP (SacB fragment ), ddH2O

- **Methods**:

1.
Mix all reagrens mentioned above ( add them in order from largest to
smallest )

2.
Place the tube inside the centrifuge machine

3.
Take out the tube and place it in a PCR machine

4.
Turn on the machine

5.
Click “file” on the screen

6.
Click “program
”

7.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 s |
| 3 | 55 | 30 s |
| 4 | 72 | 5 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 cycles

- **Result**: Achieving DNA amplification

1.
Continue to complete the remaining reverse PCR on the
21st

2.
Complete the unsuccessful process on the 21st: PCR: primmer: zliE F1 + zliS R1

3.
Restriction digestion of empty plasmid and target fragment

4.
Enzyme digestion to verify EV (empty vector )

5.
Recycle DNA

6.
Enzyme-linked plasmid fragments and target fragment

l
**PCR** (single--->double; Annealed PCR )

- **Objective**: Amplify the single-stranded gene (plac and puv) purchased from the
company into a double-stranded structure (amplify plac and puv), and use T4 ligase to help connect them.
And finally provide materials for the subsequent process.

- **Concepts:** When a single-stranded gene is heated and annealed, it is
amplified and double-stranded.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage ( μL) |
| T4 ligase buffer | 2 |
| Primer 1 | 5 |
| Primer 2 | 5 |
| ddH2O | 8 |
| Total | 20 |

- **Methods:**

1.
Mix all the reagents mentioned above in a centrifuge tube

2.
Place the tube inside the centrifuge (to avoid the occurrence of
solution hanging on the wall of the tube and mix well )

3.
Take out the tube and place the tube inside a PCR machine

4.
Turn on the machine

5.
Click “file” on the screen

6.
Click “program
”

7.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 5 min |
| 2 | 80 | 1 min |
| 3 | 70 | 1 min |
| 4 | 60 | 1 min |
| 5 | 50 | 1 min |
| 6 | 40 | 1 min |

- **Results:** Successfully changed the single-stranded structure into
double-stranded, and amplified.

l
**Digestion** (empty vector: pUC 19 )

- **Objective:** The cohesive ends on the fragments are cut out with enzymes to
facilitate fusion with other fragments later. Cut out a sticky end of pUC 19 (EV ) for building up
plasmid 1 in later process.

- **Concepts:** Cut out identical sticky ends to aid binding

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| pUC 19 (EV) | 30 |
| Enzyme 1: Sal I | 1 |
| Enzyme 2: pst I | 1 |
| rCutSmart | 5 |
| dd H 2 O | 13 |
| Total | 50 |

- **Methods:**

1.
Mix all
reagents
 mentioned above in a tube

2.
Place the test tube in a water bath at 37˚C for 30 min (This is the optimum
environment for enzyme growth )

- **Results:** Digestion was successfully completed and the cohesive ends were
cut out.

l
**Digestion** (annealed PCR product ( plac/puv) and IPCR: plasmid 4
)

- **Objective:** The cohesive ends on the fragments are cut
openby
 enzymes to facilitate fusion with other fragments
later.

- **Concepts:** The cleaved fragments have identical cohesive ends to facilitate
later joining. The gene fragment made before has no suitable restriction site, and the added primers
provide it with restriction sites. The annealed PCR product is also equivalent to providing two primers,
so that the target gene can have a site.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| IPCR product--plasmid 4/annealed PCR product--plac & puv | 10 |
| Enzyme 1: Avr II | 1 |
| Enzyme 2: Sal I | 0.5 |
| rCutSmart | 2 |
| ddH20 | 6 |

- **Methods:**

1.
Mix
reagents
 mentioned above in two tubes separately

a)
Group 1: annealed PCR product—plac + both two types of enzyme +
rCutSmart + dd
H
2
O

b)
Group 2: annealed PCR product—puv + both two types of enzyme +
rCutSmart + dd
H
2
O

2.
Group 3: IPCR product--plasmid 4+both two types of enzyme + rCutSmart +
dd
H
2
O
 Place the tubes in a water bath at 37 ˚C for 30
min

- **Results:** Digestion was successfully completed and the cohesive ends were
cut out.

l
**E****nzyme-link** (mix digested plac for plasmid 5 or digested puv for plasmid 6 with
annealed PCR product respectively )

- **Objective:** Ligate and fuse the previously cut plasmid and target gene with
the same cohesive ends.

- **Concepts:** Ligase helps gene fragments join and fuse.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| T4 ligase | 1 |
| T4 ligase buffer | 2 |

Digested plac for plasmid 5

 Digested puv for plasmid 6

 IPCR product: plasmid 4

- **Methods:**

1.
Mix digested IPCR plac, IPCR product--plasmid 4, T4 ligase, and T4
ligase buffer in a tube

2.
Mix digested IPCR puv, IPCR product--plasmid 4, T4 ligase, T4 ligase
buffer in a tube

- **Results:** Fuse the target gene and plasmid together. Build up plasmid 5 and
plasmid 6.

Day 3

1.
PCR amplify RFP (outline: prepare for RFP; plasmid 2--->plasmid
3)

2. Homologous recombination (homology arm )

3. Transform into
DH5
α
*E.coli* and grow overnight.

l
**PCR (RFP )**

- **Objective:** Amplify RFP to 687bp, prepare reagents for building up plasmid
3

- **Concepts:** At 95
℃
, the DNA helix unwinds transforme
d
 from double to single. When held at 72
℃
 for 10 min, all the single chains will become double. During the
cycle, the structure of DNA transformed repeatedly between single and double strands.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: RFP-F2 | 1 |
| Primer: TrrnB-R1 | 1 |
| DNA template: puc57-RFP | 1 |
| ddH2O | 7 |
| Total | 20 |

 Mix: Mg2+, dNTP, DNA polymerase

- **Methods:**

1.
Mix both of the two primers, DNA template, ddH2O, and the mix in a centrifuge tube (follow the order from large
quantity to small quantity )

2.
vortex and centrifuge (to avoid the occurrence of solution hanging on
the wall of the tube and mix well )

3.
Place the tube inside the PCR machine

4.
Place in PCR machine

5.
Turn on the machine

6.
Click “file” on the screen

7.
Click “program
”

8.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 sec |
| 3 | 55 | 30 sec |
| 4 | 72 | 30 sec |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30
cycle
s

- **Result:** amplify RFP to about 687 bp

l
**Homologous recombination** ( RFP and IPCR--->plasmid 3 )

- **Objective:** Fusion of plasmid 2 (IPCR product) and target gene RFP. Build up
plasmid 3.

- **Concepts:** Because there is no suitable enzyme cutting site, a homology arm
is used to forcibly connect the two gene fragments. Find Homology arms: Compare the sequence and base
pairs of two DNA fragments. Homologous recombination refers to the exchange of time that occurs between
two homologous chromosomes on a chromosome. The gene fragments are first cut with double-strand
breakase, the homologous arms are reconnected with homologous recombinase, and finally the chromosome is
connected with DNA ligase.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Template: IPCR product: plasmid 2 | 2 |
| Target gene: RFP | 1 |
| Mix | 5 |
| ddH2O | 2 |
| Total | 10 |

6000bp\*0.02=120 ---------- 2 µL template: IPCR product: plasmid
2

687bp\*0.04=28 ---------- 1 µL target gene: RFP

     Mix: dNTP, Mg2+, DNA polymerase

- **Methods:**

1.
Mix the template, the target gene, ddH2O, and the mix in a tube.

2.
Put the tube into a water bath machine at 37˚C for 5
min

3.
Place the tube on crashed ice for 3 min

- **Result:** Embedding of target gene and template gene; completely build up
plasmid 3.

l
**Transformation** (Insertion of three plasmids into *E. coli* )

- **Objective:** Let *E. coli* help increase the number of plasmid 3, 5 and 6

- **Concepts:** *E. coli* can keep growing to continuously replicate our implanted genes.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Plasmid 5/plasmid 6/RFP after homologous recombination--plasmid 3 | 20 |

Solution that contains*E.coli*

- **Methods:**

1.
Mix the reagents separately (group 1: plasmid 3 + solution that
contains *E.coli*; group 2: plasmid 5 + solution that contains *E.coli*; group 3: plasmid 6 + solution that contains *E.coli* )

2.
Place them into crashed ice for 30 min

3.
Water bath Heat shock at 42 ˚C for 45 s ( let the *E.coli* devour the three plasmids we want to implant faster and better;
only 45 s, because if it take longer than that, the *E.coli* will die due to high temperatur
e
 )

4.
Place them into crashed ice again, for only 2-3 min

- **Result:** all plasmid 3, plasmid 5, and plasmid 6 were implanted in
*E.coli* respectively.

l
**Culture** ***E.coli*****with three different plasmids**

- **Objective:** we need to get *E.coli* to help expand the production of plasmid 5, plasmid 6, and
plasmid 3.

- **Concepts:** the LB media provides a suitable environment which is beneficial
for *E.coli*to grow. (LB media: tryptophan and yeast extract provide carbon and nitrogen needed by
*E. coli.* NaCl helps maintain the osmotic pressure)

- **Materials:**

Part 1:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| LB media ( solution ) | 900 |
| Mixture 1 | / |
| Mixture 2 | / |
| Mixture 3 | / |

Ps: Mixture 1: plasmid 3 + *E.coli*

 Mixture 2: plasmid 5 + *E.coli*

 Mixture 3: plasmid 6 + *E.coli*

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Prepared solid AMP + LB media | / |
| Mixture 1 | 10 |
| Mixture 2 | 10 |
| Mixture 3 | 10 |

Ps: Mixture 1: liquid LB media+*E.coli*+plasmid 3

  Mixture 2: liquid LB media+*E.coli*+plasmid 5

      Mixture 3: liquid LB media+*E.coli*+plasmid 6

- **Methods:**

Part 1:

1.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

2.
Light the alcohol lamp

3.
Mix 900 µL LB media and *E.coli* with plasmid 3 in a new tube.

4.
Mix 900 µL LB media and *E.coli* with plasmid 5 in a new tube.

5.
Mix 900 µL LB media and*E.coli*with plasmid 6 in a new tube.

(notion: things are all made inside the sterilizing tank; from 4-6, all
process should be operated near the alcohol lamp )

6.
Place them inside a machine, keep constant shaking for 1 h

7.
Transfer the tubes to the sterilizing tank

Part 2:

1.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

2.
Light the alcohol lamp

3.
Peel off the membrane on the side containing the solid LB media petri
dish

4.
Transfer 10 µL mixture (liquid LB media + *E.coli*+ plasmid 3) onto a prepared solid LB + AMP media

5.
Transfer 10 µL mixture (liquid LB media + E.coli + plasmid 5) onto a solid LB
+ AMP media

6.
Transfer 10 µL mixture (liquid LB media + E.coli + plasmid 6) onto a solid LB
+ AMP media

7.
Use a disposable scrapeer to evenly apply the mixture to the solid LB +
AMP media’s surface respectively

8.
Seal the sides of the petri dish

9.
Turn the petri dish upside down (place the liquid coated side down ),
and place it in an incubator, waiting for *E.coli* to grow.

- **Result:** wait for tomorrows observation.

l
**PCR** (*E.coli* with plasmid 3/plasmid 1 ( RFP without its own promoter )
)

- **Objective:** amplify the *E.coli* with plasmid 1 and plasmid 3

- **Concepts:** At 95
℃
, the DNA helix unwinds transforme
d
 from double to single. When held at 72
℃
 for 10 min, all the single chains will become double. During the
cycle, the structure of DNA transformed repeatedly between single and double strands.

- **Material:**

 Group 1 ( *E.coli* with plasmid 1 )

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Mix | 10 |
| Primer 1: TrrnB-R1 | 1 |
| Primer 2: RPT-F2 | 1 |
| Template: a single strain of *E.coli* with plasmid 1 | / |
| ddH2O | 8 |
| Total | 20 |

 Group 1 ( *E.coli* with plasmid 1 )

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Mix | 10 |
| Primer 1: TSacB-F1 | 1 |
| Primer 2: zli-SacB-BkR1 | 1 |
| Template: a single strain of *E.coli* with plasmid 3 | / |
| ddH2O | 8 |
| Total | 20 |

- **Methods:**

1.
Mix ddH20, mix, and two primers in a PCR tube

2.
Select a single strain of *E.coli*

a)
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

b)
Light the alcohol lamp

c)
Peel off the membrane on the side containing the solid LB media petri
dish (both of the small one that contains *E.coli* with plasmid 1/plasmid 3 ) and the larger one that only contains
solid LB media )

d)
Use the pipette to pick up a single strain with the tip of the
pipette

e)
Transfer the single strain on to the surface of solid LB media in a
larger petri dish

f)
Draw a line on it with the tip of the pipette (Draw a marker next to
the first line )

g)
Continue to draw lines after the first line, about 7-8 or so
(withou
t
 crossing each other; remember to mark the corresponding serial
number on the outside of the Petri dish with a marker )

h)
Put the tip of the pipette back into the PCR tube, hold the tip with
your hand, and stir it

3.
Vortex and centrifuge (to avoid the occurrence of solution hanging on
the wall of the tube )

4.
Place in PCR machine

5.
Turn on the machine

6.
Click “file” on the screen

7.
Click “program
”

8.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 s |
| 3 | 55 | 30 s |
| 4 | 72 | 1 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 times

- **Result:** Achieving the amplification of *E.coli* that contains plasmid 1/plasmid 3

l
**AMP solid medium**

- **Objective:** to kill most of the*E.coli* that is not implanted with plasmad1 or plasmid 3

- **Concepts:** The implanted *E. coli* is resistant to ammonia, so only the*E. coli* implanted with plasmid 1 and plasmid 3 can survive in the presence of
AMP.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| Stock solution (AMP) | 100 mg/L |
| LB media | 35 mL |
| AMP solution | 40 µL |

Our target concentration: 100 µg/L

100mg/L stock solution (AMP)

 To achieve the target concentration: 1ml LB+1µl stock
solution (AMP )

 Total (LB+AMP ): 3 m
L
/plate\*20 plate=60 m
L
 solid LB

 70 m
L
-->70 µL AMP-->80µL AMP

 50 mL centrifuge tube+35mL LB+40µL AMP

- **Methods:**

10.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

11.
Light the alcohol lamp

12.
Mix 35 mL LB and 40 µL AMP solution in a tube

13.
Transfer 3 mL of the mixture into a new smaller tube

14.
And then let the bacterial liquid evenly distribute on the surface of
the culture medium

15.
Seal the side of the petri dish

16.
Wait for solidification

- **Result:** solidified

l
**Gel electrophoresis**

- **Objective:** To check whether the grown *E.coli* contains the correct DNA segments/fragments we need

- **Concepts:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the
correct DNA fragments we need according to the marker.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL)

4.
Pour the gel

5.
Wait until it turns into a solid gel

6.
Remove the comb

7.
Pour TAE solution into the tank

8.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

9.
Put the lid on and power on (180V, 20 min)

10.
(After 20 min) put the gel under a gel imager to document
bands

11.
Software: Genosens

12.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Results:**

1.
Plasmid 3: contains the correct DNA segments/fragments except 3-2-6, 3-2-7, 3-2-8

   Reasons (might be ):

a)
Monoclonal strains were not picked clean

b)
Some reagents are not sucked clean

c)
There is liquid hanging on the tube when transferring liquid, or gloves
pick up some liquid when opening the test tube

2.
Plasmid 1: all failed

   Reason (might be ):

a)
Enzyme inactivated

b)
The colony we picked only contains empty vector

3.
Plasmid 5 and plasmid 6: the color of the solid LB + AMP in the petri
dish turns red (because we’ve added RFP, so if the color turn red, it means that converting plasmid 5
and plasmid 6 into the *E.coli* succeed)

l
**PCR** (to amplify the promoter of RFP ( PRFP)

- **Objective:** amplify the promoter of RFP

- **Concepts:** At 95
℃
, the DNA helix unwinds transforme
d
 from double to single. When held at 72
℃
 for 10 min, all the single chains will become double. During the
cycle, the structure of DNA transformed repeatedly between single and double strands.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: pzliE-R2-MIu | 1 |
| Primer: pzliE-Fi-Sac | 1 |
| DNA template: puc57-RFP | 1 |
| ddH2O | 7 |
| Total | 20 |

Mix: Mg2+, DNA polymerase, dNTP, ddH2O

- **Methods:**

9.
Mix dd water, both of the two primers, template, and the mix in a
centrifuge tube ( follow the order from large quantity to small quantity )

10.
vortex and centrifuge (to avoid the occurrence of solution hanging on
the wall of the tube )

11.
Place the tube inside the PCR machine

12.
Place in PCR machine

13.
Turn on the machine

14.
Click “file” on the screen

15.
Click “program
”

16.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 s |
| 3 | 55 | 30 s |
| 4 | 72 | 1min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 times

**Result:** achieving the amplification of the promoter of plasmid 4
(PRFFP)

l
**PCR** (Because the DNA electrophoresis of plasmid 1 failed in the
previous test, plasmid 1 should be reconstructed )

- **Objective:** to amplify part of plasmid 1 (zliE-zliS )

- **Concepts:** At 95
℃
, the DNA helix unwinds, changing from double to single. When held at
72
℃
 for 10 minutes, all the single chains will become double. During
the cycle, the structure of DNA changes repeatedly between single and double strands.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: zliE-F1 | 1 |
| Primer: zliS-R1 | 1 |
| DNA template: *zm4* | 1 |
| ddH2O | 7 |
| Total | 20 |

Mix: Mg2+, DNA polymerase, dNTP, dd water

- **Methods:**

8.
Mix all the reagents mentioned above in a centrifuge tube ( all tubes
should be placed in crushed ice, otherwise most enzyme will become inactive, such as DNA polymerase
)

9.
Vortex and centrifuge (to avoid the occurrence of solution hanging on
the wall of the tube )

10.
Place in PCR machine

11.
Turn on the machine

12.
Click “file” on the screen

13.
Click “program
”

14.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 s |
| 3 | 55 | 30 s |
| 4 | 72 | 1min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 times

- **Result:** Achieving zliE-zliS amplification

l
**AMP**

- **Objective:** Cultivate *E. coli* with plasmid 3 while killing all *E. coli* that are not implanted with plasmid 3

- **Concepts:** The implanted *E. coli* is resistant to ammonia, so only the *E. coli* implanted with plasmamid1 and plasmid 3 can survive in the presence of
AMP.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| Stock solution (AMP) | 100 mg/L |
| LB media | 45 mL |
| AMP solution | 60 µL |

Our target concentration: 100 µg/L

100 mg/L stock solution ( AMP )

 To achieve the target concentration: 1m
L
 LB+1µ
L
 stock solution (AMP)

 Total (LB+AMP ): 5 m
L
/plate\*20 plate=60m
L
 solid LB

 70 m
L
-->70 µ
L
 AMP-->80 µ
L
 AMP

 50 m
L
 centrifuge tube + 45m
L
 LB + 60µ
L
 AMP

- **Methods:**

1.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

2.
Light the alcohol lamp

3.
Mix 45 m
L
 solid LB and 60 µL AMP in a tube

4.
Use a pipette to transfer 5 m
L
 of the mixture into a smaller tube

5.
Seal it and place it inside a machine to shake for a night

- **Result:** wait for tomorrow’s observation

l
**Gel electrophoresis**

- **Objective:** To check whether the DNA sample (plasmid 1 (zliE-zliS) and plasmid 4 (
promoter ) ) we have contains the correct DNA segments/fragments we need

- **Concepts:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the
correct DNA fragments we need according to the marker.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

2K marker

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special” )

1.
Mix all
reagents
 mentioned above

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

13.
Fix comb in the mold (thick: 50 mL; thin: 20 mL)

14.
Pour the gel

15.
Wait until it turns into a solid gel

16.
Remove the comb

17.
Pour TAE solution into the tank

18.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

19.
Put the lid on and power on (180V, 20 min)

20.
(After 20 min) put the gel under a gel imager to document
bands

21.
Software: Genosens

22.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Results:**

1.
Plasmid 1 (zliE-zliS): the line left on the gel is a little bit lower
than the 2000 bp line of marker’s. target: 1680bp

2.
Plasmid 4 (promoter): the line left on the gel is a little bit higher
than the 250 bp line of marker’s. target: 300bp

l
**Extract plasmid 3**

- **Objective:** Lysis of *E.coli* to extract plasmid 3

- **Concepts:** Use Buffer SP1 to degrade the RNA, then use Buffer SP2 to lyse
the unwanted cytoskeleton and other wastes, then use Buffer SP3 to stop the lysis, and finally extract
the Plasmid 3 in *E.coli*

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| Buffer S | 500 µL |
| Water | 2mL X2 |
| Bacteria solution (*E.coli* with plasmid 3) | 2mL X2 |
| Buffer SP1 | 250 µL |
| Buffer SP2 | 250 µL |
| Buffer SP3 | 350 µL |

- **Methods:**

1.
Add 500 µL buffer S into a tube (X2) ( Help the cells added later
to better adsorb on the membrane )

2.
Centrifuge for 1 min (12
,
000
×
g)

3.
Take out the tubes and pour all the solution into a garbage
can

4.
Add 2mL bacteria solution (*E.coli* with plasmid 3 ) into a 2mL centrifuge tube, and add 2mL water to
another tube for balance

5.
Centrifuge both of the two tubes for 1 min (12
,
000
×
g)

6.
Take out the tubes and pour all the solution into a garbage can (some
white precipitate could be observed at the bottom of the tube )

7.
Add 2 mL bacteria solution (*E.coli* with plasmid 3 ) into the same 2 mL tube

8.
Centrifuge both of the tubes for 1min (12
,
000
×
g; also the one with water; remember: Opening towards the center of the
centrifuge so that the precipitates would stay at the same spot on the tube )

9.
Take out the tubes and pour the solution in the tube with
*E.coli* into a garbage can ( some white precipitate could be observed at
the bottom of the tube )

10.
Add 250 µL buffer SP1 into the tube with *E.coli*, mix them by pipetting. (notion: Do not hit the bottom of the pipette
and do not make bubbles; since our *E.coli* is a live bacteria, centrifuge is not available ) ( SP1: degrade
RNA )

11.
Add 250 µL buffer SP2 into the same tube. Invert the tube back and
forth 5 times to mix the solution. (SP2: lysed cells )

12.
Add 350 µL buffer SP3 into the tube. Invert the tube back and
forth 5 times to mix the solution. (SP3: stop lysis; White flocs can be seen, eg: cytoskeleton
)

13.
Centrifuge for 5 min (14
,
000
×
g)

- **Result:** Separated the plasmid 3 we want from *E.coli* ( in the transparent solution )

l
**Digestion**

- **Objective:** The cohesive ends on the fragments are cut out with enzymes to
facilitate fusion with other fragments later. Cut out a sticky end of a fragment of gene for building up
a plasmid in later process.

- **Concepts:** Cut out identical sticky ends to aid binding

- **Materials:**

 Group 1:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| pUC 19 ( empty vector ) ( template for plasmid 1 ) | 30 |
| Enzyme 1: Pst I-F | 1 |
| Enzyme 2: Sal I | 1 |
| rCutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

Group 2:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| zliES ( target gene for plasmid 1 ) | 30 |
| Enzyme 1: Pst I | 1 |
| Enzyme 2: Sal I | 1 |
| rCutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

Group 3:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Promoter of RFP ( target gene for plasmid 4 ) | 30 |
| Enzyme 1: Sac I | 1 |
| Enzyme 2: Mlu l | 1 |
| rCutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

Group 4:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| SacB ( target gene of plasmid 2 ) | 30 |
| Enzyme 1: Xbal | 1 |
| Enzyme 2: Sal I | 1 |
| rCutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

Group 5:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| plasmid 3 ( the template of plasmid 4 ) | 30 |
| Enzyme 1: Mlu I | 1 |
| Enzyme 2: Sac I | 1 |
| rCutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

Group 6:

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| plasmid 1 ( the template of plasmid 2 ) | 30 |
| Enzyme 1: Xbal | 1 |
| Enzyme 2: Sal I | 1 |
| rCutSmart | 5 |
| ddH2O | 13 |
| Total | 50 |

- **Method:**

3.
Mix materials mentioned above in two tubes separately

c)
Group 1: pUC 19 + Pst I-F + Sal I + rCutSmart + ddH2O

d)
Group 2: zliES (target gene for plasmid 1) + Pst I + Sal I + rCutSmart
+ ddH2O

e)
Group 3: Promoter of RFP (target gene for plasmid 4) + Sac I + Mlu l +
rCutSmart + ddH2O

f)
Group 4: SacB (target gene of plasmid 2) + Xbal + Sal I + rCutSmart +
ddH2O

g)
Group 5: plasmid 3 (the template of plasmid 4) + Mlu I + Sac I +
rCutSmart + ddH2O

h)
 Group 6: plasmid 1 (the template of plasmid 2) + Xbal + Sal I +
rCutSmart + ddH2O

4.
Place the tubes at a temperature of 37 ˚C for 1h

    - **Result:** Digestion was successfully completed and the cohesive ends were
cut out.

l
**DNA extraction**

- **Objective:** Remove proteins and other impurities that could affect
results

- **Concept:** Elution buffer and washing buffer can help purify the genes we
really want

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage ( μL) |
| Buffer 2 | 300  X6 |
| Wash solution | 500  X6 |
| Elution buffer | 40  X6 |

Silica column    X6

- **Methods:** (note: protein: 280 nm; DNA: 260 nm )

1.
Mix the product that contains pUC 19/plasmid 1/plasmid 3/zliE-zliS/SacB/Promoter
of RFP (all of them are the product after digestion ) with 300 µL Buffer 2 in a tube

2.
Mix them with a pipette

3.
Tube (pUC 19/plasmid 1/plasmid 3/zliES/SacB/Promoter of RFP )

4.
Centrifuge ( 8,000
×
g ) for 30sec and pour off the underlying liquid ( liquid in the
transparent tube )

5.
Tube (pUC 19/plasmid 1/plasmid 3/zliES/SacB/Promoter of RFP ):+ 500
µL wash solution

6.
Centrifuge (9,000
×
g) for 30 s and pour off the underlying liquid ( liquid in the
transparent tube )

7.
Tube ( pUC 19/plasmid 1/plasmid 3/zliES/SacB/Promoter of RFP ):+ 500
µL wash solution

8.
Centrifuge (9,000
×
g) for 30 s and pour off the underlying liquid ( liquid in the
transparent tube )

9.
Centrifuge (9,000
×
g) for 1 min

10.
Place both of the solution (with the internal yellow tube ) into two
new 1.5ml tubes

11.
Add 40 µL elution buffer into two tubes

12.
Wait for 1 min

13.
Centrifuge for 1 min

14.
Turn on the machine that detects DNA concentration

15.
Wipe the instrument with paper

16.
Click “nucleic acid” on the screen

17.
Add 1 µL elution buffer into the “small hole” on the
instrument

18.
Click “empty test” + “zero”

19.
Wipe the “hole” with paper

20.
Add 1 µL solution that contains DNA into the “hole”

21.
Click “sample testing”

(note: normally: A260/A280: 1.8-2.1 )

- **Results:**

 pUC 19 (empty vector ): 40.7 ng/µL; A260/A280:
1.83

 Plasmid 1: 10.7 ng/µL; A260/A280: 1.82

 Plasmid 3: 31.25 ng/µL; A260/A280: 1.83

 zliES: 0.5 ng/µL; A260/A280: 2.5

 SacB: 4.9 ng/µL; A260/A280: 1.5

 Promoter of RFP: 13.8ng/µL; A260/A280: 1.78

l
**Enzyme link**

- **Objective:** to build up plasmid 1, plasmid 2, and plasmid 4 by using the
previous 6 DNA fragments which had been digested and recycled.

- **Concept:** Ligase helps gene fragments join and fuse. T4 ligase buffer can
help T4 ligase work better.

- **Material:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Template: (a)Plasmid 1: pUC 19 ( EV )/(b)Plasmid 2: plasmid 1/(c)plasmid 4: plasmid 3 | 10 |
| Target gene: (2)Plasmid 1: zliE-zliS/(b)Plasmid 2: SacB/(c)Plasmid 4: the promoter of RFP | 7 |
| T4 ligase ( imported ) | 1  X3 |
| T4 ligase buffer | 1  X3 |

    - **Method:**

1.
Mix the materials mentioned above in tree tubes separately.

a)
Template (a) + target gene (a) + T4 ligase + T4 ligase
buffer

b)
Template (b) + target gene (b) + T4 ligase + T4 ligase
buffer

c)
Template (c) + target gene (c) + T4 ligase + T4 ligase
buffer

2.
Mix them evenly with a pipette

3.
Place the three tubes into a water bath machine for 20min at 22℃
(Because the previous construction of plasmid 1 failed, and we assumed that it might mainly due to the
enzyme inactivation ( T4 ligase ), we replaced the domestically produced T4 ligase with imported ones,
and those imported T4 ligase only need 20 min to bind our target gene and our template together.
Additionally, the imported ones can ignore all other DNA fragments, which means that they can help build
up a plasmid directly. However, to be on the safe side, we still did a recycling)

-**Result:** fuse the target gene and plasmid together, while the final result
will have to wait for tomorrow’s observation of the growth of *E.coli* with plasmid 1/plasmid 2/plasmid 4

l
**Gel electrophoresis**

- **Objective:** To check whether our digestion for plasmid 1&2&4 is succeeded or not.

- **Concept:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the
correct DNA fragments we need according to the marker.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| pUC 19/pUC 19 after digestion/Plasmid 1/Plasmid 1 after digestion/Plasmid 3/Plasmid 3 after digestion | 1µL |
| TAE buffer | 100mL |
| Agarose | 1%-->1g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10µL |

15k marker

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL )

4.
Pour the gel

5.
Wait until it turns into a solid gel

6.
Remove the comb

7.
Pour TAE solution into the tank

8.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

9.
Put the lid on and power on (180V, 20 min)

10.
(After 20 min) put the gel under a gel imager to document
bands

11.
Software: Genosens

12.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Result:**

1.
all the 6 verification objects had shown a clear line on the
gel

2.
The lines of plasmid 1&2&4 after digestion illustrated only 1 line,
which means that they are pure, without any pUC 19 (EV) remaining.

l
**Convert**

-**Objective:** Let *E. coli* help increase the number of plasmid 1, 2 and 4

- **Concept:** *E. coli* can keep growing, so it can keep replicating our implanted genes.

- **Material:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| plasmid 1/plasmid 2/plasmid 4 after enzyme link | 20 |
| Solution that contains *E.coli* | / |

- **Method:**

5.
Mix the materials separately by pipetting 10 times (group 1: plasmid
1+*E.coli*; group 2: plasmid 2 + *E.coli*; group 3: plasmid 4+*E.coli* )

6.
Place them into crashed ice for 30 min

7.
Water bath at 42˚C for 45 s (let the*E.coli* devour the three plasmids we want to implant faster and better;
only 45sec, because if it take longer than that, the *E.coli* will die due to high temperatur
e
 )

8.
Place them on crashed ice again, for only 2-3 min (stop allowing
*E. coli* to continue to ingest the given plasmid )

9.
Then place the tubes in a 37℃ constant temperature shaker for 1 hour

- **Result:** predicted: plasmid 1, plasmid 2, and plasmid 4 were implanted in
*E.coli* respectively. However, we’ll have to wait for tomorrow’s
(26th ) observation.

l
**Gel electrophoresis** (only for plasmid 1, since the previous ones failed )

- **Goal:** To check whether the DNA sample you have contains the correct DNA
segments/fragments you need

- **Concept:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, the distance that DNA travels are also different. Then, we can obtain the
correct DNA fragments we need according to the marker.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

2k marker

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL)

4.
Pour the gel

5.
Wait until it turns into a solid gel

6.
Remove the comb

7.
Pour TAE solution into the tank

8.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

9.
Put the lid on and power on (180V, 20 min)

10.
(After 20 min) put the gel under a gel imager to document
bands

11.
Software: Genosens

12.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Result:** We poured plasmid 1 into 4 lanes and only the last one gave the
correct result

l
**Culture** ***E.coli*****with three different plasmids**

- **Objective:** we need to get *E.coli* to help expand the production of plasmid 1, plasmid 2, and
plasmid 4.

- **Concept:** the LB media provides a suitable environment which is beneficial
for *E.coli* to grow. (LB media: tryptophan and yeast extract provide carbon and nitrogen needed by
*E. coli*. NaCl helps maintain the osmotic pressure)

- **Material:**

|  |  |
| --- | --- |
| Name | Dosage (µL) |
| Mixture 1: plasmid 1 + *E.coli* | 5 |
| Mixture 2: plasmid 2 + *E.coli* | 5 |
| Mixture 3: plasmid 4 + *E.coli* | 5 |
| LB media ( including solid LB media and the one in liquid state ) | / |

- **Method:**

17.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

18.
Light the alcohol lamp

19.
Mix 900 µL LB media and *E.coli* with plasmid 1 in a new tube.

20.
Mix 900 µL LB media and *E.coli* with plasmid 2 in a new tube.

21.
Mix 900 µL LB media and *E.coli* with plasmid 4 in a new tube.

(notion: things are all made inside the sterilizing tank; from 4-6, all
process should be operated near the alcohol lamp )

22.
Place them inside a machine, keep constant shaking for 1h

23.
Transfer the tubes to the sterilizing tank

24.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

25.
Light the alcohol lamp

26.
Peel off the membrane on the side containing the solid LB media petri
dish

27.
Transfer 10 µL mixture (liquid LB media + *E.coli* + plasmid 1) onto the solid LB media

28.
Transfer 10 µL mixture (liquid LB media + *E.coli* + plasmid 2) onto the solid LB media

29.
Transfer 10 µL mixture (liquid LB media + *E.coli* + plasmid 4) onto the solid LB media

30.
Use a disposable scrapeer to evenly apply the mixture to the solid LB
surface respectively

31.
Seal the sides of the petri dish

32.
Turn the petri dish upside down (place the liquid coated side down ),
and place it in an incubator, waiting for *E.coli* to grow.

- **Result:** wait for tomorrows observation.

l
**Colony PCR** (*E.coli* with plasmid 1/plasmid 2/plasmid 4)

- **Objective:** amplify the *E.coli* with plasmid 1&plasmid 2&plasmid 4

- **Concepts:** At 95
℃
, the DNA helix unwinds transforme
d
 from double to single. When held at 72
℃
 for 10 min, all the single chains will become double. During the
cycle, the structure of DNA transformed repeatedly between single and double strands.

- **Materials:**

 Group 1:

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: zliE-F1 | 1 |
| Primer: zliS-R1 | 1 |
| DNA template: a single strain of *E.coli* that contains plasmid 1 | 1 |
| ddH2O | 7 |
| Total | 20 |

Mix: Mg2+, dNTP, DNA polymerase

Group 2:

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: SacB-F1 | 1 |
| Primer: SacB-R1 | 1 |
| DNA template: a single strain of E.coli that contains plasmid 2 | 1 |
| ddH2O | 7 |
| Total | 20 |

Group 3:

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: pzliE-F2-Sac | 1 |
| Primer: pzliE-R2-Mlu | 1 |
| DNA template: a single strain of E.coli that contains plasmid 4 | 1 |
| ddH2O | 7 |
| Total | 20 |

Ps: Sac and Mlu provided restriction sites

- **Methods:**

9.
Mix ddH2O, mix, and two primers in a PCR tube

10.
Select a single strain of *E.coli* that contains plasmid1/2/4

i)
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

j)
Light the alcohol lamp

k)
Peel off the membrane on the side containing the solid LB media petri
dish (both of the small one that contains *E.coli* with plasmid 1/plasmid 2/plasmid 4) and the larger one that only
contains solid LB media)

l)
Use the pipette to pick up a single strain with the tip of the
pipette

m)
Transfer the single strain on to the surface of solid LB media in a
larger petri dish

n)
Draw a line on it with the tip of the pipette (draw a marker next to
the first line )

o)
Continue to draw lines after the first line, about 7-8 or so (without
crossing each other; remember to mark the corresponding serial number on the outside of the Petri dish
with a marker )

p)
Put the tip of the pipette back into the PCR tube, hold the tip with
your hand, and stir it

11.
Vortex and centrifuge (to avoid the occurrence of solution hanging on
the wall of the tube )

12.
Place in PCR machine

13.
Turn on the machine

14.
Click “file” on the screen

15.
Click “program
”

16.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 s |
| 3 | 55 | 30 s |
| 4 | 72 | 1 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 times

- **Result:** Achieving the amplification of plasmid 1/plasmid 2/plasmid
4

l
**AMP** (*E.coli* with plasmid 4; prepare for the next section: IPTG)

- **Objective:** Cultivate *E. coli* with plasmid 4 while killing all *E. coli* that are not implanted with plasmid 4

- **Concepts:** The implanted *E. coli* is resistant to ammonia, so only the *E. coli* implanted with plasmid 4 can survive in the presence of
AMP.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| Stock solution (AMP) | 100 mg/L |
| LB media | 45 mL |
| AMP solution | 60 µL |

Our target concentration: 100 µg/L

100 mg/L stock solution (AMP)

 To achieve the target concentration: 1ml LB+1µl stock
solution (AMP)

 Total (LB+AMP): 5 mL/plate\*20 plate=60 mL solid LB

 70 mL-->70 µL AMP-->80 µL AMP

 50 mL centrifuge tube+45 mL LB+60 µL AMP

- **Methods:**

6.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

7.
Light the alcohol lamp

8.
Mix 45 mL solid LB and 60 µL AMP in a tube

9.
Pick up an isolated single strain with the tip of the
pipette

10.
Tap the tip of the pipette into the tube

11.
Seal the tube and place it inside a machine to shake for 1 h at 37
℃

- **Result:** achieved the amplification of *E.coli* with plasmid 4

l
**Gel electrophoresis** (PCR product with plasmid 1&2&4 )

- **Objective:** To check whether the DNA sample we have contains the correct DNA
segments/fragments we need

- **Concepts:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, then, the distance that DNA travels is also different. Then, compared to
the marker, we can find different DNA we want.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL)

4.
Pour the gel

5.
Wait until it turns into a solid gel

6.
Remove the comb

7.
Pour TAE solution into the tank

8.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

9.
Put the lid on and power on (180V, 20 min)

10.
(After 20 min) put the gel under a gel imager to document
bands

11.
Software: Genosens

12.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Results:**

 Plasmid 1: failed; so we can only use the previous
data

 Plasmid 2: 1827 bp

 Plasmid 4: 300 bp

 (the data of the promoter of plasmid 4 and the target gene of
plasmid 2 (SacB) is the same as the data of plasmid 2&4, since they all have two restriction sites
provided by the cutting enzyme, so during the PCR, only their target gene or promoter was amplified. So
the result turned out to be the data of SacB’s (1827bp ) or the promoter of plasmid 4’s
(300bp).

l
**Inverse PCR** (prepare for building up plasmid 3, amplifying plasmid 2
)

- **Objective:** to amplify plasmid 2; be prepare for the template of plasmid 3.

- **Concepts**: Because there is no suitable enzyme cutting site on this structure,
or there is a terminator blocking the normal direction, it is necessary to change the direction for PCR.
The purpose of inverse PCR is to amplify DNA flanking a known sequence, that is to say, this reaction
system synthesizes DNA not between a pair of primers but outside the primers.

- **Materials**:

|  |  |
| --- | --- |
| Name | Dosage（ μL ） |
| Mix | 10 |
| Primer: PFP-F2-GA | 1 |
| Primer: zli-SacB-BKR1-GA | 1 |
| DNA template: plasmid 2 | 1 |
| ddH2O | 7 |
| Total | 20 |

Mix: Mg2+, DNA polymerase, dNTP, ddH2O

- **Methods**:

8.
Mix all reagents mentioned above (add them in order from largest to
smallest; When some reagents are not used, they need to be placed on crushed ice. Especially mix,
because there are enzymes in it, it will be inactivated due to the high temperature)

9.
Place the tube inside the centrifuge

10.
Take out the tube and place in it inside a PCR machine

11.
Turn on the machine

12.
Click “file” on the screen

13.
Click “program
”

14.
Click “primmer start”

|  |  |  |
| --- | --- | --- |
| steps | temperature（ ℃ ） | time |
| 1 | 95 | 3 min |
| 2 | 95 | 30 s |
| 3 | 55 | 30 s |
| 4 | 72 | 5 min |
| 5 | 72 | 10 min |
| 6 | 4 | forever |

PS: Repeat 2-4 30 times

- **Result**: wait until tomorrow (27th)

l
**Ampicillin configuration**

- **Objective**: Prepare an AMP solution with an appropriate concentration for
subsequent cultivation of *E.col*with our plasmid, killing all other bacteria without the plasmid we
implanted (except pUC 19 ( EV ) ).

- **Concepts:**

 our target concentration: 100 µL/mL

 We need 10 mL AMP solution

 So we have to add 1 g Ampicillin, sodium salt

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| Ampicillin, sodium salt | 1 g |
| ddH2O | 10 mL |
| 0.22 nm filter | / |
| Syringe | / |

- **Methods:**

1.
Weigh out 1g of Ampicillin, sodium salt with an electronic
scale

2.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

3.
Light the alcohol lamp

4.
Mix the powder with 10 mL ddH2O by pipetting 8 times in a tube.

5.
Divide the mixture into 6 small tube

- **Result:** composed an AMP solution at a right concentration.

l
**IPTG & Sucrose control variable**

- **Objectives:**

1.
The purpose was to test the optical density value of E. coli with or
without the addition of IPTG in LB medium with sucrose as the sole carbon source (OD 600 nm ), enzyme
activity, colony color (colorless to red), fluorescence, colony swelling phenotype.

- **Concepts:** IPTG can induce protein expression

- **Materials:**

 Group 1:

|  |  |
| --- | --- |
| Name | Dosage |
| AMP solution | 5 µL |
| LB media | 50 mL |
| *E.coli* with plasmid 4 | 100 µL |

Group 2:

|  |  |
| --- | --- |
| Name | Dosage |
| AMP solution | 5 µL |
| LB media | 50 mL |
| *E.coli* with plasmid 4 | 100 µL |
| IPTG | 5 µL |

     Group 3:

|  |  |
| --- | --- |
| Name | Dosage |
| AMP solution | 5 µL |
| LB media | 50 mL |
| *E.coli* with plasmid 4 | 100 µL |
| sucrose | 50 g/L |

     Group 4:

|  |  |
| --- | --- |
| Name | Dosage |
| AMP solution | 5 µL |
| LB media | 50 mL |
| *E.coli* with plasmid 4 | 100 µL |
| sucrose | 50 g/L |
| IPTG | 5 µL |

- **Concepts:**

33.
Spray the gloves with alcohol and check clothing on arms, hands, etc.,
before you put your hands in the tank (to make sure no skin is exposed to the air )

34.
Light the alcohol lamp

35.
Mix the materials mentioned above (eg: group 1--->tube 1
)

36.
Place the four tubes inside a machine to shake for 16 h at 37
℃

-**Result:** wait for tomorrow’s result

l
**Gel** **electrophoresis**

- **Objective:** To check whether the DNA sample (IPCR product: plasmid 2)) we
have contains the correct DNA segments/fragments we need

- **Concept:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, then, the distance that DNA travels is also different. Then, compared to
the marker, we can find different DNA we want.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

2K marker

-**Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL)

4.
Pour the gel

5.
Wait until it turns into a solid gel

6.
Remove the comb

7.
Pour TAE solution into the tank

8.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

9.
Put the lid on and power on (180V, 20 min)

10.
(After 20 min) put the gel under a gel imager to document
bands

11.
Software: Genosens

12.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Results:**

3.
*E.coli* with Plasmid 1: the line left on the gel is a little bit lower
than the 2000bp line of marker’s. target: 1635 bp

4.
IPCR product plasmid 2: failed

l
**RM media test**

- **Objective:** Determination of fructan content by DNS method

- **Concepts:** After *E.coli* absorbs sucrose, it excretes fructan. And because we have no way
to use chemical methods to directly detect the content of levan, we can only detect the content of
fructan, so as to indirectly obtain the content of levan.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| RM solution | 5 mL |
| *E.coli* with plasmid 4 | 500 mL |
| AMP solution | 6 µL |

  RM:

|  |  |
| --- | --- |
| Name | Dosage |
| Yeast extract | 10 g/L |
| Glucose | 20 g/L |
| kH2PO4 | 2 g/L |
| (NH4)2SO4 | 1 g/L |
| MgSO4 ·7H 2O | 2 g/L |

- **Concepts:**

1.
Mix RM solution, *E.coli* with plasmid 4, and AMP solution in a tube ( make five tubes
containing this mixture )

2.
Place each tube at different temperatures for 16h to determine which
temperature is most conducive to the growth of *E.coli* ( 20 ℃, 30℃, 40 ℃, 50℃, 60 ℃ )

- **Result:** wait for tomorrow’s observation

l
**Gel electrophoresis** (Because the IPCR of plasmid 2 failed in the previous section
)

- **Objective:** To check whether the DNA sample we have contains the correct DNA
segments/fragments we need

- **Concepts:** The gel we constructed has a porous structure, which provides
small holes for DNA to run. Since DNA is negatively charged, DNA will move to a positively charged place
when electricity is applied. Moreover, different DNA fragments are of different sizes, so they move at
different speeds. Obviously, then, the distance that DNA travels is also different. Then, compared to
the marker, we can find different DNA we want.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| TAE buffer | 100 mL |
| Agarose | 1%-->1 g |
| YeaRed nucleic acid gel stain | 1/10 thousand-->10  μL |

- **Methods:** (note: the containers used in this process should be marked with
the words “TAE special”)

1.
Mix TAE buffer, agarose, and YeaRed nucleic acid gel stain in a
erlenmeyer flask

2.
Place the solution in the microwave on medium-high heat for 2
min repeatedly until the solution boils (to ensure uniform mixing)

3.
Fix comb in the mold (thick: 50 mL; thin: 20 mL)

4.
Pour the gel

5.
Wait until it turns into a solid gel

6.
Remove the comb

7.
Pour TAE solution into the tank

8.
Use the pipette to add the four reagents made in the step “PCR” and
marker to the corresponding grooves respectively.

9.
Put the lid on and power on (180V, 20 min)

10.
(After 20 min) put the gel under a gel imager to document
bands

11.
Software: Genosens

12.
Click “ultraviolet light” + “diaphragm (7)” + “automatic focus” +
“collect” + “save” + name the graph (date + reagent name)

- **Result:** Bands show the right base pair amount that we need.

l
**Extraction of sample fructan**

- **Objective:** Determination of fructan content by DNS method

- **Concepts:** Because we have no way to use chemical methods to directly detect the
content of levan, we can only detect the content of fructan, so as to indirectly obtain the content of
levan.

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| *E.coli* in the mixture of LB and AMP solution at different temperatures (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃) | 0.9 mL |
| Ethanol | 2.7 mL |

- **Methods:**

1.
Transfer 1mL mixture containing *E.coli* with plasmid 4, LB media, and AMP solution at different
temperatures (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃) from a large tube into a 1.5mL tube respectively

2.
Centrifuge the 5 tubes at 4
℃ for 5min ( 5,000
×
g )

3.
Take out the tubes from the machine and transfer 900
μL of each of them (Aspirate only the clear upper liquid (the
solution containing fructan), excluding bacteria adsorbed at the bottom of the tube) into a tube
with 2.7 mL ethanol solution

4.
Stand still

5.
Centrifuge the tubes for 5 min at 4
℃
 ( 5,000 r/min )

6.
Place the tubes in a
n
 environment at 4
℃

- **Result:** refer to the data of scant software (in the last section of today’s report
)

l
**Testing the effect of sucrose and IPTG**

- **Objective:** to find out whether adding or not adding IPTG or sucrose could provide the largest
percentage yield.

- **Concept:** IPTG can induce the expression of protein, SacB, which can cause the subsequent
RFP fluorescence. So we can use this indirect method to measure the intensity of fluorescence first, and
then speculate which one has the most products.

- **Materials:**

 Quartz cuvette

 The mixture of fructan and LB + AMP/Sucrose/IPTG solution

- Methods:

1.
Pour pure water into a quartz cuvette

2.
Turn on a machine

3.
Click “nucleic acid” on the screen

4.
Put the quartz cuvette containing water inside a machine

5.
Click “empty test” + “zero”

6.
Take out the container and clean it with dd water

7.
Pour the mixture into a quartz cuvette

8.
Click “sample testing”

(6-8 X3 times for each of the solution; Four solutions were all tested
in this way )

-**Results:**

AMP + LB: 0.467/0.47/0.472

AMP + LB + IPTG: 0.742/0.755/0.758

AMP + LB + 50 g/L sucrose: 0.736/0.744/0.743

AMP + LB + 50 g/L sucrose + IPTG: 0.798/0.806/0.805

l
**Prepare the solution for the standard curve of
fructan**

- **Objective:** Prepare the standard curve solution of fructan to compare the fructan
curves produced by the solutions that have experienced different temperatures

- **Concept:** the solutions we prepared have different concentration of glucose
(similar to fructan), so the figure would show a different gradient

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage |
| ddH2O | Determined by the amount of glucose and target concentration |
| Glucose | 0 mg/5 mg/10 mg/15 mg/20 mg/25 mg/30 mg/35 mg |
| HCl | 0.1 M |

- **Methods:**

1.
Mix different quantity of glucose with ddH2O to achieve the concentration of 0mg/mL, 2 mg/mL, 2.5 mg/mL, 3 mg/mL,
3.5 mg/mL, 4 mg/mL respectively

2.
Centrifuge the 4 tubes

3.
Keep only the turbid part below

4.
Add 0.1 M hydrochloric acid to each of the tube with the precipitate of
glucose

5.
Place the tubes inside a water bath for 30min at 100 ℃

- **Result:** achieving the target concentration

l
 **Optical density value (OD 540nm)**

- **Objective:** to directly figure out which of the material can affect the
percentage yield of levan the most obviously.

- **Concept:** Microplate reader can the microplate reader can use light to detect
which of the liquids at different temperatures has the largest yield

- **Materials:**

|  |  |
| --- | --- |
| Name | Dosage (mL) |
| The standard solution ( 0 mg/mL, 2 mg/mL, 2.5 mg/mL, 3 mg/mL, 3.5 mg/mL, 4 mg/mL ) | Each: 2 mL |
| DNS solution | 2 mL |
| ddH2O | 5 mL |
| Solution at different temperature ( 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ ) | 2 |

- **Methods:**

1.
Mix the standard solutions (0 mg/mL, 2 mg/mL, 2.5 mg/mL, 3 mg/mL, 3.5
mg/mL, 4 mg/mL ) with 2 mL of DNS solution respectively in 6 new tubes

2.
Mix the solutions at different temperature (20 ℃, 30 ℃, 40 ℃, 50 ℃, 60
℃ ) with 2 mL DNS solution respectively in 5 new tubes

3.
Add 5 mL ddH2O r to all 11 tubes

4.
Use a pipette to transfer 100 µL of each of the solution into a
96-well plate, a liquid corresponds to three consecutive holes arranged vertically

5.
Turn on a microplate reader

6.
Click “scant” (software ) on the screen

7.
Click “new program”, “layout”
(
 select all ), “protocol “selec
t
 “in” and “out”, “shock”,
fill in “540 nm”, “start”, and finally name the file.

{% endblock %}

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

![](https://static.igem.wiki/teams/4873/wiki/results.png)

**Overview**

Our project's goal is to obtain levan through a
biosynthetic pathway and investigate the effects of different promoter sequences on levan expression. In
the experimental design, we constructed an RFP (Red Fluorescent Protein) to identify which system is
more effective based on the expression level of RFP and the final production of levan. Ultimately,
through a large number of experiments, we confirmed the suitable promoter sequences.

|  |
| --- |
| **Target**  T arget 1: Construction of plasmids  1)  Amplification of DNA fragments: PCR of zliES, SacB, RFP, plac, puv5. IPCR of plasmid 1 and plasmid 2 .  2)  Using enzyme digestion and enzyme ligation to construct our 6 plasmids.       Specifically as follows:  **Plasmid 1** :  pUC-zliES  **Plasmid 2** :  pUC-zli-SacB :  C ompared to  pUC-zliES , includ ing   the SacB sequence along with the promoter sequence naturally present in SacB itself.  **Plasmid 3** : pUC-zli-SacB-RFP :RFP was constructed into  pUC-zli-SacB , however, this plasmid cannot express RFP as it lacks the corresponding promoter sequence.  **Plasmid 4** : pUC-zli-SacB-PRFPT : This part added a promoter to RFP, allowing RFP to express.  **Plasmid 5** : pUC-zli-plac-SacB-PRFPT :  Plasmid 5, as compared to Plasmid 4, replaced the promoter sequence naturally carried by SacB with plac (a different promoter sequence), in the hope of achieving improved expression of SacB.  **Plasmid 6** : pUC-zli-puv-SacB-PRFPT :Plasmid 6, compared to plasmid 5, replaced the plac promoter with the puv promoter, altering the promoter, to compare the difference in expression of SacB. |
| Target 2: DNA extraction  1)  To separate the gel and our DNA fragment.  2)  Figure out the concentration of DNA we constructed. |
| Target 3:  1)  Testing the productivity of levan at different temperature.  2)  Compare the result brought by plasmid  4,  plasmid 5 and plasmid 6. |

**Detailed****results**

**Construction of Plasmid 1,2,3,4,5,6**

![](https://static.igem.wiki/teams/4873/wiki/results-1.png)

Fig1. Work flow of construction of Plasmid 1,2,3,4,5,6

1)

We successfully obtained the target fragment, such as zliE-zliS, promoter,
SacB, RFP, through PCR amplification. As shown in Figure 1, we confirmed their consistent sizes
using DNA gel electrophoresis.

![](https://static.igem.wiki/teams/4873/wiki/results-2.png)

Fig 2.  Analysis of DNA gel electrophoresis for the identification of
the target fragment

2)
Construction of plasmid 1(pUC-zliES)

In order to construct plasmid 1, we used PCR to
amplify the EV(empty vector) pUC19 and zliES.

Then we used enzyme digestion to cut a same
shape of
the end of the DNA of pUC19 and zliES respectively. Finally, they were combined together by T4-DNA
ligase.

|  |  |
| --- | --- |
|  | ![](https://static.igem.wiki/teams/4873/wiki/results-3-1.png) |

Fig 3.1.
Monoclonal cultivation

![](https://static.igem.wiki/teams/4873/wiki/results-3-2.png)

Fig 3.2. Sequencing results indicate the successful construction of
pUC-zliES

3)

C
onstruction of plasmid 2 (pUC-zli-SacB)

By using PCR to amplify SacB and IPCR to amplify
plasmid 1, we had successfully obtaied recombiant plasmid.
Afterwards, we still used the enzyme digestion
to cut
the same shape of the end of SacB and plasmid 1. Through Figures 4.1 and 4.2, we have successfully
constructed pUC-zli-SacB.

![](https://static.igem.wiki/teams/4873/wiki/results-4-1.png)

Fig. 4.1 Monoclonal cultivation

![](https://static.igem.wiki/teams/4873/wiki/results-4-2.png)

Fig.
4.2
  Identification of the SacB fragment after enzyme
digestion through DNA gel electrophoresis

![](https://static.igem.wiki/teams/4873/wiki/results-4-3.png)

Fig.4.3 Sequencing results indicate the successful construction of
pUC-zliES

4)

C
onstruction of plasmid 3
(pUC-zli-SacB-RFP)

Similar as the previous sections, we used PCR to
amplify plasmid 2 and RFP, while instead of using enzyme digestion and ligation, we use homologous
recombination to combined plasmid 2 and RFP. As shown in Figures 5.1 and
5.2, we
have successfully constructed pUC-Zli-SacB-RFP.

![](https://static.igem.wiki/teams/4873/wiki/results-5-1.png)

Fig.5.1 Identification of the RFP fragment after enzyme digestion through
DNA gel electrophoresis

![](https://static.igem.wiki/teams/4873/wiki/results-5-2.png)

Fig 5.2 Sequencing results indicate the successful construction of
pUC-Zli-SacB-RFP

5)

C
onstruction of plasmid 4 (pUC-zli-SacB-PRFPT)

In order to add a promoter for RFP to help the
plasmid
to express its red fluorescence, we used PCR to amplify the promoter of RFP and IPCR to amplify plasmid
3. After that, we used enzyme digestion and T4-DNA ligase to combined them and construct plasmid 4. As
shown in Figure 6, we have successfully obtained pUC-Zli-SacB-RFP-T.

![](https://static.igem.wiki/teams/4873/wiki/results-6-1.png)

Fig 6.1 Monoclonal cultivation

![](https://static.igem.wiki/teams/4873/wiki/results-6-2.png)

Fig.6.2 Identification of the promoter fragment after enzyme digestion
through DNA gel electrophoresis, with a fragment size of approximately 300 bp.

![](https://static.igem.wiki/teams/4873/wiki/results-6-3.png)

Fig.
6.3
Sequencing results indicate the successful
construction of pUC-Zli-SacB-
P
RFPT.

6)
Construction of plasmid 5 and plasmid 6
(pUC-zli-plac-SacB-PRFP and
pUC-zli-puv-SacB-PRFP)

By using PCR, we successfully amplified plac,
puv5 and
plasmid 4. Then a series of enzyme digestion and enzyme ligation (to link plac with plasmid 4 and to
link puv5 with plasmid 4 respectively.) were operated, and finally plasmid 5 and plasmid 6 were
constructed
.
Figure 7.1 displays a monoclonal culture
containing
plasmid 5, Figure 7.2 displays a monoclonal culture containing plasmid 6, and Figures 7.3/7.4 are the
respective sequencing identification results.

![](https://static.igem.wiki/teams/4873/wiki/results-7-1.png)

Fig7.1 Monoclonal culture containing plasmid 5

![](https://static.igem.wiki/teams/4873/wiki/results-7-2.png)

Fig7.2 Monoclonal culture containing plasmid 6

![](https://static.igem.wiki/teams/4873/wiki/results-7-3.png)

Fig7.3 Sequencing results indicate the successful construction of
pUC-zli-plac-SacB-PRFP

![](https://static.igem.wiki/teams/4873/wiki/results-7-4.png)

Fig7.4 The sequencing results confirm the successful construction of
pUC-zli-puv-SacB-PRFP

**Functional Test**

1.

In order to investigate the expression levels of SacB in different promoter
systems (plasmid 4/5/6), we first compared the fluorescence intensity of RFP. We observed that in
the Plasmid 5 system, SacB exhibited the highest red fluorescence intensity. Subsequently, we
measured the fluorescence intensity values of RFP, as shown in Figure 8, which also confirmed that
Plasmid 5 had higher values, indicating that SacB has a higher expression level in the Plasmid 5
system.

|  |
| --- |
|  |
|  | ![](https://static.igem.wiki/teams/4873/wiki/results-8.png) |

Fig 8.  In the Plasmid 4/5/6 expression systems, when comparing the
brightness of RFP, Plasmid 5 exhibits stronger RFP expression.

We conducted qualitative (RFP color brightness) and quantitative analyses
of RFP expression levels in different plasmid systems. Figure 9 demonstrates that in the Plasmid 5
system, there is also a higher level of RFP expression.

![](https://static.igem.wiki/teams/4873/wiki/results-9.png)

Fig.9.  Quantitative analysis of RFP expression levels in three
different systems, with the vertical axis representing the fluorescence intensity of RFP.

2.

In order to validate which biosynthetic pathway results in higher levan
production, we compared the levan yields in the plasmid 4/5/6 systems. The average levan production
was higher in the plasmid 5/6 systems, as shown in Figure 10-B/C/D. Additionally, we also tested
levan production at different temperatures. In all three systems, levan production was higher at
around 30
°
C.

![](https://static.igem.wiki/teams/4873/wiki/results-10.png)

Fig10. pUCzli-SacB-PRFP, pUCzli-PlacsacBRFP, pUCzli-PUVsac-BRFP plasmids
’
expression and functional identification.

**Future plans**

In the future, we hope to use the data from this
experiment and the highest-yield plasmid 5 to mass-produce levan. Moreover, we hope to conduct more
explorations into the production of levosan, making levosan more practical and allowing it to be used in
various fields. For example, regarding cancer treatment, it is integrated into health products and skin
care products to prevent aging.

{% endblock %}