Team:BIT/Bio/Addtional Work
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Additional Work
We optimized the JACOB system last year. The principle of the system is to convert the macromolecule to be detected into a small molecule signal by using an aptamer. The small molecule activates the engineering bacteria to express fluorescence, and detects the macromolecule by detecting the fluorescent signal. JACOB chose lysine as a small molecule that allows downstream lysine-deficient E. coli to grow and express fluorescence. But not all lysine has been used to grow engineered bacteria, and there are uncertainties for the synthesis of fluorescent proteins, which adds to the complexity of the system.
In the process of improving JACOB, we have deepened our thinking about the whole system, which has had a very important impact on our proposed JACOB 2.0.
Modification
We modify the small molecules from lysine to S-adenosine methionine (SAM) to solve the problem of the endogenous lysine, and then through the thiol and crosslinking agent of amino SPDP connect SAM molecules with thiol complementary strand. When the complementary strand is decoupled by CK-MB, the disulfide bond with SAM can be reduced by DTT to achieve the signal transmission from protein to SAM.
In the downstream expression part, we designed a system based on SAM ribosome switch. When there was no SAM, the ribosome switch formed a terminator to terminate transcription, while there was a certain amount of SAM, the ribosome switch formed anti-terminating subsequence and the bacteria began to express fluorescence
First we incubated SAM with SPDP, then added a complementary chain reaction with mercaptogenic modification, and purified the product using an ultrafiltration tube. Then we measured the mass spectra, and the results showed that we successfully connected the small molecule of SAM.
Test
Then, we used the culture medium of different concentrations of SAM to cultivate our engineered bacteria to test the expression system(BBa_K23708003), whose fluorescence intensity should be inversely proportional to the concentration of SAM
The results of these two experiments showed that the system work very well, engineering bacteria expressed different fluorescence intensity in different concentrations of SAM environment
We selected the data for the ninth hour, with SAM concentration as the abscissa, and the fluorescence reduction of the control at this time relative to 0 μg/ml as the ordinate.
It can be seen that at the ninth hour, as the concentration of SAM increases, the amount of decrease in fluorescence intensity increases, and the system works effectively. That’s to say our improvement is effective
After completing the improvement of JACOB last year, we consider more about this system. We noticed that although the whole system is effective, the process from biomarkers to fluorescent signals is from biomarker to small molecules to engineering bacteria. This process produces unavoidable losses and reduces the system the accuracy of system. So we think about how to reduce the reaction steps and avoid unnecessary losses. We began to consider whether we can directly convert from the signal of disease markers to the signals of engineering bacteria, which promoted our JACOB 2.0.
References:
[1] Weston L A, Bauer K M, Skube S B, et al. Selective, Bead-Based Global Peptide Capture Using a Bifunctional Cross-Linker[J]. Analytical Chemistry, 2013, 85(22):10675-10679.
[2] Serganov A, Patel D J. Amino acid recognition and gene regulation by riboswitches[J]. BBA - Gene Regulatory Mechanisms, 2009, 1789(9):592-611.
[3] Christopher W, Pablo C, Polaski J T, et al. A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning[J]. Journal of Molecular Biology, 2015, 427(22):3473-3490.
