iGEM 2023 — Team rec-chennai

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## NOTEBOOK

## OUR DAILY PROGRESS

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## ENGINEERING

iGEM REC Chennai aims to tackle climate change with Project RECOVER, a hybrid carbon capture and biofuel production system. Our project involves the development of a system to capture flue gas and efficiently separate CO2 for utilization
by engineered *E. coli* K12, producing Isobutanol in a reactor compatible with marine vessels. Achieving this milestone required several iterations in the engineering cycle consisting of the four stages below:

**Design:** Engineering principles are used to specify a biological system with an intended function, and models are used to help refine the initial design.
**Build:** The desired DNA sequence encoding the biological system or a part of the biological system is constructed and introduced to a chassis organism.
**Test:** The function of the engineered biological system is assayed.
**Learn:** The differences between the desired and observed function are analyzed, frequently with quantitative data, to develop an improved system.

## CYCLE 1

### RECOMBINANT EXPRESSION OF ENZYME PRK AND RUBISCO IN E.COLI K12 FOR CO2 FIXATION.

##### DESIGN

In the design phase of our engineering cycle, we were committed to addressing the climate crisis by leveraging the power of genetically engineered *E. coli* K12. Our goal is to reduce carbon dioxide (CO2) emissions through the
expression of specific enzymes, namely Phosphoribulokinase and Rubisco, within K12 that will actively utilize CO2. This critical step involves carefully planning and designing the genetic modifications that will enable *E. coli* K12 to efficiently Fix CO2, contributing to our mission of combating climate change.

##### BUILD

To express PRK and RuBisCO in *E. coli*, we first codon-optimized the gene sequences of Synechococcus elongatus PRK and RuBisCO for RFC[10] to ensure efficient protein expression. Next, we performed restriction digestion and ligation, integrating
the genes encoding PRK and RuBisCO into the pSB3K3 plasmid under the T7 promoter. Finally, the resulting plasmid was transformed into *E. coli*, completing this crucial step in our project.

##### TEST

We conducted the Xylose consumption growth curve for engineered *E. coli* that has RuBisCo(without chaperone - rbcX) and prk.

We initially observed the increase in growth curve made evidential that prk has been expressed and the xylose has been consumed and through the pentose phosphate pathway.By convention it generated Ribulose-5 phosphate. The expressed enzyme phosphoribulokinase
which will convert Ribulose-5 phosphate into Ribulose-1,5 Bisphosphate which serves as a substrate for RuBisCO. We found growth arrest in *E. coli* over a period of time .

##### LEARN

We had inferred from this experiment that the absence of functionality of RuBisCo lead to the accumulation of RuBP in the cell that lead to the cell death. Through the inputs from Dr. Natarajan we had been informed that the folding of proteins is
important for the efficient functioning of an expressed enzyme.

## CYCLE 2 :

### RECOMBINANT EXPRESSION OF E.*coli* K12 WITH THE CHAPERONE SUBUNIT FOR EFFICIENT FUNCTIONING OF RuBisCo

##### DESIGN

In this design phase of our engineering cycle, our primary goal is to produce a fully functional RuBisCo enzyme along within K12 that will actively utilize CO2. This critical step was accomplished with the addition of a chaperone subunit
in RuBisCo enzyme along with the large and small subunit

##### BUILD

The codon optimized sequences of prk and the RuBisCo with chaperone was engineered into the pSB1C3 backbone.

##### TEST

We had successfully produced the RuBisCo enzyme and the functionality was proved by the Xylose utilization and efficient conversion of the xylose to pyruvate which was observed using a growth curve. The comparison between the engineered and control
strain led us to a conclusion that addition of RuBisCo chaperone led to the functioning of the enzyme.

We initially observed the increase in growth curve made evidential that prk has been expressed and the xylose has been consumed and through the pentose phosphate pathway.By convention it generated Ribulose-5 phosphate. The expressed enzyme phosphoribulokinase
which will convert Ribulose-5 phosphate into Ribulose-1,5 Bisphosphate which serves as a substrate for RuBisCO. We found growth arrest in *E. coli* over a period of time .

##### LEARN

Lack of chaperone subunit is the reason for the problem in functionality of RuBisCo.

## CYCLE 3 :

##### DESIGN

The addition of kivD and adhA parts in the plasmid for isobutanol production.

##### BUILD

We engineered the dual plasmid prk +RuBisCo in psB1C3 backbone kivD+ adhA in psB3K3 backbone

##### TEST

We observed the production of isobutanol over a period of time which can be seen in the results

##### LEARN

Efficient production of isobutanol was accomplished.

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## RESULTS

#### SDS Page:

After DNA fragments were assembled and ligated, DNA gel analysis was used to estimate the size. Additionally, the compatibility of the size and protein expression was confirmed by the SDS-PAGE analysis.
 The results of the SDS-PAGE study
performed on our engineered *E. coli* are shown in the attached figure.

![ ](https://static.igem.wiki/teams/4689/wiki/results-page/res-1.jpg)

The study illustrates the production of prk, rbCS, rbCX, and rbcL at molecular weights of 35 kDa, 10 kDa, 14.4 kDa, and 48 kDa, respectively. As it is the case of the dual plasmid, the production of kivD and adhA were also confirmed separately,
with molecular weights of 61 kDa and 35 kDa, respectively.

![ ](https://static.igem.wiki/teams/4689/wiki/results-page/res-2.jpg)

### [1]TIME VS XYLOSE CONSUMPTION

A growth curve was monitored for Engineered *E.coli* K12 containing prk and RuBisCo, showing a rapid and increasing uptake of xylose over time.In engineered *E.coli*, there are two primary sources for CO
2 metabolism, namely xylose and CO2 itself. Our experiments were carried out using approximately under 5% CO2 and 1% xylose.

![](https://static.igem.wiki/teams/4689/wiki/results-page/xylose-consumption-engineered-e-coli-k12-prk-rubisco.jpg)

![](https://static.igem.wiki/teams/4689/wiki/results-page/xylose-consumption-e-coli-k12-control.jpg)

![](https://static.igem.wiki/teams/4689/wiki/results-page/average-xylose-consumption.jpg)

### [2] TIME VS RUBISCO+PRK

Batch fermentation profile of *E. coli* K12 engineered strain containing prk and RuBisCo was conducted.Bacterial strain was cultivated in M9 minimal media with xylose in a bioreactor to favor growth of recombinant bacteria supplemented
with 1% Xylose and supplied with 5% CO2 at 37°C under pH control.The optimal pyruvate production was observed in the engineered bacteria which is confirmed using the Methyl red test .

![](https://static.igem.wiki/teams/4689/wiki/results-page/time-vs-od-at-600nm-engineered-e-coli-k12-prk-rubisco.jpg)

![](https://static.igem.wiki/teams/4689/wiki/results-page/negative-control-e-coli-k12.jpg)

![](https://static.igem.wiki/teams/4689/wiki/results-page/growth-curve-engineered-k12-prk-rubisco.jpg)

#### [3] TIME VS ISOBUTANOL PRODUCTION

Batch fermentation profile of *E. coli* K12 engineered strain containing prk,RuBisCo,kivD and adhA.Bacterial strain was cultivated in M9 minimal media with xylose in a bioreactor to favor growth of recombinant bacteria supplemented
with 1% Xylose and supplied with 5% CO2 at 37°C under pH control.Isobutanol is an extracellular product confirmed by the Lucas test to check for it’s presence at the end of the engineered cycle.

![ ](https://static.igem.wiki/teams/4689/wiki/results-page/time-vs-isobutanol.jpg)

### [4] PRK TOXICITY TEST

PRK Toxicity test was conducted to understand the function of prk.The PRK product RuBP, cannot be metabolized by wild-type E.coli.Accumulation of RuBP depletes sugar from the natural pentose phosphate pathway.Due to the lack of carbon source, the growth
of this strain may be inhibited. We incubated the prk expressing strain and the plasmid-free control strain in M9 medium and M9 medium modified with 4 (g/l) xylose as the sole carbon source.In normal M9 medium, glucose will not be converted
into RuBP.In modified M9 medium, xylose will go through the natural pathway and be converted to RuBP.The PRK strain must be observed to stop growing. After 12 hours, the plasmid-free strain could grow to 1.4 O.D.600 in modified M9 xylose medium.The
prk expressing strain can also grow up to 0.75 O.D.600 in normal M9 medium. In contrast, prk strain grown in modified M9 xylose medium did not grow.The results showed that prk could repress/inhibit growth, which was consistent with our expectations.
The PRK expressing strain grown in altered M9 xylose showed merely no growth, which proves the function of PRK.

![](https://static.igem.wiki/teams/4689/wiki/results-page/prk-toxi.jpg)

![](https://static.igem.wiki/teams/4689/wiki/results-page/res-3.png)

#### CONFIRMATORY TESTS

##### DNS Test (prk rubisco)

In this DNS test, we measure the xylose quantitatively in order to distinguish between our engineered *E. coli* strain, which shows lower xylose uptake owing to its effective xylose consumption, and the regular K12 E.coli strain, which has
a high xylose content because it doesn't utilize xylose effectively like the engineered strain.

![](https://static.igem.wiki/teams/4689/wiki/results-page/dns-1.jpg)

### Methyl red test(prk rubisco)

In the Methyl Red test, our engineered *E. coli*, which has prk rubisco part produces a significant amount of pyruvate, exhibits a strong red color with the methyl red reagent. In contrast, the control, represented by the regular *E. coli* K12 strain appears orange, indicating the less amount of pyruvate production when compared to our engineered strain.

![](https://static.igem.wiki/teams/4689/wiki/results-page/mr-test-1.jpg)

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