Difference between revisions of "Part:BBa K4156101"

Revision as of 01:48, 12 October 2022

pLldR

An Operon unit consisting of a collection of LldR-regulated promoters, and is capable of responding to lactate concentration.

Usage and Biology

pLldR is composed of the lldR regulatory protein ( BBa_K1847001 ), the terminator B0012, and the LldPRD promoter（ BBa_M36021 ）. At the lldprD promoter, there are two operator sites O1 and O2 , which are able to bind to the lldR protein and block subsequent gene transcription. The presence of lactic acid molecules can unblock this blocking phenomenon.^[1-2] Thus, the pLldR can specifically respond to lactate and activate downstream genes. In our experiments, we used pLldR to adapt to the high lactate characteristics of the tumor cell microenvironment. Enabling our therapeutic strains to rapidly target to tumor cells and achieve targeted therapy.

Improvement

We built on the previously created parts lldPRD operon（ BBa_M36021 ) and lldR ( BBa_K1847001 ) by introducing a combination of hyper-spank and RBS spoVG in its head and an optimized sequence of RiboJ in its tail, once created a new pLldR promoter. Most importantly, we combined the constructed pLldR with the TP901-mediated XOR logic gate, which greatly improved the performance of pLldR.

The optimized pLldR promoter improves the signal output level while the signal output level is more stable. This is undoubtedly beneficial for the subsequent construction. More notably, we introduced the TP901-XOR gate system, which helps to coordinate many different inducible operons.We characterized our optimized system using pLldR-mRFP as a control as follows.

Characterization

Initial Testing of lactate Promoter

To Characterize part，we first added mRFP after the promoter and wanted to initially test the response of this promoter to different lactate concerntartions based on the fluorescence intensity. E. coli Nissle 1917 was used as chassis.Details of the characterization and test results can be found at BBa_K4156114

We constructed a lactate reporter consisting of the lactate-inducible promoter pPepT+mRFP. To test its performance, we added reporter in different chassis organisms.Fig 1 indicates that pLldR induces the expression of the downstream gene mRFP with the decrease of O2. Thus, it can be seen that the lactate reporter can work properly.

Stability improvement

Then，amplifying genetic switches and Boolean logic gates based on serine integrase (TP901) are used in the design of biosensor systems ^[3]. These genetic devices enable bacteria to perform reliable detection, multiplex logic and data storage of clinical biomarkers in human clinical samples ^[4-5] to meet the requirements of medical testing. For characterization, we added switch, which is TP901 and XOR gate, then followed with mRFP. Details of the characterization and test results can be found at BBa_K4156104

1.In vitro characterization and data analysis of the reported strains

To improve signaling stability as well as accuracy, we added Amplifying genetic switches based on serine integrase (TP901) to the R reporter( BBa_K4156115 ) to construct the AR reporter. Fig 1 indicates lactate (plldR) induced AR reporter with homogenized fluorescence intensity (mRFP/Cell). Comparing Fig1, 2, it can be seen that the fluorescence intensity of the AR reporter decreased significantly at a lactate concentration of 0 mM, and its expression was more stable over time. The fluorescence intensity of the AR reporter was also greater at other concentrations of lactate induction, and the difference between the fluorescence intensity after lactate induction at each concentration was more pronounced. This result indicates that the addition of amplifying genetic switch enhances the reporter intensity and robustness of the lactate biosensor.

2.Engineered strain co-incubated with RKO cells

Details of this section can be found in the next column "Addition of lysis genes"

Addition of lysis genes

Because we have therapeutic proteins that cannot be exocytosed, it is not enough to simply stabilize the response signal, and we intend to add bacteriophage lysis gene phiX174E parts that will enable bacteria lysis.So next we added phiX174E to the above genetic parts. Details of the characterization and test results can be found at BBa_K4156105

References

1 Aguilera L, Campos E, Giménez R, Badía J, Aguilar J, Baldoma L. Dual role of LldR in regulation of the lldPRD operon, involved in L-lactate metabolism in Escherichia coli. J Bacteriol. Apr 2008;190(8):2997-3005. doi:10.1128/jb.02013-07

2 Weghoff MC, Bertsch J, Müller V. A novel mode of lactate metabolism in strictly anaerobic bacteria. Environ Microbiol. Mar 2015;17(3):670-7. doi:10.1111/1462-2920.12493

3 Courbet A, Endy D, Renard E, Molina F, Bonnet J. Detection of pathological biomarkers in human clinical samples via amplifying genetic switches and logic gates. Sci Transl Med. May 27 2015;7(289):289ra83. doi:10.1126/scitranslmed.aaa3601

4 Benenson Y. Biomolecular computing systems: principles, progress and potential. Nat Rev Genet. Jun 12 2012;13(7):455-68. doi:10.1038/nrg3197

5 Bonnet J, Yin P, Ortiz ME, Subsoontorn P, Endy D. Amplifying genetic logic gates. Science. May 3 2013;340(6132):599-603. doi:10.1126/science.1232758

Sequence and Features