iGEM 2023 — Team shsid-china

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### **7.31**

l ice
breaker and self introductions

l laboratory
safety training

### **8.1**

l preparing
to construct plasmid pTRIP with pTrac99k+RIP

¡ make
nucleic acid gel

¡ PCR,
gel electrophoresis, and DNA gel extraction for both vector and target gene

n failed
-> multiple trials with different annealing temperatures

l preparing
both liquid and solid LB culture medium

### **8.2**

l constructing
plasmid pTRIP

¡ Test
the DNA concentration with Nanodrop

¡ homologous
recombination of pTrac99k and RIP to obtain plasmid pTRIP

¡ pTRIP
transformed into DH5α

### **8.3**

l run
another gel electrophoresis of pTRIP

l Send
the company to sequencing

l EGFP and cddB
,pTRIP-E and pTRIP-C to
varify the lengths

¡ DNA
gel extraction of the correct strips

l tranform
pTRIP-EGFP into DH5α and pTRIP-ccdB into DB3.1

l plating
of DH5α-pTRIP-EGFP and DB3.1-pTRIP-ccdB

l leave
the samples in the isothermic incubator at 37°C overnight

### **8.4**

l Monoclonal
antibodies were selected and verified, and
put into kanamycin-included LB medium, incubate at 37°C, shaking for 12-16h

### **8.5**

l plasmid
extraction of bacterial fluids

¡ test
the concentration with Nanodrop

¡ gel
electrophoresis for varification

¡ Send
the company to sequencing

l inducing
protein expression

l prepare
protein gel for SDS-PAGE using Sangon 12.5%
SDS-PAGE Color Preparation kit

### **8.6**

l Ultrasonic
crushing, protein concentration(pTRIP-EGFP
and pTRIP-ccdB)

### **8.7**

l protein
extraction

l SDS-PAGE (pTRIP-EGFP
and pTRIP-ccdB)

### **8.8**

l The
fluorescence intensity of EGFP was measured

l The
reporter gene EGFP was
observed under microscope

###

### **8.9**

l  The
pTRIP-ccdB was transformed into*E.coil* DH5α
and BL21 ( DE3 )

### **8.10**

l Monoclonal
antibody verification of pTRIP-ccdB in*E.coil* DH5α
and BL21 ( DE3 )

### **8.11**

l Detection
of pTRIP-ccdB growth ability in*E.coil* DH5α
and BL21 ( DE3 )

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![](https://static.igem.wiki/teams/4942/wiki/engineering.png)

Overview

Some
genetically modified microorganisms used in the production of engineered probiotics or industrial
fermentation strains require special precautions for biosafety1.
It is important to prevent the unintentional release, multiplication, and spread of these genetically
modified microorganisms into the environment, which could lead to unpredictable biological
contamination2.
This project has designed a simple and user-friendly "safety lock" for engineered microorganisms. Under
normal conditions at 37℃ (the temperature inside the human body, which is also the working temperature for
probiotics and commonly used in industrial microbial fermentation), the "safety lock" remains inactive,
allowing the host microorganism to reproduce and function normally. However, at 22℃ (a temperature closer
to natural environmental conditions, excluding tropical regions and extremely hot summers), the "safety
lock" becomes active, expressing a toxic protein that leads to the self-destruction of the host
microorganism, thereby preventing the release of the engineered microorganisms3-5. We constructed a temperature control system（pTRIP）, and introduced a visual
reporting gene(EGFP), into the system to monitor its functionality and optimize its sensitivity. Lastly,
we developed a plasmid, pTRIP-ccdB, which contains a toxic protein, (ccdB), that can kill bacteria upon
expression, thereby preventing the leakage of engineered microorganisms(Figure 1).

.
![](https://static.igem.wiki/teams/4942/wiki/engineering/1.png)

Figure
1. The engineering design schematic diagram.

Part 1 pTRIP-EGFP (BBa\_K4942008)

**Design:**

pTRIP plasmid was constructed by synthetic RIP from Genscript and
the vector ppTrc99k.provided by SubCat.

The template sequences RIP include luxR2, luxI, PluxI, and
PluxCDABEG. Homologous recombination method was employed for the construction of pTRIP
(ppTrc99k-RIP).

The pTRIP functions as a temperature-controlled switch and
consists of two fundamental regulatory proteins, LuxI and LuxR2. LuxI is a synthesis enzyme for the
autoinducer, which generates signal molecules known as AHL or HSL. LuxR, on the other hand, serves as
both a receiver for cytoplasmic signal molecules and a DNA-binding transcriptional regulator. These two
proteins jointly regulate the expression of the bioluminescence-related fluorescent protein manipulator,
PluxCDBEG. Consequently, it allows for the control of both the PluxCDBEG module and protein expression.

In order to measure the function effectivity of our system, we
induced the reporter EGFP for visible fluoresces which was also provided by SubCat. Using pTRIP plasmid
as a template, the EGFP fragment was homologously recombined with a linearized plasmid to construct the
pTRIP-EGFP plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/2.png)

Figure 2. The
plasmid map of pTRIP-EGFP

**Build:**

1.1 **pTRIP**

We
constructed pTRIP using homologous recombination. The RIP sequence was amplified by PCR, with a length of
2065 bp. The Figure 3A indicates
the band
consistent
with the results. The pTrc99k plasmid was used as a template for PCR amplification, resulting in a
fragment of 3408bp for
pTrc. The Figure 3B shows
a band consistent with the target size. It indicating
successful amplification of RIP and linearization of the pTrc plasmid. After
DpnI
digestion and gel recovery, the pRIP fragment was obtained.

![](https://static.igem.wiki/teams/4942/wiki/engineering/3.png)

Figure 3. The gel
electrophoresis validation of RIP and pTrc nucleic acids.

The plasmids were
transformed into *E.Coil*DH5α .
Figure4 A shows the presence of single colonies on
the plate. We selected colonies 1-10 and sent them directly for sequencing. According to the results shown
in Figure 4B and
C, the
pTRIP was successfully
ligated to the pTrc vector without any apparent mutations, confirming the successful construction of the
pTRIP plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/4.png)

Figure 4. The Monoclonal
antibody validation and sequencing of pTRIP (DH5α) .

Note：

A. Transformation
plate of pTRIP:

B.  Sequencing
results of pTRIP

C.  Base
comparison of a specific region within pTRIP

1.2 **pTRIP-EGFP**

We constructed the pTRIP-EGFP plasmid using homologous
recombination. The EGFP sequence, which is 720bp in length, was amplified by PCR. The Figure 5 A showed
that matched the expected size, indicating successful amplification of the EGFP sequence. Furthermore,
using the pTRIP plasmid as a template, we performed PCR amplification and obtained 5473bp fragment of
pTRIP-E. The Figure 5 B showed bands that matched the expected size, indicating successful amplification
of the linearized pTRIP-E plasmid. We can conclude that we successfully amplified the EGFP sequence and
linearized pTRIP-E plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/5.png)

Figure 5. The gel
electrophoresis validation of EGFP and pTRIP-E .

We transformed the plasmid pTRIP-EGFP into DH5α, and Figure 6 A and
B showed the growth of single clones. We selected clones 1-6 and performed antibody verification.The
expected size of 720bp for the EGFP sequence. Additionally, Figure 6 C showed bands that matched the
expected size, indicating successful transformation. Subsequently, we sent clones 1-6 for sequencing.
The sequencing results in Figure 6 D showed a 100% match with the nucleotide sequence of EGFP,
confirming the successful integration of the EGFP fragment into the pTRIP-E plasmid and further
validating the successful construction of the plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/6.png)

Figure 6. The Monoclonal
antibody validation and sequencing of pTRIP-EGFP
(*E.Coil* DH5α) .

Note:

A.B shows the plate
images of pTRIP-EGFP transformed into *E.Coil* DH5α cells.

C is the image showing
the verification of single clone colonies.

D is the sequencing
image of pTRIP-EGFP plasmid.

**Test:**

**1.****Protein expression**

The target protein
EGFP has a size of 26.9kDa. Protein expression was induced at a concentration of 0.6mmol AI
(AutoInducers, herein it refers to N-(3-oxohexanoyl)-L-homoserine lactone), and induction was performed
at 37 oC and 22
oC. According to the
figure 9, in the control group (line 1), no EGFP protein is present. Under the condition of 37
oC (line 1 to line
3), EGFP protein was not observed. However, under the condition of 22 oC (line 4 to line
5), there is a clear presence of EGFP protein. This indicates that EGFP expression is not
express or occurring
at a lower level at 37 oC, while there is
substantial expression at 22 oC. Therefore, it can
be concluded that our temperature control system is activated state at 22oC and deactivated
state at 37 oC.

![](https://static.igem.wiki/teams/4942/wiki/engineering/7.png)

Figure 7.The SDS-PAGE
protein gel of EGFP at different temperatures

Note:

line
1: pTRIP(DH5α)

line
2：pTRIP-EGFP-37 oC(DH5α)

line
3：pTRIP-EGFP-37 oC(DH5α)

line
4：pTRIP-EGFP-22 oC(DH5α)

line
5：pTRIP-EGFP-22 oCo(DH5α)

2. **Functional Test**

3.1.1 Detection of fluorescence intensity EGFP

Protein expression was induced by AI at a
concentration of 0.6 mmol and at different temperatures. As shown
in Figure 8, the fluorescence intensity of pTRIP-EGFP
increased first and then decreased with the increase of temperature. At 22 oC, the fluorescence intensity of pTRIP-EGFP was
the strongest. At 37 oC, the fluorescence intensity of pTRIP-EGFP was
the weakest. However, no fluorescence signal was detected in the control group pTRIP. This shows that the
temperature control system of our subject is controlled by temperature. At 22 oC, in the open state ; At 37 oC, it is close to the closed state.

![](https://static.igem.wiki/teams/4942/wiki/engineering/8.png)

Figure 8. The
fluorescence intensity of reporter gene EGFP at different temperatures

In order to observe
the fluorescence signal of pTRIP-EGFP more clearly, we made the glass slides of pTRIP-EGFP bacteria and
control group ( pTRIP ) cultured at 22 oC and 37
oC. According to the
Figure9, A (colonies under white light) and B (pTRIP-EGFP at 37
oC) have no
fluorescence signal, C and D (pTRIP-EGFP at 22
oC) have obvious
fluorescence signal. It shows that there is no fluorescence signal at 37 oC, and obvious
fluorescence signal can be seen at 22 oC.

![](https://static.igem.wiki/teams/4942/wiki/engineering/9.png)

Figure 9.The
fluorescence signal of report gene EGFP under microscope

Note:

A : Colonies under white
light

B. Fluorescence reporter
gene of pTRIP-EGFP plasmid at 37 °C

C. 22 ° fluorescence
reporter gene of pTRIP-EGFP plasmid ( 10 × 40 )

D. 22 ° fluorescence
reporter gene of pTRIP-EGFP plasmid ( 10 × 100 )

**Learn:**

Through this cycle that evaluated the function of the pTRIP-EGFP
system, we can basically predict that the temperature-based “switch” works so that
we can step further to construct the temperature-based “kill-switch” system.

Besides, we also encourage future iGEM teams to further explore
and optimize pTRIP-EGFP, such as whether it is a self-induced temperature control system or a system
that requires exogenous induction, which requires more experimental data for more precise evaluation,
for example, the relationship between EGFP fluorescence intensity and temperature can be more refined,
the temperature gradient can be more intensive, and the optimal induction time also needs to be further
explored.

Part 2 pTRIP-ccdB(BBa\_K4942010)

**Design:**

The ccdB is a toxic protein that needs to be transformed into
*E.coli* DB3.1 competent cells. *E.coli* DB3.1 competent cells are anti-toxic. In this cycle, we will
replace EGFP with ccdB to upgrade the temperature-based “switch” to a “kill-switch”(Figure 10).

After the first cycle, we successfully obtained pTRIP plasmid
which functions as a temperature-controlled switch.The pTRIP-C linearized plasmid was obtained by using
pTRIP plasmid as template.

![](https://static.igem.wiki/teams/4942/wiki/engineering/10.png)

Figure 10 . The
plasmid map of pTRIP-ccdB

**Build:**

2.1 **pTRIP-ccdB**

We constructed the pTRIP-ccdB plasmid using homologous
recombination. The PCR amplification of the ccdB sequence resulted in a fragment of 306bp in length. The
figure 11 indicates that the amplified band matches the expected size, confirming the successful
amplification of the ccdB sequence from the linearized plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/11.png)

Figure 11. The gel
electrophoresis validation of ccdB.

By using the pTRIP plasmid as a template, we performed PCR
amplification to obtain a 5473kb fragment referred to as pTRIP-C. The gel electrophoresis image in
Figure 12 shows that the amplified band matches the expected size,
indicating the successful amplification of the linearized pTRIP-C plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/12.png)

Figure 12. The gel
electrophoresis validation of pTRIP-C.

The pTRIP-ccdB plasmid was transformed into
*E. coli* DB3.1. The single clone colony growth on plates is
shown in Figure 13 A and B. Clones 1-8 were selected for
antibody verification, and the results in Figure 15C demonstrate clear bands, confirming the
presence of the ccdB sequence with a length of 306bp. The gel electrophoresis image in Figure 15C matches the expected band, indicating the
successful transformation.

Next, colonies 1-8 were sent for sequencing,
and the sequencing results in Figure 13 D showed a 100% match with the ccdB
nucleotide sequence. This confirms the successful integration of the ccdB fragment into the pTRIP-E
plasmid. It further validates the successful construction of the pTRIP-ccdB plasmid.

![](https://static.igem.wiki/teams/4942/wiki/engineering/13.png)

Figure 13. The monoclonal
antibody validation and sequencing of pTRIP-ccdB
(*E.Coil* DB3.1) .

**Test:**

**1.****Protein expression**

The size of the ccdB protein, the target protein, is 11.7 kDa.
Protein expression was induced at a concentration of 0.6mmol AI, and induction was performed at 37
oC and 22 oC. According to the figure 14, in the control group (line 1 -line
2 and line 9 -line 10), ccdB protein was not observed. Under the condition of 37 oC (line 3 to line 4), ccdB protein was not
observed. However, under the condition of 22 oC (line 5 to line 8), a weak presence of ccdB protein is detected.
This indicates that ccdB expression does not occur at 37 oC, while there is limited expression at 22
oC. Therefore, it can be concluded that our temperature control
system is in the activated state at 22 oC and in the deactivated state at 37 oC.

![](https://static.igem.wiki/teams/4942/wiki/engineering/14.png)

Figure
14 :
The SDS-PAGE of ccdB protein

Note:

1-2:
 *E.coil* DB3.1(control)

3-4：37oC-pTRIP-ccdB*(E.coil* DB3.1)

5-8：22oC-pTRIP-ccdB(*E.coil* DB3.1)

9-10: *E.coil* DB3.1(control
)

**2.** **The growth ability****test** **of
pTRIP-ccdB** **(****DH5α****)**

**A. The growth
ability of pTRIP-ccdB****(*****E.coil*****DH5α****)****at 22
° C**

According to Figure
15 A to
E, given
the temperature 22 °C,
the OD600 of E.coil in the control group increased first and
then gradually tended to be stabilized over
time. While
there was no significant
increase
in OD600 of pTRIP-ccdB
at the experimental group over
time. Therefore,
compared with the wild DH5α (the control
group), we can conclude that the ccdB did kill bacteria, the host
at 22 °C upon
expression.

![](https://static.igem.wiki/teams/4942/wiki/engineering/15.png)

Figure 15.The growth ability of pTRIP-ccdB
( *E.coil* DH5α ) in different AI concentrations at 22 ° C

B. **The growth ability of
pTRIP-ccdB****(*****E.coil*****DH5α****)****at****37** **° C**

According to Figure16A-E,
the pTRIP-ccdB E.coil
DH5α has a
very similar growth trend
with the control group, the wild E.coil DH5α at 37 °C which means the “switch”
pTRIP-ccdB has little effect on the strain’s growth since no
expression of ccdB at 37
°C.

![](https://static.igem.wiki/teams/4942/wiki/engineering/16.png)

Figure 16.The growth ability of pTRIP-ccdB (
*E.coi*l DH5α ) in different AI concentrations at 27 ° C

C. **Compar****ison of
t****he
growth ability of pTRIP-ccdB****(*****E.coil*****DH5α****)****at****37** **°C** **and****22****°C**

According to Figure 17, in two groups
with
AI concentration of 0.6 mmol, the OD600 of pTRIP-ccdB at 37 °C increased firstly and then tended to
be stabilized
over time, while
there was almost no
significant change
of that
at 22 °C. It is seen that
pTRIP-ccdB (E.coil
DH5α) grew much better at 37 °C than
22 °C where it
indicated little growth over time. This further supports the conclusion that the
bacterial strain grows normally at 37°C, while the presence of ccdB at 22°C leads to bacterial
cell death.

.

![](https://static.igem.wiki/teams/4942/wiki/engineering/17.png)

Figure 17.
Comparison of OD600
at
37°C and 22°C pTRIP-ccdB (*E.coi*l DH5α ) with
AI concentration of 0.6 at different times

**2.2** **The growth ability****test** **of pTRIP-ccdB
in*****E.coil*** **BL21 (DE3).**

Comparisons of growth capabilities were made
at 37°C and 22°C with an AI concentration of 0.6 mmol.The *E.coil* BL21 was the
control group. According to
Figure 18, it is evident that the OD600 of BL21 (the control
group) is
significantly higher than that of
BL21(pTRIP-ccdB) at
22°C while the OD600 of BL21 and BL21(pTRIP-ccdB) have similar value.
This test result is consistent with that for DH5α as discussed
previously and this also back up the engineering success of our temperature-based “kill
switch”, the plasmid
pTRIP-ccdB in E. coli host.

![](https://static.igem.wiki/teams/4942/wiki/engineering/18.png)

Figure 18.
The Growth ability of pTRIP-ccdB
in *E.coil* BL21 ( DE3 )

**Learn:**

1. Based on the
growth ability tests, it indicates that the experimental strains with pTRIP-ccdB could grow normally
at 37 °C but stop
growing at 22 °C which
validates the engineering success of our temperature-based “kill-switch” as
expected.

2. In the experiment, we only conducted sensitivity
testing of the temperature-controlled switch in *E. coli*BL21(DE3) and *E. coli* DH5α. In the future, we need to
transform it into more bacterial strains to perform sensitivity tests and further optimize the sensitivity
of our temperature-controlled switch based on the test results. We also need to explore the temperature
range of 22-37°C for the temperature-controlled switch and validate its states through additional
verification methods.

3. Our temperature-controlled switch requires the
addition of exogenous inducers (N-(3-oxohexanoyl) -L-homoserine lactone) for better expression. When no inducer is added,
the expression level is relatively low, and this limitation needs to be addressed in practical
applications.

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![](https://static.igem.wiki/teams/4942/wiki/experiments.png)

## 1. **Disposition of LB Culture Base**

Goal:
To create a functional and nutritional liquid environment for bacteria to grow. Liquid media used for
various purposes such as propagation of microogranims or othe tests.

Materials:

1）
Sterlized
duran bottle, cap

2）
Autoclave

3）
Tryptone

4）
Yeast
Extract

5）
Sodium
Chloride

6）
Double
distilled H2O

7）
Lab
Refrigerator

Procedures:

1）
Create
LB Medium (Used for maintaining and cultivating recombinant straints of E.coli):

2）
Prepare
14g of premix LB broth powder (Tryptone: Yeast extract: Sodium chloride= 2:1:1)

3）
Add
into sterlized 1L duran bottle

4）
Add
double distilled H2O to total volume of 700ml

5）
Close
the cap and autoclave it for 20 minutes at least

6）
Wait
until it cools

7）
Store
at 4 degree Celcius

2.
**The construction
of plasmid**

**2.1** **Construction of pTRIP**

### **A.****Obtaining target gene fragments** **by** **PCR**

### Goal: To amplify pTracK99 and RIP fragments for later recombination

Materials:

1）
2xPrimeMixStar\*

2）
pTracK99
and RIP gene template

3）
double
distilled water (ddH2O）

4）
Primer-R

5）
Primer-F

6）
centrifuge
tube

7)
PCR
thermal cycler

\*
PrimeMixStar includes buffer and dNTPs for PCR. The extention rate for PrimestarMix (2x) is 2000bp/min;
the time varies with different templates

Procedure:

1）
Add
2xPrimerMixStar 25µl, pTracK99 template 1µl, primer-R 1µl, and primer-F 1µl to
centrifuge tube. Add ddH2O to the centrifuge tube until reaching 50µl. If bubbles are observed in
the mixture, centrifuge the mixture for a few seconds to remove the bubbles

2）
Insert
mixture into a PCR thermal cycler. Denature the fragments at 95˚C for 3 minutes

3）
Perform
30 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C
extension.

4）
Extend
the fragments for 5 minutes at 72˚C

5）
If
not use immediately, preserve the fragments at 4˚C

6）
Repeat
step 1-5 for RIP. Replace pTracK99 template and the corresponding primers for those of RIP. Annealing
temperature for pTracK99 is 48˚C

### B. **Agarose gel construction, deployment and recycle**

Goal:
To produce a functional and well-shaped gel for electrophoresis

Materials:

1）
Casting
Tray

2）
Well
comb

3）
Microwave

4）
1x
TAE

5）
Agarose
M

6）
10000x
Nucleic
acid gel stain

7）
Erlenmeyer
flask

Procedure:

1.
Dissolve
0.3g Agarose in 30mL Erlenmeyer flask

2.
Add
TAE (Running buffer)

3.
Heat
the material with microwave

4.
Melt
the mixture at 30 sec interval

5.
See
whether bubbles are forming, if did succesfully mixed

6.
Add
3μl
nucleic acid gel stain (10000x)

7.
Let
it cool for 5~10 minutes

8.
Pour
the agarose mixture into the casting tray

9.
Place
the appropriate well comb to create the wells

10.
Wait
to solidify 7~10 minutes

11.
Remove
the comb and place the gel in the gel box

C.
**A****garose gel electrophoresis**

Goal:
To verify the DNA bands' lengths and thus preparing for the next step of gel extraction to extract the
bands that are of the correct length (corresponding with the length of pTRIP), eliminating the genetic
fragments that didn't successfully undergo PCR.

Materials:

1）
Sample

|  |  |
| --- | --- |
| Sample | Amount |
| RIP (PCR result) | 100+ |
| pTrac-99K (PCR result) | 200+ |

2)
Marker
(DNA ladder)

3)
Prepared
gel (from previous step)

4)
10x
Loading buffer (dye)

5)
Horizontal
gel electrophoresis system

Procedure:

1)
extract
50µl of every sample; add 5µl of loading buffer (10x) to each.

2)
Place
the gel in the horizontal electrophoresis system

3)
Extract
50µl of the mixed solution containing the sample, mix them till their colors are almost
uniform

4)
Add
the marker to the first well and load 50µl of each sample into the wells

5)
The
procedure runs at 160V for 30min

6)
Afterwards,
take out the gel from the device and proceed to following steps

### D. **DNA Gel Extraction**

Goal:
To recover DNA fragments in agarose gel.

Materials

l
agarose
gel with pTRIP

l
clean
scalpel

l
1.5
mL Eppendorf (EP) tube

l
pipettes
and sterilized pipette heads

l
hot
water bath

l
buffer
B2

l
purification
column and collection tube

l
centrifuge

l
wash
solution containing pure ethanol

l
elution
buffer

Procedure

1)
Cut
the slice of gel containing pTRIP, cutting off as much unneeded gel as possible, and place it in an 1.5 mL
Eppendorf (EP) tube.

2)
Add
500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely
melted.

3)
Transfer
the solution containing melted gel into a purification column that's in a collection tube and perform
centrifugation at 8000 g for 30 seconds.

4)
Empty
the collection tube, put the purification column back in, and add 500 microliters of wash solution
containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.

5)
Repeat
above.

6)
Perform
centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one
minute to allow the ethanol to evaporate.

7)
Transfer
purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the
purification column.

8)
Cap
the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9)
Discard
the purification column. Store DNA in the EP tube at 4°C.

### E. **Homologous recombination of pTrac99k with RIP**

Goal:
creating the vector pTRIP as template for homologous recombination with EGFP and ccdB

Materials
& Apparatus:

l
ClonExpress
Mix (2x)

l
double
distilled water (ddH2O)

l
PCR
products of pTrac99k & RIP

l
Nanodrop

l
water
bath

Procedure:

1.
test
the concentration on pTrac99k and RIP using Nanodrop

2.
prepare
the system (20μl)

|  |  |
| --- | --- |
| ClonExpress Mix(2x) | 10μl |
| vector (pTrac99k) | 30fM |
| target gene (RIP) | 60fM |
| ddH2O | fill to 20μl |

3.
place
the system in 50°C water bath for 10min

## F. **T****ransformation**

Materials

Ligated
samples, LB
Agar plates and liquid solution

Procedure:

a）Ligated
samples are added to*E.Coli*
samples

b）Placed
on ice for 20 minutes.

c）Heat
at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.

d）Add
900μL LB Agar solution, incubate for 30 minutes

e）Centrifuge
at 5000xg for 3 minutes, extract 900μL of supernatant then discard

f）Apply
end product to agar plates, incubate for 12-16hr at 37 degrees Celsius.

\*Possible
mistake: It’s required to spread bacteria mildly, since harsh physical shock will damage their
structure.

G.
**Monoclonal
Antibody Validation**

\*purpose:
DH5alpha
bacteria containing constructed plasmids has multiplied. Monoclonal Antibody Validation is used for
ensuring that the plasmids have replicated during this process.

Procedure

a）Within
sterilized fume hood workstation, remove a colony of preprepared bacteria from the agar plate and add to
1.5mL centrifuge tube (Note: choose larger colonies but also be aware that colonies near the sides of the
petri dish are most likely to be contaminated or are undesired bacteria and are less ideal)

|  |  |  |
| --- | --- | --- |
| PCR program | Temp. (celsius degrees) | Period |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Repetition-Denaturation,Annealing,Elongation | / | 30times |

Procedure

A.
Obtain a conical flask. Use measuring cylinder to measure 100ml of (1x)TAE solution and transfer it to the
conical flask.

B.
Place the agarose sample on the weighing paper, Use digital balance and spatula to measure 1g of agarose
sample and add it to the flask.

C.
Place the flask in the microwave oven and heat in high temperature for 2 to 3 minutes.

D.
Use pipette to measure 5μl of Nucleic acid dye(Gel red) and add it to the flask.

E.
Add the sample in the flask to the mode of electrophoresis instrument.

F.
Wait for approximately 20 minutes till the gel coagulates.

## **2.2****Construction of pTRIP-EGFP**

### **A.****PCR of linear pTRIP vector** **and** **EGFP**

Goal:
To amplify pTRIP and EGFP fragments for later recombination

Materials:

1)
2xPrimeMixStar\*

2)
pTRIP
and EGFP gene template

3)
double
distilled water (ddH2O)

4)
Primer-R

5)
Primer-F

6)
centrifuge
tube

7)
PCR
thermal cycler

\*
PrimeMixStar includes buffer and dNTPs for PCR. The extention rate for PrimestarMix (2x) is 2000bp/min;
the time varies with different templates

Procedure:

1)
Add
2xPrimerMixStar 25µl, EGFP template 1µl, primer-R 1µl, and primer-F 1µl to
centrifuge tube. Add ddH2O to the centrifuge tube until reaching 50µl. If bubbles are observed in
the mixture, centrifuge the mixture for a few seconds to remove the bubbles

2)
Insert
mixture into a PCR thermal cycler. Denature the fragments at 95˚C for 3 minutes

3)
Perform
30 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C
extension.

4)
Extend
the fragments for 5 minutes at 72˚C

5)
If
not use immediately, preserve the fragments at 4˚C

6)
Repeat
step 1-5 to amplify pTRIP. Replace EGFP template and corresponding primers to those of pTRIP's. Instead of
annealing at 55˚C, pTRIP anneals at 50˚C

7)

### B. **Homologous Recombination of pTRIP with EGFP**

Goal:
add target gene into vector so that the gene could be transformed into competent cells for expression in
the future

Materials
& Apparatus:

l
ClonExpress
Mix (2x)

l
double
distilled water (ddH2O)

l
PCR
products of pTRIP & EGFP

l
Nanodrop

l
water
bath

Procedure:

1.
test
the concentration on pTRIP and EGFP using Nanodrop

2.
prepare
the system (20μl)

|  |  |
| --- | --- |
| ClonExpress Mix(2x) | 10μl |
| vector (pTRIP) | 30fM |
| target gene (EGFP) | 60fM |
| ddH2O | fill to 20μl |

3.
place
the system in 50°C water bath for 10min

### C. **Transformation of pTRIP-EGFP into DH5α via heatshock**

##### Goal

##### By transforming the plasmids into the competent cells, quick replication of the plasmid copies are enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we are able to utilize the resistance gene initially designed on the plasmid to filter out the competent cells which don't have the needed transformed plasmids.

Transformation
is conducted via the heatshock method.

##### Materials

The
materials used are similar to the ones in the former transformation process

l
LB
liquid culture medium (K-)
(1ml)

l
LB
solid culture medium with kanamycin (K+)

l
A
box of ice

l
DH5α
(50μl)

l
pTRIP-EGFP
(10μl)

Equipments

l
Water
bath machine

l
EP
tubes

l
Shaking
incubator

l
Mini-centrifuge

l
Clean
bench

##### Procedures

1.
Take
out *E.
coli*
DH5α
competent
cells from -80 ℃ refrigerator;

2.
Add
10μl
of pTRIP-EGFP and 50μl of DH5α
cells
into an EP tube,
and
10μl
of pTRIP-ccdB
and
50μl of DB3.1
cells
into another
DH5α
tube.
Process
repeated for 2 times in total so 2 samples of each are acquired.

3.
Mix
the EP tubes with the mini-centrifuge for 5 seconds

4.
Put
the EP tubes on ice for 30 minutes

5.
Heat
shock the samples at 42'C for 60 seconds in the waterbath

6.
Put
the EP tubes in ice for 2 minutes

7.
Transfer
the bacteria samples from the EP tubes into K-
liquid
LB
medium. This step is performed in the clean bench.

8.
Shake
the cultures for 1 hour at 37°C, at 220rpm

9.
Spread
coat 50μl
on
the petri dishes with K+
LB
solid culture medium, respectively. This step is also performed in the clean bench.

10.
Inverted
culture at 37 ℃ and/or 22℃ for 12-16 hours. This process will be explained in latter sections

### D. **Plating and culturing of DH5α-pTRIP-EGFP**

Goal:
Bacterial culture serves various roles in scientific research. In our project, our team performed sample
plating to grow modified bacteria that are later going to be extracted to obtain the desired plasmid. In
this case, we will culture DH5α-pTRIP-EGFP.

Materials

1)
Disposable
latex gloves

2)
Pipette

3)
Pipette
tubes

4)
1L
Duran bottle

5)
Alcohol

6)
LB
medium

7)
Black
permanent marker

8)
Sterile
bacteria swabs

9)
Sterile
bacteria agar plates

10)
Sterile
collection tubes

11)
Vertical
clean flow bench

12)
Bacteriological
Incubator

Procedures

1)
Label
all the materials used correctly (empty plates, tube)

2)
Activate
the UV light in vertical clean flow bench to sterlize the environment inside

3)
All
the materials, including the gloves used, going into vertical clean flow bench sterlized through
alcohol

4)
Set
up a bunsen burner--> creating sterilzed area created by the updraft of flame--> place to the right
side of bench--> everytime using something, place the equipments near the fire

5)
Use
pipette to pour 10ml of LB medium to plates, wait until it becomes solid

6)
The
0.l ml of bacteria sample (DH5α-pTRIP-EGFP) pipetted onto the surface of cooled agar plate.

7)
Use
sterilized bacteria swabs or spreader to spread the sample evenly over the surface of agar plate

8)
Use
bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria

E.
**Bacterium
picking ( monoclonal ) and
shaking**

Goal:
To identify if the bacteria is correct and amplify the amount of the valid strain.

l
Samples
collected:

EGFP
1, 2, 3, 4

l
Performed
under a Vertical
flow
clean
bench.

l
Protocol:

1.
Add
5 ml of kanamycin-included LB medium into a 15ml incubation tube.

2.
Pick
colonies using small pipette tips, and eject the tips into the incubation tubes contianing LB medium.

3.
Incubate,
shaking, at 37℃
for
12-16 hours.

### F. **Plasmid Extraction**

Goal:
preparing the sample so that it can go through electrophorosis to see whether it's correct

Materials
& Apparatus:

l
bacterial
fluid of DH5α-pTRIP-EGFP

l
centrifuge

l
various
1.5mL ep tubes and absorption columns

l
Sangon
plasmid extraction kit

l
pipette
& pipette tubes

Procedures:

1.
Take
all bacterial fluid into centrifuge for 2 minutes at 8000g and abandon all supernatant

2.
Add
250ul Buffer P1 to the bacteria and let it resuspend

3.
Add
250 ul Buffer P2
and
gently turn the centrifuge
tube upside
down for
5-10 times to
let it mix.
Leave the centrifuge tube at room temperature for 2-4min.

4.
Add
350 ul Buffer P3, immediately and gently invert the centrifuge tube for 5-10 times to fully mix.

5.
Centrifuge
the tube at 12,000xg
for 5-10 min, and carefully move all the supernatant into the adsorption column and centrifuge at
8,000xg
for 30 sec. Drain the liquid in the collection tube.

6.
Add
500ul Wash Solution into the adsorption column, centrifuge at 9,000xg for 30 sec. Drain the liquid in the
collection tube.

7.
repeat
step 6 once

8.
Put
empty adsorption column and the collection tube together and centrifuge them at 9,000xg for 1 min.

9.
Add
50-100ul Elution Buffer to the center of the adsorption membrane. Leave
the
tube at room temperature for 1-2min. Centrifuge it at 9,000xg for 1min.

G.
**Verfication of
Extracted
Plasmids**

Goal:
To produce a functional and well-shaped gel for electrophoresis

Materials:

1)
Casting
Tray

2)
Well
comb

3)
Microwave

4)
TAE

5)
Agarose

6)
Nucleic
acid gel stain

7)
Erlenmeyer
flask

Procedure:

1.
Dissolve
0.3g Agarose in 30mL Erlenmeyer flask

2.
Add
TAE (Running buffer)

3.
Heat
the material with microwave

4.
Melt
the mixture at 30 sec interval

5.
See
whether bubbles are forming, if did succesfully mixed

6.
Add
3μl
nucleic acid gel stain (10000x)

7.
Let
it cool for 5~10 minutes

8.
Pour
the agarose mixture into the casting tray

9.
Place
the appropriate well comb to create the wells

10.
Wait
to solidify 7~10 minutes

H.
**Agarose gel
electrophoresis**

Goal:
To verify the DNA bands' lengths of the plasmids extracted from the competent cells, and thus
preparing for the next step of gel extraction to extract the bands that are of the correct length
(corresponding with the length of pTRIP-EGFP).

Materials:

1)
Sample

|  |  |
| --- | --- |
| Sample | Amount (µl) |
| pTRIP-EGFP (Plasmid extraction result) | 300+ |

2)
Marker
(DNA ladder)

3)
Prepared
gel (from previous step)

4)
10x
Loading buffer (dye)

5)
Horizontal
gel electrophoresis system

Gel
electrophoresis

1)
extract
50µl of every sample; add 5µl of loading buffer (10x) to each.

2)
Place
the gel in the horizontal electrophoresis system

3)
Extract
50µl of the mixed solution containing the sample, mix them till their colors are almost
uniform

4)
Add
the marker to the first well and load 50µl of each sample into the wells

5)
The
procedure runs at 160V for 30min

6)
Afterwards,
take out the gel from the device and proceed to following steps

###

### I. **DNA Gel Extraction**

Goal:
To recover DNA fragments in agarose gel.

Materials

l
agarose
gel with pTRIP-EGFP

l
clean
scalpel

l
1.5
mL Eppendorf (EP) tube

l
pipettes
and sterilized pipette heads

l
hot
water bath

l
buffer
B2

l
purification
column and collection tube

l
centrifuge

l
wash
solution containing pure ethanol

l
elution
buffer

Procedure

1)
Cut
the slice of gel containing pTRIP-EGFP, cutting off as much unneeded gel as possible, and place it in an
1.5 mL Eppendorf (EP) tube.

2)
Add
500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely
melted.

3)
Transfer
the solution containing melted gel into a purification column that's in a collection tube and perform
centrifugation at 8000 g for 30 seconds.

4)
Empty
the collection tube, put the purification column back in, and add 500 microliters of wash solution
containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.

5)
Repeat
above.

6)
Perform
centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one
minute to allow the ethanol to evaporate.

7)
Transfer
purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the
purification column.

8)
Cap
the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9)
Discard
the purification column. Store DNA in the EP tube at 4°C.

## **2.3.****Construction of pTRIP-ccdB**

### **A.****PCR of linear pTRIP vector** **and** **ccdB**

Goal:
To amplify pTRIP and ccdB fragments for recombination

Materials:

1)
2xPrimeMixStar\*

2)
pTRIP
and ccdB gene template

3)
double
distilled water (ddH2O)

4)
Primer-R

5)
Primer-F

6)
centrifuge
tube

7)
PCR
thermal cycler

\*
PrimeMixStar includes buffer and dNTPs for PCR. The extention rate for PrimestarMix (2x) is 2000bp/min;
the time varies with different templates

Procedure:

1)
Add
2xPrimerMixStar 25µl, ccdB template 1µl, primer-R 1µl, and primer-F 1µl to
centrifuge tube. Add ddH2O to the centrifuge tube until reaching 50µl. If bubbles are observed in
the mixture, centrifuge the mixture for a few seconds to remove the bubbles

2)
Insert
mixture into a PCR thermal cycler. Denature the fragments at 95˚C for 3 minutes

3)
Perform
30 cycles of 30 seconds 95˚C denaturing, 30 seconds 55˚C annealing, and 2 minutes extension 72˚C
extension.

4)
Extend
the fragments for 5 minutes at 72˚C

5)
If
not use immediately, preserve the fragments at 4˚C

6)
Repeat
step 1-5 to amplify pTRIP. Replace ccdB template and corresponding primers to those of pTRIP's. Instead of
annealing at 55˚C, pTRIP anneals at 50˚C

### B. **Homologous Recombination of pTRIP with ccdB**

Goal:
add target gene into vector so that the gene could be transformed into competent cells for expression in
the future

Materials
& Apparatus:

l
ClonExpress
Mix (2x)

l
double
distilled water (ddH2O)

l
PCR
products of pTRIP & ccdB

l
Nanodrop

l
water
bath

Procedure:

1.
test
the concentration on pTRIP and ccdB using Nanodrop

2.
prepare
the system (20μl)

|  |  |
| --- | --- |
| ClonExpress Mix (2x) | 10μl |
| vector (pTRIP) | 30fM |
| target gene (ccdB) | 60fM |
| ddH2O | fill to 20μl |

3.
place
the system in 50°C water bath for 10min

### C. **Transformation of pTRIP-ccdB into DB3.1 via heatshock**

##### Goal

##### By transforming the plasmids into the competent cells, quick replication of the plasmid copies are enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we are able to utilize the resistance gene initially designed on the plasmid to filter out the competent cells which don't have the needed transformed plasmids. Since DH5α cannot stand the toxicity the ccdB gene induces, DB3.1, an alternative competent cell that is able to withstand the toxicity, is used. Transformation is conducted via the heatshock method, again.

##### Materials

The
materials used are similar to the ones in the former transformation process

l
LB
liquid culture medium (K-)
(1ml)

l
LB
solid culture medium with kanamycin (K+)

l
A
box of ice

l
DB3.1
(50μl)

l
pTRIP-EGFP
(10μl)

Equipments

l
Water
bath machine

l
EP
tubes

l
Shaking
incubator

l
Mini-centrifuge

l
Clean
bench

##### Procedures

1.
Take
out *E.
coli*
DB3.1
competent
cells from -80 ℃ refrigerator;

2.
Add
10μl
of pTRIP-EGFP and 50μl of DB3.1 cells into an EP tube,
and
10μl
of pTRIP-ccdB
and
50μl of DB3.1
cells
into another
DB3.1
tube.
Process
repeated for 2 times in total so 2 samples of each are acquired.

3.
Mix
the EP tubes with the mini-centrifuge for 5 seconds

4.
Put
the EP tubes on ice for 30 minutes

5.
Heat
shock the samples at 42'C for 60 seconds in the waterbath

6.
Put
the EP tubes in ice for 2 minutes

7.
Transfer
the bacteria samples from the EP tubes into K-
liquid
LB
medium. This step is performed in the clean bench.

8.
Shake
the cultures for 1 hour at 37°C, at 220rpm

9.
Spread
coat 50μl
on
the petri dishes with K+
LB
solid culture medium, respectively. This step is also performed in the clean bench.

10.
Inverted
culture at 37 ℃ and/or 22℃ for 12-16 hours. This process will be explained in latter sections

### D. **Plating and culturing of DB3.1-pTRIP-ccdB**

Goal:
Goal: Bacterial culture serves various roles in scientific research. In our project, our team performed
sample plating to grow modified bacteria that are later going to be extracted to obtain the desired
plasmid. In this case, we will culture DB3.1-pTRIP-ccdB.

Materials

1)
Disposable
latex gloves

2)
Pipette

3)
Pipette
tubes

4)
1L
Duran bottle

5)
Alcohol

6)
LB
medium

7)
Black
permanent marker

8)
Sterile
bacteria swabs

9)
Sterile
bacteria agar plates

10)
Sterile
collection tubes

11)
Bunsen
burner

12)
Vertical
clean flow bench

13)
Bacteriological
Incubator

Procedures

1)
Label
all the materials used correctly (empty plates, tube)

2)
Activate
the UV light in vertical clean flow bench to sterlize the environment inside

3)
All
the materials, including the gloves used, going into vertical clean flow bench sterlized through
alcohol

4)
Set
up a bunsen burner--> creating sterilzed area created by the updraft of flame--> place to the right
side of bench--> everytime using something, place the equipments near the fire

5)
Use
pipette to pour 10ml of LB medium to plates, wait until it becomes solid

6)
The
0.l ml of bacteria sample (DB3.1-pTRIP-ccdB) pipetted onto the surface of cooled agar plate.

7)
Use
sterilized bacteria swabs or spreader to spread the sample evenly over the surface of agar plate

8)
Use
bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria

E.
**Bacterium
picking and shaking**

Goal:
To identify if the bacteria is correct and amplify the amount of the valid strain.

Samples
collected:

ccdB:
1,2,3,4

Performed
under a Vertical
flow
clean
bench.

Protocol:

1.
Add
5 ml of kanamycin-included LB medium into a 15ml incubation tube.

2.
Pick
colonies using small pipette tips, and eject the tips into the incubation tubes contianing LB medium.

3.
Incubate,
shaking, at 37℃
for
12-16 hours.

### F. **Plasmid Extraction**

Goal:
preparing the sample so that it can go through electrophorosis to see whether it's correct

Materials
& Apparatus:

l
bacterial
fluid of DB3.1-pTRIP-ccdB

l
centrifuge

l
various
1.5mL ep tubes and absorption columns

l
Sangon
plasmid extraction kit

l
pipette
& pipette tubes

Procedures:

1.
Take
all bacterial fluid into centrifuge for 2 minutes at 8000g and abandon all supernatant

2.
Add
250ul Buffer P1 to the bacteria and let it resuspend

3.
Add
250 ul Buffer P2
and
gently turn the centrifuge
tube upside
down for
5-10 times to
let it mix.
Leave the centrifuge tube at room temperature for 2-4min.

4.
Add
350 ul Buffer P3, immediately and gently invert the centrifuge tube for 5-10 times to fully mix.

5.
Centrifuge
the tube at 12,000xg
for 5-10 min, and carefully move all the supernatant into the adsorption column and centrifuge at
8,000xg
for 30 sec. Drain the liquid in the collection tube.

6.
Add
500ul Wash Solution into the adsorption column, centrifuge at 9,000xg for 30 sec. Drain the liquid in the
collection tube.

7.
repeat
step 6 once

8.
Put
empty adsorption column and the collection tube together and centrifuge them at 9,000xg for 1 min.

9.
Add
50-100ul Elution Buffer to the center of the adsorption membrane. Leave
the
tube at room temperature for 1-2min. Centrifuge it at 9,000xg for 1min.

### G. **Verfication of Extracted Plasmids**

Goal:
To produce a functional and well-shaped gel for electrophoresis

Materials:

1)
Casting
Tray

2)
Well
comb

3)
Microwave

4)
TAE

5)
Agarose

6)
Nucleic
acid gel stain

7)
Erlenmeyer
flask

Procedure:

1.
Dissolve
0.3g Agarose in 30mL Erlenmeyer flask

2.
Add
TAE (Running buffer)

3.
Heat
the material with microwave

4.
Melt
the mixture at 30 sec interval

5.
See
whether bubbles are forming, if did succesfully mixed

6.
Add
3μl
nucleic acid gel stain (10000x)

7.
Let
it cool for 5~10 minutes

8.
Pour
the agarose mixture into the casting tray

9.
Place
the appropriate well comb to create the wells

10.
Wait
to solidify 7~10 minutes

11.
Remove
the comb and place the gel in the gel box

#### H. **A****garose gel electrophoresis**

#### Goal: To verify the DNA bands' lengths of the plasmids extracted from the competent cells, and thus preparing for the next step of gel extraction to extract the bands that are of the correct length (corresponding with the length of pTRIP-ccdB).

Materials:

1)
Sample

|  |  |
| --- | --- |
| Sample | Amount (µl) |
| pTRIP-ccdB (Plasmid extraction result) | 400+ |

2)
Marker
(DNA ladder)

3)
Prepared
gel (from previous step)

4)
10x
Loading buffer (dye)

5)
Horizontal
gel electrophoresis system

Gel
electrophoresis

1)
extract
50µl of every sample; add 5µl of loading buffer (10x) to each.

2)
Place
the gel in the horizontal electrophoresis system

3)
Extract
50µl of the mixed solution containing the sample, mix them till their colors are almost
uniform

4)
Add
the marker to the first well and load 50µl of each sample into the wells

5)
The
procedure runs at 160V for 30min

6)
Afterwards,
take out the gel from the device and proceed to following steps

#### I. **Snap the gel to identify the length of the PCR fragment**

Goal:
After running electrophoresis, we need to visualize the position of the DNA bands on the gel. To do so, we
used a gel transilluminator to visualize the bands and to collect the image of the gel.

Materials:

l
Agarose
Gel containing pTRIP-ccdB samples

l
Gel
transilluminator

Procedure:

1.
Place
the Gel inside the transilluminator

2.
On
a computer with a software connected to the transilluminator, visualize the results

3.
On
the software, take the image of the gel under UV light and save the image

4.
Take
the gel out and dispose it with regards to safety requirements.

5.

### J. **DNA Gel Extraction**

Goal:
To recover DNA fragments in agarose gel.

Materials

l
agarose
gel with pTRIP-ccdB

l
clean
scalpel

l
1.5
mL Eppendorf (EP) tube

l
pipettes
and sterilized pipette heads

l
hot
water bath

l
buffer
B2

l
purification
column and collection tube

l
centrifuge

l
wash
solution containing pure ethanol

l
elution
buffer

Procedure

1）
Cut
the slice of gel containing pTRIP-ccdB, cutting off as much unneeded gel as possible, and place it in an
1.5 mL Eppendorf (EP） tube.

2）
Add
500 microliters of buffer B2 and put the tube into a 50°C hot water bath until gel has completely
melted.

3）
Transfer
the solution containing melted gel into a purification column that's in a collection tube and perform
centrifugation at 8000 g for 30 seconds.

4）
Empty
the collection tube, put the purification column back in, and add 500 microliters of wash solution
containing pure ethanol. Perform centrifugation at 9000 g for 30 seconds, and empty collection tube again.

5）
Repeat
above.

6）
Perform
centrifugation one more time at 9000 g for 1 minute, then open the cap of the tube and let it sit for one
minute to allow the ethanol to evaporate.

7）
Transfer
purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the
purification column.

8）
Cap
the lid and let the tube sit for 1 minute. Perform centrifugation at 9000 g for 1 minute.

9)
Discard
the purification column. Store DNA in the EP tube at 4°C.

##

### 3. **Induction of protein expression**

**3.1** **pTRIP-EGFP**

A. Amplification culture

Goal:
to acquire large amounts of target proteins for SDS-PAGE

Materials
& Apparatus:

l
liquid
LB

l
pTRIP-EGFP
bacterial fluid

l
kana

l
inducer:
N-(3-oxohexanoyl)-L-homoserine
lactone

l
isothermic
shaker

l
microplate
reader

Procedure:

1.
Add
100μl of pTRIP-EGFP bacterial fluid and 100μl of kana into 100mL of liquid LB

2.
place
the sample in the isothermic shaker 0.5h for culturing

3.
Add
inducers to the sample with concentrations of 0.2mmol, 0.4mmol, 0.6mmol, 0.8mmol, and 1mmol
respectively

4.
Samples
for each concentration are cultured at 22°C and 37°C for 2.5h, 5h, 10h, 20h, and 30h
respectively

5.
Test
the concentration of all samples under the microplate reader

###

### B. **Protein Extraction and SDS-PAGE**

Goal:
to verify the expression of desired proteins

Protein
Extraction:

Materials

l
bacteria
culture containing desired protein

l
50
mL centrifuge tubes

l
beaker
containing ice

l
centrifuge

l
sonicator

Procedure

1)
add
samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.

2)
partially
bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.

3)
sonicate
for 15 minutes.

4)
repeat
steps 2 and 3 for all samples.

5)
perform
centrifugation of samples at 4000 rpm for 15 minutes.

6)
remove
the cell fragments that have precipitated at the bottom of the tubes.

7)
the
remaining liquid will contain desired proteins.

C.
**SDS-PAGE:**

Materials：

l
Sangon
12.5%
SDS-PAGE Color Preparation kit

l
pipette

l
distilled
water

l
electrophoresis
buffer

l
protein
ladder

l
vertical
electrophoresis system

l
Coomassie
blue

Procedure：

1）
using
a pipette, add and mix 2.2 mL of 2X separating gel solution, 2.2 mL of 2X separating gel buffer, and 44
μL of the catalyst in a plastic cup.

2）
slowly
inject mixture into casting stand and frame to avoid bubbles.

3）
add
1 mL water to flatten out the top.

4）
wait
8 minutes for the gel to set.

5）
discard
the water that was added previously.

6）
using
a pipette, add and mix 825 μL of 2X stacking gel solution, 825 μL of 2X stacking gel buffer, and 11
μL of catalyst in a plastic cup.

7）
add
stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any
air bubbles.

8）
wait
12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.

9）
add
the protein ladder into the first well, then load samples in each successive well.

10）
transfer
gel into vertical electrophoresis system.

11）
set
the voltage to 120V and run it for 90 minutes.

12）
stain
the gel with Coomassie blue by submerging it for 30 minutes.

13）
EGFP
is a protein that is 25 kDa; compare the protein strips to the ladder to see how well the protein has
expressed.

**3.2** **pTRIP-****ccdB**

**A.
A****mplification culture**

Goal:
to acquire large amounts of target proteins for SDS-PAGE

Materials
& Apparatus:

l
liquid
LB

l
pTRIP-ccdB
bacterial fluid

l
kana

l
inducer:
N-(3-oxohexanoyl)-L-homoserine
lactone

l
isothermic
shaker

l
microplate
reader

Procedure:

1.
Add
100μl of pTRIP-ccdB bacterial fluid and 100μl of kana into 100mL of liquid LB

2.
place
the sample in the isothermic shaker 0.5h for culturing

3.
Add
inducers to the sample with concentrations of 0.2mmol, 0.4mmol, 0.6mmol, 0.8mmol, and 1mmol
respectively

4.
Samples
for each concentration are cultured at 22°C and 37°C for 2.5h, 5h, 10h, 20h, and 30h
respectively

5.
Test
the concentration for all samples of bacterial fluid under the microplate reader

### **B.****Protein Extraction and SDS-PAGE**

Goal:
to verify the expression of desired proteins

Materials

l
bacteria
culture containing desired protein

l
50
mL centrifuge tubes

l
beaker
containing ice

l
centrifuge

l
sonicator

Procedure

1)
add
samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.

2)
partially
bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.

3)
sonicate
for 15 minutes.

4)
repeat
steps 2 and 3 for all samples.

5)
perform
centrifugation of samples at 4000 rpm for 15 minutes.

6)
remove
the cell fragments that have precipitated at the bottom of the tubes.

7)
the
remaining liquid will contain desired proteins.

**C.****SDS-PAGE:**

Materials

l
Sangon
12.5%
SDS-PAGE Color Preparation kit

¡
SDS-page
gel casting frame, stand, and comb

¡
2X
stacking gel solution

¡
2X
stacking gel buffer

¡
2X
separating gel solution

¡
2X
separating gel buffer

¡
catalyst
for gel polymerization

¡
two
single-use small plastic cups

l
pipette

l
distilled
water

l
electrophoresis
buffer

l
protein
ladder

l
vertical
electrophoresis system

l
Coomassie
blue

Procedure

1)
using
a pipette, add and mix 2.2 mL of 2X separating gel solution, 2.2 mL of 2X separating gel buffer, and 44
μL of the catalyst in a plastic cup.

2)
slowly
inject mixture into casting stand and frame to avoid bubbles.

3)
add
1 mL water to flatten out the top.

4)
wait
8 minutes for the gel to set.

5)
discard
the water that was added previously.

6)
using
a pipette, add and mix 825 μL of 2X stacking gel solution, 825 μL of 2X stacking gel buffer, and 11
μL of catalyst in a plastic cup.

7)
add
stacking gel mixture until the cast is completely filled, then slowly insert comb without producing any
air bubbles.

8)
wait
12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.

9)
add
the protein ladder into the first well, then load samples in each successive well.

10)
transfer
gel into vertical electrophoresis system.

11)
set
the voltage to 120V and run it for 90 minutes.

12)
stain
the gel with Coomassie blue by submerging it for 30 minutes.

13)
ccdB
is a protein that is 15 kDa; compare the protein strips to the ladder to see how well the protein has
expressed.

4.
**Functional
testing**

**4.1**
**pTRIP-EGFP**

A.
**GFP
determination**

Procedure

1.
In
100ml LB medium, 100μL pTRIP-EGFP and
pRIP
( DH5α ) bacterial solution was added, and 100μL kan was added. When the concentration was
0.5-0.8, 0.6mmol inducer ( N- ( 3-oxohexanoyl ) -L-homoserine lactone ( AI ) ) was added, and the
temperature was 16 degrees, 18 degrees, 22 degrees, 26 degrees, 34 degrees, 37 degrees ; after 10 hours of
culture.

2.
100ul
protein solution was added dropwise to the microplate, and the fluorescence intensity was measured at the
excitation wavelength of 488 nm and the fluorescence detection wavelength of 525 nm.

B.
**Microscopic
observation of fluorescent reporter
gene**

Procedure

1.Operation steps

2. Fluorescence observation

3. Open the bright field power switch

4. Turn on the mercury lamp switch

5. Put the sample on the stage

6. Open the fluorescent light path shutter, and close the shutter
when the sample needs to be protected.

7. Adjust the optical path selection knob to the observation
position

8. According to the labeling of the sample, the fluorescent filter
block is rotated to the corresponding position.1.Blue light ; 2. Green light ; 3.UV ;

9. the excitation light intensity is adjusted by two groups of
light-reducing filters.

10. Starting from the low power lens observation, focusing, find
the pre observation field of vision.

11.In turn, change to a high-power lens to observe the sample

12.When taking pictures, the optical path selection knob is
adjusted to the camera position

**4.2**
**pTRIP-****ccdB**

### **A.****Transformation of pTRIP-ccdB into*****E.coil*****DH5α****、****BL21**

##### Goal

##### In order to detect the effect of temperature control system in *E.coil* DH5α and BL21.

##### Materials

The
materials used are similar to the ones in the former transformation process

l
LB
liquid culture medium (K-)
(1ml)

l
LB
solid culture medium with kanamycin (K+)

l
A
box of ice

l
*E.coil*
DH5α、BL21
(50μl)

l
pTRIP-ccdB(10μl)

Equipments

l
Water
bath machine

l
EP
tubes

l
Shaking
incubator

l
Mini-centrifuge

l
Clean
bench

##### Procedures

##### 1.Take out *E. coli* DB3.1 competent cells from -80 ℃ refrigerator;

2.Add
10μl
of pTRIP-ccdB
and
50μl of *E.coil*
DH5α、BL21
.
Process
repeated for 2 times in total so 2 samples of each are acquired.

3.Mix
the EP tubes with the mini-centrifuge for 5 seconds

4.Put
the EP tubes on ice for 30 minutes

5.Heat
shock the samples at 42'C for 60 seconds in the waterbath

6.Put
the EP tubes in ice for 2 minutes

7.Transfer
the bacteria samples from the EP tubes into K-
liquid
LB
medium. This step is performed in the clean bench.

8.Shake
the cultures for 1 hour at 37°C, at 220rpm

9.Spread
coat 50μl
on
the petri dishes with K+
LB
solid culture medium, respectively. This step is also performed in the clean bench.

10.Inverted
culture at 37 ℃ and/or 22℃ for 12-16 hours. This process will be explained in latter sections

**B.Monoclonal verification**

Samples
collected:

ccdB:
1,
2,
3,
4, 5, 6, 7,8

Performed
under a Vertical
flow
clean
bench.

Protocol:

4.
Add
5 ml of kanamycin-included LB medium into a 15ml incubation tube.

5.
Pick
colonies using small pipette tips, and eject the tips into the incubation tubes contianing LB medium.

6.
Incubate,
shaking, at 37℃
for
12-16 hours.

Procedure I:

|  |  |
| --- | --- |
| pTRIP-ccdB | Amount |
| 2mix | 10μl |
| ccdB-R1 | 1μl |
| ccdB-F1 | 1μl |
| pTRIP-ccdB | 1μl |
| ddwater | 7μl |
| total | 20μl |

Procedure II:

|  |  |  |
| --- | --- | --- |
| PCR program | Temp. (celsius degrees) | Period |
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Elongation | 72 | 30sec. |

C.
**Agarose gel
electrophoresis**

The
steps for agarose gel electrophoresis are as follows:

1.
Prepare the agarose gel: Dissolve the agarose powder in a suitable buffer solution according to the
recommended concentration. Most agarose gels need to be prepared in a buffer solution.

2.
Load the samples: Mix the biological macromolecule mixture (e.g., DNA samples) with a loading buffer and
carefully pipette it into the wells of the prepared agarose gel.

3.
Electrophoresis: Submerge the agarose gel in an electrophoresis tank filled with an appropriate
electrophoresis buffer, ensuring that the samples are fully immersed. Then, connect the power supply and
run the electrophoresis at the desired voltage and time.

4.
Visualization and recording of results: Choose the appropriate staining method, such as DNA or protein
stains, based on the type of biological macromolecules being separated. Stain the gel by immersing it in a
staining solution. Finally, observe and record the migration of the biological macromolecules on the gel
using a transilluminator or fluorescence imaging system.

D.
**The growth ability
of pTRIP-cdBZ in** ***E.coil*** **DH5α and
BL21.**

##### Materials

l
pTRIP-ccdB
*E.coil*
DH5α、BL21
(1000μl)

##### Procedures

1.In
100 ml LB medium, add 100μL pTRIP-ccdB bacteria liquid.

2.Add
100μLkan.

3.Culture
0D equal to 0.5 hours.

4.Adding
different concentrations of inducers ( N- ( 3-oxohexanoyl ) -L-homoserine lactone ( AI ) ), the
concentrations were 0.2mmol, 0.4mmol, 0.6mmol, 0.8mmol, 1mmol ;

5.Cultured
at 22 degrees and 37 degrees for 2.5h, 5h, 10h, 20h, 30h ; the concentration of bacterial solution was
measured by microplate reader at OD600.

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![](https://static.igem.wiki/teams/4942/wiki/results.png)

Overview

Some
genetically modified microorganisms used in the production of engineered probiotics or industrial
fermentation strains require special precautions for biosafety1.
It is important to prevent the unintentional release, multiplication, and spread of these genetically
modified microorganisms into the environment, which could lead to unpredictable biological
contamination2.
This project has designed a simple and user-friendly "safety lock" for engineered microorganisms. Under
normal conditions at 37℃ (the temperature inside the human body, which is also the working temperature for
probiotics and commonly used in industrial microbial fermentation), the "safety lock" remains inactive,
allowing the host microorganism to reproduce and function normally. However, at 22℃ (a temperature closer
to natural environmental conditions, excluding tropical regions and extremely hot summers), the "safety
lock" becomes active, expressing a toxic protein that leads to the self-destruction of the host
microorganism, thereby preventing the release of the engineered microorganisms3-5. We constructed a temperature control system（pTRIP）, and introduced a visual
reporting gene(EGFP), into the system to monitor its functionality and optimize its sensitivity. Lastly,
we developed a plasmid, pTRIP-ccdB, which codes a toxic protein, (ccdB), that can kill bacteria upon
expression, thereby preventing the leakage of engineered microorganisms(Figure 1).

.
![](https://static.igem.wiki/teams/4942/wiki/results/1.png)

Figure
1. The engineering design schematic diagram.

**Results of****O****ur** **W****ork**

**1.****The construction of plasmid**

1.1 pTRIP (pTrc99k-RIP)

We
constructed pTRIP using homologous recombination. The RIP sequence was amplified by PCR, with a length of
2065 bp. The Figure 2A indicates
the band
consistent
with the results. The pTrc99k plasmid was used as a template for PCR amplification, resulting in a
fragment of 3408bp for
pTrc. The Figure 2B shows
a band consistent with the target size. It indicating
successful amplification of RIP and linearization of the pTrc plasmid. After
DpnI
digestion and gel recovery, the pRIP fragment was obtained.

![](https://static.igem.wiki/teams/4942/wiki/results/2.png)

Figure 2. The gel
electrophoresis validation of RIP and pTrc nucleic acids.

The plasmids were
transformed into *E.Coil*DH5α .
Figure3 A shows the presence of single colonies on
the plate. We selected colonies 1-10 and sent them directly for sequencing. According to the results shown
in Figure 3B and
C, the
RIP was successfully ligated
to the pTrc vector without any apparent mutations, confirming the successful construction of the pTRIP
plasmid.

![](https://static.igem.wiki/teams/4942/wiki/results/3.png)

Figure 3. The Monoclonal
antibody validation and sequencing of pTRIP (DH5α) .

Note：

A. Transformation
plate of pTRIP:

B.  Sequencing
results of pTRIP

C.  Base
comparison of a specific region within pTRIP

1.2 pTRIP-EGFP

We constructed the pTRIP-EGFP plasmid using homologous
recombination. The EGFP sequence, which is 720bp in length, was amplified by PCR. The Figure 4 A showed
that matched the expected size, indicating successful amplification of the EGFP sequence. Furthermore,
using the pTRIP plasmid as a template, we performed PCR amplification and obtained 5473bp fragment of
pTRIP-E. The Figure 4 B showed bands that matched the expected size, indicating successful amplification
of the linearized pTRIP-E plasmid. We can conclude that we successfully amplified the EGFP sequence and
linearized pTRIP-E plasmid.

![](https://static.igem.wiki/teams/4942/wiki/results/4.png)

Figure 4. The gel
electrophoresis validation of EGFP and pTRIP-E .

We transformed the plasmid pTRIP-EGFP into DH5α, and Figure 5 A and
B showed the growth of single clones. We selected clones 1-6 and performed antibody verification.The
expected size of 720bp for the EGFP sequence. Additionally, Figure 5 C showed bands that matched the
expected size, indicating successful transformation. Subsequently, we sent clones 1-6 for sequencing.
The sequencing results in Figure 5 D showed a 100% match with the nucleotide sequence of EGFP,
confirming the successful integration of the EGFP fragment into the pTRIP-E plasmid and further
validating the successful construction of the plasmid.

![](https://static.igem.wiki/teams/4942/wiki/results/5.png)

Figure 5. The Monoclonal
antibody validation and sequencing of pTRIP-EGFP
(*E.Coil* DH5α) .

Note:

A.B shows the plate
images of pTRIP-EGFP transformed into *E.Coil* DH5α cells.

C is the image showing
the verification of single clone colonies.

D is the sequencing
image of pTRIP-EGFP plasmid.

1.3 pTRIP-ccdB

We constructed the pTRIP-ccdB plasmid using homologous
recombination. The PCR amplification of the ccdB sequence resulted in a fragment of 306bp in length. The
figure 6 indicates that the amplified band matches the expected size, confirming the successful
amplification of the ccdB sequence from the linearized plasmid.

![](https://static.igem.wiki/teams/4942/wiki/results/6.png)

Figure 6. The gel
electrophoresis validation of ccdB.

By using the pTRIP plasmid as a template, we performed PCR
amplification to obtain a 5473kb fragment referred to as pTRIP-C. The gel electrophoresis image in
Figure 7 shows that the amplified band matches the expected size, indicating the successful
amplification of the linearized pTRIP-C plasmid.

![](https://static.igem.wiki/teams/4942/wiki/results/7.png)

Figure 7. The gel
electrophoresis validation of pTRIP-C.

The pTRIP-ccdB plasmid was transformed into
*E. coli* DB3.1. The single clone colony growth on plates is
shown in Figure 8 A and B. Clones 1-8 were selected for antibody verification, and the results in Figure 8
C demonstrate clear bands, confirming the presence of the ccdB sequence with a length of 306bp. The gel
electrophoresis image in Figure 8C matches the expected band, indicating the successful
transformation.

Next, colonies 1-8 were sent for sequencing,
and the sequencing results in Figure 8 D showed a 100% match with the ccdB nucleotide sequence. This
confirms the successful integration of the ccdB fragment into the pTRIP-E plasmid. It further validates
the successful construction of the pTRIP-ccdB plasmid.

![](https://static.igem.wiki/teams/4942/wiki/results/8.png)

Figure 8. The monoclonal
antibody validation and sequencing of pTRIP-ccdB
(*E.Coil* DB3.1) .

**2. Protein
expression**

2.1 pTRIP-EGFP

The target protein
EGFP has a size of 26.9kDa. Protein expression was induced at a concentration of 0.6mmol AI
(AutoInducers, herein it refers to N-(3-oxohexanoyl)-L-homoserine lactone), and induction was performed
at 37 oC and 22
oC. According to the
figure 9, in the control group (line 1), no EGFP protein is present. Under the condition of 37
oC (line 1 to line
3), EGFP protein was not observed. However, under the condition of 22 oC (line 4 to line
5),  there is a clear presence of EGFP protein. This indicates that EGFP expression is not
express or occurring
at a lower level at 37 oC, while there is
substantial expression at 22 oC. Therefore, it can
be concluded that our temperature control system is activated state at 22oC and deactivated
state at 37 oC.

![](https://static.igem.wiki/teams/4942/wiki/results/9.png)

Figure 9.The SDS-PAGE
protein gel of EGFP at different temperatures

Note:

line
1: pTRIP(DH5α)

line
2：pTRIP-EGFP-37oC(DH5α)

line
3：pTRIP-EGFP-37oC(DH5α)

line
4：pTRIP-EGFP-22oC(DH5α)

line
5：pTRIP-EGFP-22oC(DH5α)

2.2 pTRIP-ccdB

The size of the ccdB protein, the target protein, is 11.7 kDa.
Protein expression was induced at a concentration of 0.6mmol AI, and induction was performed at 37
oC and 22 oC. According to the figure 10, in the control group (line 1 -line
2 and line 9 -line 10), ccdB protein was not observed. Under the condition of 37 oC (line 3 to line 4), ccdB protein was not
observed. However, under the condition of 22 oC (line 5 to line 8), a weak presence of ccdB protein is detected.
This indicates that ccdB expression does not occur at 37 oC, while there is limited expression at 22
oC. Therefore, it can be concluded that our temperature control
system is in the activated state at 22 oC and in the deactivated state at 37 oC.

![](https://static.igem.wiki/teams/4942/wiki/results/10.png)

Figure
10 : The SDS-PAGE of ccdB protein

Note:

1-2:
 *E.coil* DB3.1(control)

3-4：37oC-pTRIP-ccdB*(E.coil* DB3.1)

5-8：22oC-pTRIP-ccdB(*E.coil* DB3.1)

9-10: *E.coil* DB3.1(control
)

**3. Functional
Test**

3.1 pTRIP-EGFP

3.1.1 Detection of fluorescence intensity EGFP

Protein expression was induced by AI at a
concentration of 0.6 mmol and at different temperatures. According to Table 1 and Figure 11, the
fluorescence intensity of pTRIP-EGFP increased first and then decreased with the increase of temperature.
At 22 oC, the fluorescence intensity of pTRIP-EGFP was
the strongest. At 37 oC, the fluorescence intensity of pTRIP-EGFP was
the weakest. However, no fluorescence signal was detected in the control group pTRIP. This shows that the
temperature control system of our subject is controlled by temperature. At 22 oC, in the “open” state ; At 37 oC, it is close to the “closed”
state.

Table 1. The
fluorescence intensity of reporter gene EGFP at
different temperatures

|  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- |
| Temperature | pTRIP | pTRIP-EGFP | | | Average | SD |
| 16 | 0.00E+00 | 3.82E+03 | 4.52E+03 | 5.01E+03 | 4.45E+03 | 4.88E+02 |
| 19 | 0.00E+00 | 1.62E+04 | 1.83E+04 | 2.01E+04 | 1.82E+04 | 1.59E+03 |
| 22 | 0.00E+00 | 4.10E+04 | 3.80E+04 | 4.08E+04 | 3.99E+04 | 1.37E+03 |
| 26 | 0.00E+00 | 2.31E+04 | 1.81E+04 | 2.20E+04 | 2.11E+04 | 2.15E+03 |
| 32 | 0.00E+00 | 3.13E+03 | 4.32E+03 | 3.33E+03 | 3.59E+03 | 5.20E+02 |
| 37 | 0.00E+00 | 7.67E+02 | 9.13E+02 | 8.59E+02 | 8.46E+02 | 6.03E+01 |

![](https://static.igem.wiki/teams/4942/wiki/results/11.png)

Figure 11. The
fluorescence intensity of reporter gene EGFP at different temperatures

In order to observe
the fluorescence signal of pTRIP-EGFP more clearly, we made the glass slides of pTRIP-EGFP bacteria and
control group ( pTRIP ) cultured at 22 oC and 37
oC. According to the
Figure12, A (colonies under white light) and B (pTRIP-EGFP at 37
oC) have no
fluorescence signal, C and D (pTRIP-EGFP at 22
oC) have obvious
fluorescence signal. It shows that there is no fluorescence signal at 37 oC, and obvious
fluorescence signal can be seen at 22 oC.

![](https://static.igem.wiki/teams/4942/wiki/results/12.png)

Figure12.The
fluorescence signal of report gene EGFP under microscope

Note:

A : pTRIP-EGFP colonies under white light

B. Fluorescence reporter
gene of pTRIP-EGFP plasmid at 37 °C

C. 22 ° fluorescence
reporter gene of pTRIP-EGFP plasmid ( 10 × 40 )

D. 22 ° fluorescence
reporter gene of pTRIP-EGFP plasmid ( 10 × 100 )

**3.2** **pTRIP-ccdB**

3.2.1 The growth ability of pTRIP-ccdB (DH5α)

**A.****The** **pTRIP-ccdB****was****transfor****m****in****to DH5α**

In order to verify the sensitivity of our
temperature control system, we transferred the plasmid pTRIP-ccdB into DH5α. The length of ccdB is 306
bp, and Figure 13A proves that the colony growth is successful. The line
1-9 in Figure 13B are
consistent with the expected results, which proves that pTRIP-ccdB has been successfully transformed into
*DH5α*.

![](https://static.igem.wiki/teams/4942/wiki/results/13.png)

Figure 13. Verification of the pTRIP-ccdB
(E.coil) monoclonal antibody

**B.The pTRIP-ccdB(*****E.coil*****DH5α****) growth
ability**

A. The growth ability of
pTRIP-ccdB (*E.coil* DH5α)
at 22 °C

Firstly, it can be seen from these 5 graphs in Figure 14 that
basically AI
induction concentration at 0.2mmol to
1mmol has very
little effect on
the
growth trend of the E. coli in the control group and experimental group.

According to Figure 14
A to E, given
the temperature 22 °C,
the OD600 of E.coil in the control group increased first and
then gradually tended to be stabilized over
time. While
there was no significant
increase
in OD600 of pTRIP-ccdB
at the experimental group over
time. Therefore,
compared with the wild DH5α (the control
group), we can conclude that the ccdB did kill bacteria, the host
at 22 °C upon
expression.

![](https://static.igem.wiki/teams/4942/wiki/results/14.png)

Figure 14.The growth ability of pTRIP-ccdB (
E.coil DH5α ) in different AI concentrations at 22 °C

C. **The growth ability of
pTRIP-ccdB****(*****E.coil*****DH5α****)****at****37****° C**

According to Figure15A,
when the temperature was 37 °C and AI was 0.2 mmol, the OD600 of the control group DH5α and the
experimental group pTRIP-ccdB increased first and then gradually stabilized over time. The control group
was slightly higher than the experimental group after 20 minutes but following they went to a similar
level after another 10 minutes.

According to Figure 15B
and 15C, when the temperature was 37 °C and AI was 0.4 mmol and 0.6 mmol, respectively, these two
groups have very similar values and trend upon time duration that the OD600 increased first and then gradually stabilized
over time.

According to Figure15D,
when the temperature was 37 °C and AI was 0.8 mmol, the OD600 of the control group DH5α and the
experimental group pTRIP-ccdB increased first and then gradually stabilized over time, and the
experimental group was slightly lower than the control group.

According to Figure15E,
when the temperature was 37 °C and AI was 1 mmol, the OD600 of the control group DH5α and the
experimental group pTRIP-ccdB increased first and then gradually stabilized over time. The control group
was slightly higher than the experimental group after 10 minutes but following they went to a similar
level after another 20 minutes.

In summary, the pTRIP-ccdB E.coil DH5α has a
very similar growth trend
with the control group, the wild E.coil DH5α at 37 °C which means the “switch”
pTRIP-ccdB has little effect on the strain’s growth since no
expression of ccdB at 37
°C.

![](https://static.igem.wiki/teams/4942/wiki/results/15.png)

Figure 15.The growth ability of pTRIP-ccdB (
*E.coi*l DH5α ) in different AI concentrations at 37 ° C.

**Note: in*****C, two curves
coincide*****.**

D. **Compar****ison of
t****he
growth ability of pTRIP-ccdB****(*****E.coil*****DH5α****)****at****37****°C****and****22****°C**

Since
we can see that the control group has
the
same growth trend
at 22 °C and 37 °C, we decided to compare the growth trend of
pTRIP-ccdB (E.coil DH5α) at 22 °C and 37 °C, respectively, to
evaluate the engineering of ccdB.

According to Figure 16, in two groups
with
AI concentration of 0.6 mmol, the OD600 of pTRIP-ccdB at 37 °C increased firstly and then tended to
be stabilized
over time, while
there was almost no
significant change
of that
at 22 °C. It is seen that
pTRIP-ccdB (E.coil
DH5α) grew much better at 37 °C than
22 °C where it
indicated little growth over time. This further supports the conclusion that the
bacterial strain grows normally at 37°C, while the presence of ccdB at 22°C leads to bacterial
cell death.

![](https://static.igem.wiki/teams/4942/wiki/results/16.png)

Figure16.
Comparison of OD600
at
37°C and 22°C pTRIP-ccdB (*E.coi*l DH5α ) with
AI concentration of 0.6 at different times

**3.2.2** **The growth ability of pTRIP-ccdB
in*****E.coil*** **BL21 (DE3).**

**A. The pTRIP-ccdB was transform into*****E.coil*****BL21(DE3).**

In order to verify the sensitivity of our
temperature control system, we transferred the plasmid pTRIP-ccdB into *E.coil* BL21 (DE3). The length of ccdB is 306 bp, and Figure 17A
proves that the colony growth is successful. The line 1-5 in Figure 17 B are consistent with the expected
results, which proves that pTRIP-ccdB has been successfully transformed into *E.coil* BL21 (DE3).

![](https://static.igem.wiki/teams/4942/wiki/results/17.png)

Figure 17. Verification of the pTRIP-ccdB
(*E.coil* BL21 (DE3)) monoclonal antibody

**B.The****Growth ability of pTRIP-ccdB
in*****E.coil*****BL21 ( DE3
)**

Comparisons of growth capabilities were made
at 37°C and 22°C with an AI concentration of 0.6 mmol.The*E.coil* BL21 was the
control group. According to
Figure 18, it is evident that the OD600 of BL21 (the control
group) is
significantly higher than that of
BL21(pTRIP-ccdB) at
22°C while the OD600 of BL21 and BL21(pTRIP-ccdB) have similar
value at 37°C. This test result
is consistent with that for DH5α as discussed
previously and this also back up the engineering success of our temperature-based “kill
switch”, the plasmid
pTRIP-ccdB in E. coli host.

![](https://static.igem.wiki/teams/4942/wiki/results/18.png)

Figure 18.
The Growth ability of pTRIP-ccdB
in*E.coil* BL21comparing to the
control group BL21

**Future
plans**

1. In the experiment, we only conducted
sensitivity testing of the temperature-controlled switch in *E. coli*BL21(DE3) and *E. coli* DH5α. In the future, we need to
transform it into more bacterial strains to perform sensitivity tests and further optimize the sensitivity
of our temperature-controlled switch based on the test results. We also need to explore the temperature
range of 22-37°C for the temperature-controlled switch and validate its states through additional
verification methods.

2. Our temperature-controlled switch requires the
addition of exogenous inducers (N-(3-oxohexanoyl) -L-homoserine lactone) for better expression. When no inducer is
added, the expression level is relatively low, and this limitation needs to be addressed in practical
applications.

References

[1] Oscar
Z,B. I C,E. Y K, et al. Traditional fermented foods of Benin Republic: microbiological safety and health
benefits[J]. Discover Food,2023,3(1).

[2] Obeng A
P,Awere E,Obeng A P, et al. Usage and Microbial Safety of Shared and Unshared Excreta Disposal Facilities
in Developing Countries: The Case of a Ghanaian Rural District[J]. Sustainability,2023,15(13).

[3] Bazhenov,
S.V., Scheglova, E.S., Utkina, A.A. et al. New temperature-switchable acyl homoserine lactone-regulated
expression vector. Appl Microbiol Biotechnol 107, 807–818 (2023). <https://doi.org/10.1007/s00253-022-12341-y>

[4] Nocadello,
S., Swennen, E.F. The new pLAI (lux regulon based auto-inducible) expression system for recombinant
protein production in Escherichia coli. Microb Cell Fact 11, 3 (2012). <https://doi.org/10.1186/1475-2859-11-3>

[5] Hoffmann
SA, Diggans J, Densmore D, Dai J, Knight T, Leproust E, Boeke JD, Wheeler N, Cai Y. Safety by design:
Biosafety and biosecurity in the age of synthetic genomics. iScience. 2023 Feb 10;26(3):106165. doi:
10.1016/j.isci.2023.106165.

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