iGEM 2018 — Team NCKU_Tainan

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# Team:NCKU Tainan/Notebook

[NCKU Tainan](https://2018.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2018.igem.org/Team%3ANCKU_Tainan/Description)[Design](https://2018.igem.org/Team%3ANCKU_Tainan/Design)
  [Improve](https://2018.igem.org/Team%3ANCKU_Tainan/Improve)
  [Results](https://2018.igem.org/Team%3ANCKU_Tainan/Results)
  [Protocol](https://2018.igem.org/Team%3ANCKU_Tainan/Protocol)
  [Measurement](https://2018.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Parts](https://2018.igem.org/Team%3ANCKU_Tainan/Parts)
* Model

  [Model](https://2018.igem.org/Team%3ANCKU_Tainan/Model)
  [Kinetic Law](https://2018.igem.org/Team%3ANCKU_Tainan/Kinetic_Law)
  [Analysis](https://2018.igem.org/Team%3ANCKU_Tainan/Analysis)
* Device

  [Software](https://2018.igem.org/Team%3ANCKU_Tainan/Software)
  [Hardware](https://2018.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Demonstrate](https://2018.igem.org/Team%3ANCKU_Tainan/Demonstrate)[pH Meter](https://2018.igem.org/Team%3ANCKU_Tainan/pH_meter)
  [Thermometer](https://2018.igem.org/Team%3ANCKU_Tainan/Temperature)
  [Wi-Fi Module](https://2018.igem.org/Team%3ANCKU_Tainan/wi_fi)
  [CO2 Sensor](https://2018.igem.org/Team%3ANCKU_Tainan/CO2)
* [Safety](https://2018.igem.org/Team%3ANCKU_Tainan/Safety)
* Human Practices

  [About](https://2018.igem.org/Team%3ANCKU_Tainan/Human_Practices)[Integrated Human Practices](https://2018.igem.org/Team%3ANCKU_Tainan/Integrated_Human_Practices)
  [Education & Public Engagement](https://2018.igem.org/Team%3ANCKU_Tainan/Public_Engagement)
* Product Design

  [Product design](https://2018.igem.org/Team%3ANCKU_Tainan/Applied_Design)
  [Entrepreneurship](https://2018.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
* Team

  [Team Members](https://2018.igem.org/Team%3ANCKU_Tainan/Team)
  [Collaborations](https://2018.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Attributions](https://2018.igem.org/Team%3ANCKU_Tainan/Attributions)
* [Notebook](https://2018.igem.org/Team%3ANCKU_Tainan/Notebook)
* [Awards](https://2018.igem.org/Team%3ANCKU_Tainan/Judge)

# Notebook

###### Of Blood, Sweat and Tears

[Pasr and PgadA](#Pasr_and_Pgada)
[pH Sensor Test](#pH_sensor_test)
[CA](#CA)
[RbcL](#RbcL)
[RbcXS](#RbcXS)
[PRK](#PRK)
[Whole Construction](#Whole_Construction)
[Total Solution](#Total_Solution)

### Construction of Pasr and PgadA

4/26~4/27

* PCR Pasr and PgadA from *E. coli* MG1655 chromosome. Confirmed and extracted
  products by DNA gel.

4/28

* Digested the pSB1C3-GFP plasmid. Ligase the plasmid with Pasr / PgadA and
  RiboJ by PCR. Confirmed by DNA gel electrophoresis. Transformed the gene in DH5
  alpha and spread the bacteria on LB plates.

4/30~5/1

* Confirmed the transformation by colony PCR ,enzyme digestion and DNA gel
  electrophoresis. Selected the correct colonies for sequencing and cultured them
  on LB plates.

5/9

* Store the correct colonies in glycerol in -80°C.

6/1

* Transformed each plasmid into BL21(DE3)

### The pH sensor test

5/9

* Prepared the M9 medium in different pH value.

5/11

* Pre-cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain.

5/12

* Cultured the DH5 alpha-pSB1C3 -Pasr-sfGFP strain in M9 medium in different pH
  value.
  Measured the O.D. 600 value and fluorescence every hour.

5/18

* Pre-cultured DH5 alpha-pSB1C3 –PgadA-RiboJ-sfGFP strain.
* Prepared the M9 medium in different pH value.

5/19

* Cultured the DH5 alpha-pSB1C3 –PgadA-RiboJ-sfGFP strain in M9 medium in different
  pH
  value. Measured the O.D. 600 value and fluorescence every hour.

6/2~6/3

* Repeated the experiment on 5/11~5/12

7/29

* Pre-culture the BL21(DE3)-pSB1C3 –Pasr-sfGFP strain

7/30

* Cultured the BL21(DE3)-pSB1C3 –Pasr -sfGFP strain on 96 well plate in different pH
  value. Measure the fluorescence every 5 minutes in 30 minutes.

9/4

* Pre-culture the BL21(DE3)-pSB1C3 –PgadA-RiboJ-sfGFP strain

9/5

* Cultured the BL21(DE3)-pSB1C3 –PgadA-RiboJ-sfGFP strain strain on 96 well plate in
  different pH value. Measure the fluorescence every 5 minutes in 30 minutes.

### Construction of CA

7/2

* PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
  Extracted the gene from DNA gel.

7/5

* PCR the CA. Confirmed by DNA gel electrophoresis and extracted the gene from DNA
  gel.

7/6

* Digested and ligase the pSB1C3 plasmid and CA gene.

7/10~7/11

* Repeated the PCR of CA, digestion, ligation.

7/12

* Transformed the pSB1C3-PT7-CA into DH5 alpha.

7/19

* Send the plasmid to sequencing

### Construction of RbcL

7/2

* PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
* Extracted the gene from DNA gel.

7/11

* PCR the RbcL with new primer.

7/20

* PCR the RbcL with old and new primer.

7/21~24

* Repeated the PCR of RbcL

8/1

* Ligate the RbcL with PLacI.
* Transformed the pSB1C3-PT7-RbcL and pSB1C3-PLacI-RbcL into DH5 alpha.

8/3

* Confirmed the pSB1C3-PT7-RbcL construct by colony PCR.
* Sent the gene to sequencing

### Construction of RbcXS

7/2

* Prepared the M9 medium in different pH value.

7/3~7/21

* PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
* Extracted the gene from DNA gel.

7/23

* PCR normal/mutant RbcXS with old and new primer.

7/24

* Confirmed by DNA gel.

### Construction of PRK

7/2

* PCR IDT products (RbcL, RbcXS, PRK, CA). Confirmed by DNA gel electrophoresis.
* Extracted the gene from DNA gel.

7/3~8/2

* PCR PRK.

8/1

* Ligated PRK with PLacI and PT7.
* Transformed into DH5 alpha.

8/2

* Confirmed by colony PCR. Selected the colony with correct length and sent to
  sequencing.

### Whole construction

8/8

* Ligated PT7-CA with PLacI-PRK on pSB1C3

8/9

* Ligated PT7-RbcL with PT7-RbcXS on pSB1C3

8/28

* Inserted PT7-CA with PLacI-PRK into pSB3K3

### SDS PAGE, Functional test and Total solution

7/25~7/30, 8/4~8/5, 8/11~8/12

* Ran SDS-PAGE of CA, normal-RbcXS

8/7, 8/9, 8/10

* Ran SDS-PAGE of RbcL and PRK

8/16

* Ran the SDS-PAGE of RbcL, RbcXS and PRK

8/18~8/19

* Activity test of CA

9/2

* Ran SDS-PAGE of pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-PLacI-PRK-PT7-CA

9/3

* Transformed pSB1C3-PT7-RbcL-PT7-RbcXS and pSB3K3-PLacI-PRK-PT7-CA into W3110

9/5~9/17

* Total solution.

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---

##### Contact us

igem.ncku.tainan@gmail.com

No.1, Daxue Rd., East Dist., Tainan City 701, Taiwan (R.O.C.)


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# Team:NCKU Tainan/Experiments

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TEAM

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HUMAN PRACTICES

[Human Practices](https://2018.igem.org/Team%3ANCKU_Tainan/Human_Practices)
[Education & Engagement](https://2018.igem.org/Team%3ANCKU_Tainan/Public_Engagement)

AWARDS

[Applied Design](https://2018.igem.org/Team%3ANCKU_Tainan/Applied_Design)
[Entrepreneurship](https://2018.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
[Hardware](https://2018.igem.org/Team%3ANCKU_Tainan/Hardware)
[Measurement](https://2018.igem.org/Team%3ANCKU_Tainan/Measurement)
[Model](https://2018.igem.org/Team%3ANCKU_Tainan/Model)
[Plant](https://2018.igem.org/Team%3ANCKU_Tainan/Plant)
[Software](https://2018.igem.org/Team%3ANCKU_Tainan/Software)

[JUDGING FORM ⇗](https://igem.org/2018_Judging_Form?team=NCKU_Tainan)

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:NCKU Tainan/Results

[NCKU Tainan](https://2018.igem.org/Team%3ANCKU_Tainan)

* Project

  [Description](https://2018.igem.org/Team%3ANCKU_Tainan/Description)[Design](https://2018.igem.org/Team%3ANCKU_Tainan/Design)
  [Improve](https://2018.igem.org/Team%3ANCKU_Tainan/Improve)
  [Results](https://2018.igem.org/Team%3ANCKU_Tainan/Results)
  [Protocol](https://2018.igem.org/Team%3ANCKU_Tainan/Protocol)
  [Measurement](https://2018.igem.org/Team%3ANCKU_Tainan/Measurement)
  [Parts](https://2018.igem.org/Team%3ANCKU_Tainan/Parts)
* Model

  [Model](https://2018.igem.org/Team%3ANCKU_Tainan/Model)
  [Kinetic Law](https://2018.igem.org/Team%3ANCKU_Tainan/Kinetic_Law)
  [Analysis](https://2018.igem.org/Team%3ANCKU_Tainan/Analysis)
* Device

  [Software](https://2018.igem.org/Team%3ANCKU_Tainan/Software)
  [Hardware](https://2018.igem.org/Team%3ANCKU_Tainan/Hardware)
  [Demonstrate](https://2018.igem.org/Team%3ANCKU_Tainan/Demonstrate)[pH Meter](https://2018.igem.org/Team%3ANCKU_Tainan/pH_meter)
  [Thermometer](https://2018.igem.org/Team%3ANCKU_Tainan/Temperature)
  [Wi-Fi Module](https://2018.igem.org/Team%3ANCKU_Tainan/wi_fi)
  [CO2 Sensor](https://2018.igem.org/Team%3ANCKU_Tainan/CO2)
* [Safety](https://2018.igem.org/Team%3ANCKU_Tainan/Safety)
* Human Practices

  [About](https://2018.igem.org/Team%3ANCKU_Tainan/Human_Practices)[Integrated Human Practices](https://2018.igem.org/Team%3ANCKU_Tainan/Integrated_Human_Practices)
  [Education & Public Engagement](https://2018.igem.org/Team%3ANCKU_Tainan/Public_Engagement)
* Product Design

  [Product design](https://2018.igem.org/Team%3ANCKU_Tainan/Applied_Design)
  [Entrepreneurship](https://2018.igem.org/Team%3ANCKU_Tainan/Entrepreneurship)
* Team

  [Team Members](https://2018.igem.org/Team%3ANCKU_Tainan/Team)
  [Collaborations](https://2018.igem.org/Team%3ANCKU_Tainan/Collaborations)
  [Attributions](https://2018.igem.org/Team%3ANCKU_Tainan/Attributions)
* [Notebook](https://2018.igem.org/Team%3ANCKU_Tainan/Notebook)
* [Awards](https://2018.igem.org/Team%3ANCKU_Tainan/Judge)

# Results

###### Hard Work Pays Off

[Overview](#Overview)
[Construction](#Construction)
[Total solution](#Total_solution)
[Carbon fixation](#Carbon_fixation)
[pH Sensing System](#pH_Senesing)
[References](#References)

### Overview of the result

1. Construct each part and test the function of CA, and PRK.
2. Develop a new measurement approach to determine the carbon fixation ability of
   each strain.
3. Estimate the carbon fixation amount with our experiment result.
4. Characterize the pH sensing promoter Pasr ([BBa\_K1231000](http://parts.igem.org/Part%3ABBa_K1231000)), and improve PgadA biobrick ([BBa\_K1962013](http://parts.igem.org/Part%3ABBa_K1962013)).

### Construction and functional test

###### PRK (PHOSPHORIBULOKINASE)

Achievements:

1. Construction and digestion of DNA gel shows that the size of it was
   right
2. The SDS-PAGE of PRK showed that the expression of PRK in the expected
   protein size
3. PRK toxicity test proves that the function of it varies when cloned
   into
   different plasmids

We constructed *prk* fragments ([BBa\_K2762007](http://parts.igem.org/Part%3ABBa_K2762007)) from IDT DNA synthesis. After PCR
amplification,
*prk* is then cloned into pSB1C3 and transformed into DH5 alpha. SDS-PAGE ensured that
the protein expression was as expected. The results are shown below:

![](https://static.igem.org/mediawiki/2018/8/80/T--NCKU_Tainan--Results_fig_1.jpg)

Fig 1. Confirmation of *prk* digestion.
Fig 2. Confirmation of PRK expression in DH5 alpha. The expected protein size is
37.7kDa.

We initially decided to test its function by HPLC to measure the amount of RuBP
inside the cell. Our instructors pointed out some difficulties in HPLC
measurement such as excessive noise signal in our sample. We, therefore,
determined to test its function with a toxicity test. The product of PRK, RuBP,
cannot be metabolized by wild-type *E. coli*. The accumulation of RuBP
depletes
the sugar from the native pentose phosphate pathway. Lack of carbon source, the
growth of that strain may be repressed. We incubate the PRK expressing strain
and control stain that contains no plasmid in M9 medium and altered M9 medium
with 4 (g/l) xylose as its sole carbon source. In normal M9 medium, glucose will
not be converted into RuBP. In altered M9 medium, xylose will go through the
native pathway and be converted into RuBP. Growth arrest of PRK strain should
be observed.

We tested PRK in different strains. We first cloned *prk* into pSB1C3 and
transformed into BL21 (DE3). After 12 hours, the strain without plasmid could
grow up to 1.4 O.D.600 in altered M9 xylose medium. The strain that contains
PRK can grow up to 0.75 O.D.600 in normal M9 medium either. In contrast, the
PRK strain that grew in altered M9 xylose medium showed no growth at all. The
result shows that PRK can suppress/inhibit the growth, which matches to our
expectation.

![](https://static.igem.org/mediawiki/2018/3/3d/T--NCKU_Tainan--Results_fig_3.png)

Fig 3. The result of PRK test in BL21 (DE3). The PRK expressing strain is
incubated
in both normal M9 medium and altered M9 xylose medium to compare with the
strain
without plasmid. The PRK expressing strain grown in altered M9 xylose showed
merely
no growth, which proves the function of PRK.

Although the function of PRK has been confirmed, we would like to lower the
expression of it to minimize the growth arrest. We thus cloned the part into
pSB3K3, a low copy number plasmid to lower its protein expression. We then
compare
the growth under high and low copy number plasmid. We found out that pSB3K3
shows a
little growth arrest comparing to the strain without plasmid. The growth of it
exceeds that of PRK expressed in pSB1C3. We can regulate the expression of PRK
via
high or low copy number plasmid to optimize the growth and carbon fixation
efficiency of the bacteria.

![](https://static.igem.org/mediawiki/2018/3/3a/T--NCKU_Tainan--Results_fig_4.PNG)

Fig 4. Compares the growth in M9 xylose medium of PRK expressing strain which prk is cloned into
high
and low copy number plasmids respectively. The low copy number plasmid, pSB3K3, shows a
little bit of growth retard compare to the control strain. However, its
toxicity is much less than that expressed in high copy number.

We also transformed pSB3K3-*prk* into W3110 strain. W3110 is reported to have
higher pressure tolerance. The trend of the results is similar to that of the
BL21 (DE3), but the difference between experiement and control group is not obvious . We deduce that PRK can still function in W3110 since the
trend matches our expectation. As pSB3K3 is a low copy number plasmid, the
expression of the protein may be lower than that of high copy number plasmid.
The
pressure tolerance of W3110 strain may also lessen the influence of toxicity by
PRK.

![](https://static.igem.org/mediawiki/2018/f/f6/T--NCKU_Tainan--Results_fig_4_2.PNG)

Fig 5. The result of PRK test in W3110

###### CA (Carbonic Anhydrase)

Achievements:

1. Construct the *ca* and transform it into BL21 (DE3)
2. Run the SDS-PAGE to confirm its expression
3. Measure the activity of CA enzyme

We cloned the DNA fragments ([BBa\_K2762008](http://parts.igem.org/Part%3ABBa_K2762008)) into pSB1C3 plasmid after the gene
is amplified
with PCR. We transform the plasmid into DH5 alpha and BL21 (DE3). Next, we
confirm its protein expression with SDS-PAGE.

![](https://static.igem.org/mediawiki/2018/0/04/T--NCKU_Tainan--Results_fig_5.jpg)

Fig 6. Confirmation of *ca* digestion
Fig 7. Confirmation of CA expression in BL21 (DE3). The expected protein size is
27.9kDa.

We then ran the activity test of CA. In our bypass pathway, the function of CA
is to
convert proton and bicarbonate into water and carbon dioxide. CA activity was determined using the Wilbur-Anderson assay. Briefly, 9 mL ice-cold Tris−HCl (20
mM, pH8.3) buffer and 0.2 mL enzyme were mixed and transferred to a 20 mL
sample bottle, with further incubation at 0 °C with stirring. Then, 6 mL of
ice-cold CO2-saturated solution was added immediately into the sample bottle
and the time course (sec) of pH decrease from 8.3 to 6.3 was recorded. CA
activity was calculated using a Wilbur–Anderson unit (WAU) per milliliter of
sample. The definition for WAU is (T0-T)/ (T0) in which T0
and T was the time required for the pH drop from 8.3 to 6.3, with and without
CA, respectively. The enzyme activity of our CA is 21.8
unit/liter.
To confirm the contribution of the CA to the whole pathway, we also ran the
total
solution which will be described in the following content.

###### Rubisco

Achievements:

1. Construction and digestion of DNA gel shows that the size of it was right
2. The SDS-PAGE of Rubisco showed that the expression of Rubisco in the expected size

We constructed rubisco fragments from IDT DNA synthesis. After PCR amplification of
the three subunits, rubisco is then cloned into pSB1C3 and transformed into
DH5 alpha. SDS-PAGE ensured that the protein expression was as expected. The results
are shown below:

![](https://static.igem.org/mediawiki/2018/c/cb/T--NCKU_Tainan--Results_fig_9.jpg)

Fig 8. Confirmation of *rbcX* and *rbcS* digestion
Fig 9. Confirmation of *rbcX* and *rbcS* expression in BL21 (DE3). The expected protein
size is 15.3 kDA and 13.8 kDA respectively.

![](https://static.igem.org/mediawiki/2018/0/0d/T--NCKU_Tainan--Results_fig_7.jpg)

Fig 10. Confirmation of *rbcL* digestion.
Fig 11. Confirmation of *rbcL* expression in DH5 alpha. The expected protein size is
52.37 kDa.

After mining a lot of information from the publications, we found out a method
to determine the activity of Rubisco by thin-layer chromatographic has been
reported. However, due to time concern, we are not capable of measuring the
enzyme activity of Rubisco with this method. We finally confirm its function
from the results of total solution test.

### Total solution

###### Rubisco

Achievements:

1. Develop an index to evaluate the carbon fixation ability of each
   constructed strain
2. Confirm the importance of Rubisco enzyme in bypass pathway
3. Check the growth and carbon fixation enhancement of CA enzyme
4. Compare the carbon fixation rate of W3110 and BL21 (DE3) *E. coli*
   strains
5. Compare different CO2 incubation environment

[Principle and Mechanism of Xylose Utilzation Index](#Total_solution_overview)

In the total solution experiment, we strive to measure the carbon fixation
amount of each sample. After reading numerous publications, we found out that
previous researches determine the efficiency of carbon fixation via measuring
the decrease of carbon dioxide concentration in the closed system or measure
the weight percentage of 14C radioisotope in the dry cell. However, due to biosafety
constrain of our lab, we can barely use the radioisotope. Measuring the
decrease of carbon dioxide concentration in the closed system is also
impractical for us since we have too much test samples. A new method to measure
multiple samples in the short period of time is developed by our team. We are
able to evaluate the fixation efficiency of each sample with the optical
density
O.D. 600 and xylose consumption. We have measure various construction to prove
that all the enzymes in our design is necessary for carbon fixation.

The bacteria samples in total solution test were incubated in an altered M9 medium which substitutes
glucose to xylose. 1/1000 of LB medium was added to support the trace elements.
Since the concentration of LB medium is too low, it doesn’t contribute the
carbon source of the bacteria.

We defined a new index, Xylose Utilization Index, to describe the potential of
carbon fixation. We can compare this index of each strain to find out the
strain that has the highest capacity of carbon fixation.

To define the XUI, we firstly made two assumptions:

1. O.D. 600 of the sample has a linear relationship to dry cell weight
   (biomass). Optical density is frequently used as a means of describing the
   cell density in the broth. We measured the dry cell weight of samples in
   different O.D. value and discovered that it has a linear relationship. We
   conclude that we can utilize O.D. value to estimate the dry cell weight. 1
   0.D. of BL21 (DE3) strain per litter yields the dry cell weight of 0.8 gram.

![](https://static.igem.org/mediawiki/2018/6/64/T--NCKU_Tainan--Results_fig_10.png)

Fig 12. shows the dry cell weight of BL21 (DE3) incubated in altered M9 xylose
medium.
A linear relationship between O.D. and dry cell weight is observed.

2. The elemental formula of *E. coli* should be fixed or varies within a
   small range. Although the formula may have variations in different
   growth condition, we assume that such error can be ignored during the
   following calculation.

After these two assumptions, the Xylose Utilization Index is designed to
evaluate
the carbon fixation ability of each strain. The definition of the index is
xylose
consumption over O.D. 600. O.D. 600 measurement can be viewed as the weight of
carbon of the bacteria. The index shows the ratio of xylose consumption per
biomass. For wild-type *E. coli*, it only consumes xylose (the sole carbon
source
provided in our medium) as its carbon source. Although some native *E. coli*
pathway
may utilize CO2 (such as lipid synthesis), the amount is too small
to consider.
As
for engineered strain, carbon dioxide can be utilized as it’s carbon source. By
producing the same amount of carbon biomass, it requires less xylose. We can
thus
compare the XUI of each strain to determine the strain that fixes
carbon.
The less the XUI in the sample, the more possibility that it fixes carbon.

$${XUI = {{xylose \ consumption \ (g/l)} \over {O.D. 600}}}$$

![](https://static.igem.org/mediawiki/2018/1/1b/T--NCKU_Tainan--CO2_results.gif)

We use the Dinitrosalicylic Acid (DNS) reducing sugar assay to measure the
xylose
concentration in the medium. Under base solution, DNS will turn to brown color
while reacting with reductive sugar in high temperature. In the specific
temperature range, the color will have a linear relationship with the reductive
sugar
concentration. We can thus measure the xylose concentration at O.D.540.

![](https://static.igem.org/mediawiki/2018/3/3f/T--NCKU_Tainan--Results_Fig_10.PNG)

Fig 13. Shows the calibration line of DNS assay kit.

Before measuring the XUI, we observe the growth curve of each strain. We found
out
that W3110 (L5T7) constructed strain cannot grow in altered M9 solution.
W3110 (L5T7)
is a newly constructed strain, we are not quite certain its characteristic. We
eliminate this strain from the following experiment. BL21 (DE3) and W3110
constructed strains show little growth after 24 hours.

![](https://static.igem.org/mediawiki/2018/5/51/T--NCKU_Tainan--Results_Fig_11.PNG)

Fig 14. shows the growth of engineered (contains Rubisco and PRK) W3110 (L5T7), BL21 (DE3), W3110 incubated in normal
incubator for 24 hours. The growth of W3110 (L5T7) is not obvious while other
strains show growth after 24hours.

###### Total solution check: Function of Rubisco

We then utilized XUI to evaluate the function of each enzyme in the pathway. We
first check the function of Rubisco in BL21 (DE3) strain. Rubisco enzyme with
promoter PT7 ([BBa\_K2762011](http://parts.igem.org/Part%3ABBa_K2762011)) was cloned into pSB1C3 and PRK
with promoter PLacI ([BBa\_K2762007](http://parts.igem.org/Part%3ABBa_K2762007)) was cloned into pSB3K3. Both
plasmids were then co-transformed into BL21 (DE3). We
measured the XUI of the strain and compared them with the control group that IPTG was not
added and BL21 (DE3) without plasmid. IPTG can induce the promoter
PT7 to produce the downstream enzyme. The growth of each strain is
first examined. The IPTG induced strain showed growth retard. We assume the
cause of growth retard is due to the pressure from overexpressing the protein
Rubisco. The control strain without IPTG induction produce less Rubisco enzyme
than the experiment and has less pressure. We then compare the XUI of each
strain and discovered that control strain without IPTG induction produces less
Rubisco enzyme than the experiment. Without Rubisco, the bypass pathway is not
capable of using CO2. We found out that the strain without Rubisco
has higher
XUI, symbolizing that Rubisco is essential in the carbon fixation pathway.

![](https://static.igem.org/mediawiki/2018/3/35/T--NCKU_Tainan--Results_fig_13.png)
![](https://static.igem.org/mediawiki/2018/0/0e/T--NCKU_Tainan--Results_fig_14.png)

Fig 15. Shows the growth and XUI measured in 5% CO2 incubation for 12
hours
respectively. The strain that contains PRK and Rubisco shows little growth. The XUI of the strain
that contains both Rubisco and PRK shows statistically significant decrease
compare to strain without both enzymes.

###### Total solution check: Function of CA

From the above results, we discovered that although Rubisco and
PRK alone can enhance the utilization rate of carbon dioxide, the growth and
utilization ability didn’t meet our expectations. The third important enzyme
came into play: CA enzyme. We cloned Rubisco ([BBa\_K2762011](http://parts.igem.org/Part%3ABBa_K2762011)) into pSB1C3 and
cloned PRK with PLacI promoter and CA with PT7
promoter ([BBa\_K2762013](http://parts.igem.org/Part%3ABBa_K2762013))
into pSB3K3. Two plasmids are then co-transformed into BL21 (DE3). We measured
the
XUI of this strain and compare with the previous strain that only contains PRK
and Rubisco. We found out that CA can raise the growth and lower the XUI. We
infer that CA can enhance the intracellular CO2 concentration and
thus increase
the carbon flux of the bypass pathway. The efficiency of the bypass pathway is
thus been increased.

![](https://static.igem.org/mediawiki/2018/9/99/T--NCKU_Tainan--Results_fig_15.png)
![](https://static.igem.org/mediawiki/2018/5/54/T--NCKU_Tainan--Results_fig_16.png)

Fig 16. Shows the growth and XUI comparison of each strain. All the tested
strains are incubated in 5% CO2 incubator for 12 hr. 0.1mM of IPTG
was added to
induce protein expression. We can observe that growth speed of the
construction has been increased with the CA and the XUI of the strain that
contains complete three enzymes was the lowest compared to the strain without
plasmid or the strain that only contains PRK and Rubisco, stating that three
enzymes are required to optimize the carbon fixing bypass pathway.

###### XUI Comparison between BL21 (DE3) and W3110

We then compare the XUI value between BL21 (DE3) and W3110 constructed strain.
When we design our IDT sequence, we link the CA directly to the promoter PLacI,
so we could not transform CA construct into W3110 strain. We thus compare the
XUI of strains that only contains Rubisco and PRK ([BBa\_K2762011](http://parts.igem.org/Part%3ABBa_K2762011)),
([BBa\_K2762007](http://parts.igem.org/Part%3ABBa_K2762007)).
We found out that both strains show similar trend: the XUI will
be lower with the expression of the constructed protein. The growth condition
of both constructed strains is similar for the first 12 hours. We then compare
the difference of XUI between two *E. coli* strain. We found out that both
strain
shows similar trend: the XUI will be lower with the expression of the constructed protein. However, W3110 has a higher XUI compared with BL21 (DE3) in
constructed strain as well as the strain without plasmid. We infer two reasons
that cause the difference of XUI:

1. W3110 “wildtype” strain has more flexible metabolic network. The carbon flux to pentose phosphate pathway of W3110 is more than that of BL21 (DE3) and thus consumes
   more xylose compare to lab strains such as BL21 (DE3).
2. The constructed protein expression in W3110 may be less than BL21 (DE3) lab
   strain. BL21 (DE3) is commonly used to express protein. We inferred that with
   more protein been expressed, the bypass pathway in BL21 (DE3) will be more
   favored than the W3110 strain.

![](https://static.igem.org/mediawiki/2018/0/0c/T--NCKU_Tainan--Results_fig_17.png)
![](https://static.igem.org/mediawiki/2018/5/52/T--NCKU_Tainan--Results_fig_18.png)

Fig 17. Shows the growth and the XUI of BL21 (DE3) and W3110 strains.

We finally concluded that the efficiency of the bypass pathway in BL21 (DE3) is
better than that in the W3110 strain.

###### Incubation under different CO2 concentration

Finally, we compare the XUI under different CO2 concentration. We
incubated the engineered
bacteria in normal incubator without CO2 input and the cell culture
incubator
that maintains 5% CO2 concentration. We observed that the strain in
5% CO2
incubator has lower the XUI. The supply of sufficient CO2 can
increase the
efficiency of the bypass pathway and enhance the growth. We can conclude that
our constructed pathway can utilize carbon dioxide as one of its carbon
source from this result.

![](https://static.igem.org/mediawiki/2018/6/67/T--NCKU_Tainan--Results_Fig_15.PNG)
![](https://static.igem.org/mediawiki/2018/7/71/T--NCKU_Tainan--Results_fig_20.png)

Fig 18. The comparison of the growth and the XUI of the BL21 (DE3) that contains
all three enzymes in normal incubator and 5% CO2 incubator. The
strain grown in
CO2 incubator showed better growth and lower XUI, which indicates
that our
strain can use CO2 as a carbon source in the presence of high CO2
level.

### Carbon fixation

[Amount of Carbon Fixed: 0.575 mg / l \* hr](#Estimation_Carbon_fixation)

To find out how much and how efficient genetically engineered *E. coli*
can fix
carbon dioxide, we use the material balance concept to evaluate the
heterotrophic CO2 fixation process. Consider a system composed of a
single
component, the general material balance can be written as:
$${\{Input\ to\ the\ system\}\ –\ \{Output\ to\ the\ system\}\ =\
\{Accumulation\ in\ the\ system\}}$$
A system can be defined as an arbitrary portion of a process considered for
analysis, in which in this case, is an engineered carbon capturing *E. coli*.

![]()

The engineered *E. coli* BL21 (DE3) is cultured in M9 medium with formula
adjusted so that xylose is the sole carbon source. The aforementioned M9 Medium
contains
4 (g/l) xylose and 1/1000 LB medium (the carbon consumed from LB medium can be
ignored). By applying the law of conservation of mass, which states that mass
may neither be created nor destroyed, the material balance for carbon in an
engineered *E. coli* may simply be written as
$${\{C\_{CO\_2}\ in\}\ +\ \{C\_{xylose}\}\ -\ \{C\_{CO\_2}\ out\}\ -\ \{C\_{waste}\}\
=\ \{C\_{biomass}\}...(1)}$$
Considering the difficulties in measuring carbon in *E. coli* metabolic
waste and
that Cwaste would be positive, the equation reduces to
$${\{C\_{CO\_2}\ in\}\ -\ \{C\_{CO\_2}\ out\}\ ≥\ \{C\_{biomass}\}\ -\
\{C\_{xylose}\}...(2)}$$
Let {CCO2 net}= {CCO2 in} - {CCO2
out}, equation (2) further simplifies to
$${\{C\_{CO\_2}\ net\}\ ≥\ \{C\_{biomass}\}\ -\ \{C\_{xylose}\}...(3)}$$
If Cwaste is very small and negligible, we can obtain the net amount
of carbon
dioxide fixed over time. If, on the contrary, Cwaste cannot be
neglected,
equation (3) allows us to estimate the minimum net amount of carbon dioxide
fixed.

Cbiomass can be calculate by multiplying O.D. 600 to DCW and mass
percent of carbon in *E. coli* biomass. The O.D. 600 of engineered *E.
coli* is
measured after a 12-hour cultivation and the result obtained is 0.45O.D. . Yin
Li et al. reported that dry cell weight (DCW) of *E. coli* is
$${0.35g\over L ∙ 𝑂.𝐷. 600}$$
, determined by experiment. *E. coli* biomass contains 48% of carbon by
mass.
$${C\_{biomass}\ =\ 0.4511\ ×\ 0.35\ ×\ 48\%}$$
$${=\ 0.0758\ g/L}$$

On the other hand, Cxylose can be calculated by multiplying the
amount of
xylose consumed per unit volume of broth to the mass percent of carbon in
xylose. Xylose consumption is calculated by using a DNS kit that measures the
concentration of reducing sugar and the result obtained is 0.1723g of xylose
consumed per liter of M9 medium. Carbon mass percentage of xylose
is 40%.
$${C\_{xylose}\ =\ 0.1723\ ×\ 40\%\ =\ 0.0689\ g/L}$$
By equation (3)
$${C\_{CO\_2\ net}\ =\ 0.0758\ -\ 0.0689}$$
$${=\ 0.0069\ g/L}$$
Since the *E. coli* has been cultured for 12 hours, we can calculate the
rate of
carbon fixation by
$${Rate\ of\ carbon\ fixation\ =\ {𝐶\_{𝐶𝑂\_2\ 𝑛𝑒𝑡}\over 12}}$$
$${=\ {0.0069\over 12}}$$
$${=\ 0.575\ {mg\over L ∙hr}}$$
To find out how much carbon in biomass comes from the carbon in CO2 captured by the heterotrophic microbes, we can divide equation (3) by the mass percentage of carbon in biomass:

$${{{ \{ CO\_{2 net}} \} \over \{ {C\_{biomass}} \} } \geq {1 -
{ \{ {C\_{xylose}} \} \over \{ {C\_{biomass}} \} }}}$$

We can thus calculate the ratio with our experiment results:

$${{Ratio \ of \ carbon \ in \ CO\_2 \ fixed \ to \ carbon \ in \ biomass} = {1 -{0.0689 \over 0.0758}} = 9.1 \%}$$

### pH sensing system

###### Achievements:

1. Construct the pH sensing system
2. Measure and characterize the short-term fluorescence intensity of Pasr ([BBa\_K1231000](http://parts.igem.org/Part%3ABBa_K1231000))
3. Measure and characterize the long-term fluorescence intensity of PgadA ([BBa\_K1962013](http://parts.igem.org/Part%3ABBa_K1962013))
4. Improve the previous biobrick of PgadA ([BBa\_K1962013](http://parts.igem.org/Part%3ABBa_K1962013))

###### Construction of the pH sensing system

We construct both promoters with PCR, using primer as templates since the size
of it is small. For the construction of Pasr, we cloned into the
plasmid that
contains both rbs and GFP. For the construction of PgadA, we took
the
constructed part from 2016 Dundee iGEM team as a reference. Both parts were
then cloned into pSB1C3 plasmid and transformed into BL21 (DE3).

###### Fluorescence intensity measurement of Pasr

Pasr ([BBa\_K1231000](http://parts.igem.org/Part%3ABBa_K1231000)) is reported to be induced in acidic condition. We think that it
can be used
to report the abnormal acidity of the medium. We thus determine to measure the
fluorescence intensity in a short period of time. We first incubated the
bacteria to log phase (within 2 hour) with LB medium. We then centrifuged the
broth and suspended the pellet with pH modified M9 medium (the pH value is
modified with 1M HCl). We then took the sample and incubate in the 96 well and
measure its fluorescence intensity for every 3 minutes. We found out that the
promoter Pasr will be induced at the pH value below four within 30 minutes. The
different fluorescence intensity can be observed within 30 minutes. The
fluorescence had the peak at pH value of 4.25.

![](https://static.igem.org/mediawiki/2018/4/44/T--NCKU_Tainan--Results_fig_21.png)

Fig 19. The data shows the fluorescence intensity (absorbance: 485 nm,
excitation:
535 nm) expressed by Pasr in different pH.

Based on the data has shown above, we could conclude that Pasr is an acidic
promoter as it has a high expression of fluorescence at pH 4.25 and pH 5. The
results show that Pasr constructed pH sensing system can be used as an alert.
When the medium turns acidic, fluorescence can be easily observed. We believe
that this system can also be applied to various bio-detection system.

###### Fluorescence intensity measurement of PgadA

PgadA ([BBa\_K1962013](http://parts.igem.org/Part%3ABBa_K1962013)) was previously reported to be induced under neutral and mild
acidic
environment. We measured the fluorescence intensity for 14 hours. We pre-cultured
the strain and incubate the strain with pH modified M9 medium (the pH value is
modified with 1M HCl). The induction of PgadA is observed under
neutral and
weak acidic environment.

![](https://static.igem.org/mediawiki/2018/a/a3/T--NCKU_Tainan--wifi_pgada_flrescent.png)

Fig 20. The data shows the fluorescence intensity (absorbance: 485 nm,
excitation: 535 nm) expressed by PgadA in different pH.

###### Improvement of PgadA

We found out that the fluorescence intensity of PgadA is much lower
than the
Pasr and would like to improve the sensitivity of this biobrick. We
thus add a
RiboJ sequence at the downstream of PgadA. RiboJ sequence is reported to increase the expression of downstream protein. We thus compare the fluorescence of previous and improved biobrick. We discover that with RiboJ, the protein of down-stream reporter protein is increased.

![](https://static.igem.org/mediawiki/2018/f/fd/T--NCKU_Tainan--PGADA_OMPARISON.png)

Fig 21. The data compares the fluorescence of PgadA and PgadA with RiboJ sequence.

For more information,
please check
the [Improvement](https://2018.igem.org/Team%3ANCKU_Tainan/Improve) page.

### References

1. F. Gong, G. Liu, X. Zhai, J. Zhou, Z. Cai, Y. Li,
   Quantitative analysis of an engineered CO2-fixing Escherichia Coli
   reveals great potential of heterotrophic CO2 fixation. Biotechnology for
   Biofuels,8(1). doi:10.1186/s13068-015-0268-1
2. U. V. Stockar, J. Liu, Does microbial life always
   feed on negative entropy? Thermodynamic analysis of microbial growth.
   Biochimica Et Biophysica Acta (BBA) - Bioenergetics,1412(3), 191-211.
   doi:10.1016/s0005-2728(99)00065-1
3. 2016 Dundee iGEM team
4. S. Chakrabarti, S. Bhattacharya, S. K. Bhattacharya,
   A nonradioactive assay method for determination of enzymatic
   activity of d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco).
   Journal of Biochemical and Biophysical Methods,52(3), 179-187.
   doi:10.1016/s0165-022x(02)00072-6

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