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.

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.


[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.


[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 .



[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.

[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.



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.


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.