Syn­thet­ic Genet­ic Clock Checks the Thermometer

Genet­ic sys­tems run like clock­work, attuned to tem­per­at­ure, time of day and many oth­er factors as they reg­u­late liv­ing organ­isms. Sci­ent­ists at Rice Uni­ver­sity and the Uni­ver­sity of Hou­s­ton have opened a win­dow onto one aspect of the pro­cess that has con­foun­ded research­ers for dec­ades: the mech­an­ism by which genet­ic reg­u­lat­ors adjust to chan­ging tem­per­at­ure. Until now, syn­thet­ic bio­lo­gists have not been able to duplic­ate this mar­vel, but Rice bio­chem­ist Mat­thew Ben­nett and his team developed a robust syn­thet­ic genet­ic clock that allows Escheri­chia coli bac­teria to accur­ately keep time in a wide tem­per­at­ure range. The clock, which reg­u­lates the pro­duc­tion of pro­teins, does not speed up or slow down with chan­ging tem­per­at­ures, and offers one pos­sible solu­tion to a prob­lem that has hindered the advance of syn­thet­ic bio­logy. The res­ults were pub­lished this week in the Pro­ceed­ings of the Nation­al Academy of Sci­ences. The rev­el­a­tion will be of interest to bio­lo­gists who study reg­u­lat­ory sys­tems, par­tic­u­larly cir­ca­di­an rhythms, but it may be most valu­able to syn­thet­ic bio­lo­gists who wish to repro­gram cel­lu­lar reg­u­lat­ory mech­an­isms for bio­tech­no­logy, Ben­nett said. ​“One of the prob­lems we’ve had is that the genet­ic cir­cuits we build are fra­gile,” he said. ​”We can build sys­tems that do what we want, but they often do not work well in oth­er people’s hands, or if we change the media or tem­per­at­ure. We wanted to cre­ate a sys­tem that should work inde­pend­ently of the para­met­ers that might be hard for a syn­thet­ic bio­lo­gist to con­trol. We want to show we can build robust cir­cuits, not just by mak­ing the archi­tec­ture of the sys­tem more com­plic­ated, but by using the right pro­teins.” The abil­ity to reg­u­late for tem­per­at­ure comes nat­ur­ally in mam­mals, but not all life is warm-blooded, and tem­per­at­ure gen­er­ally affects bio­chem­istry. ​“The warm­er things are, the more bio­chem­istry speeds up,” Ben­nett said. ​“This mani­fests in a lot of ways: Enzymes work faster and bio­chem­ic­al rates are faster.” He said that E. coli, for instance, shows dra­mat­ic changes in beha­vi­or even with­in its com­fort zone of about 30 to 41 degrees Celsi­us. ​“For every 10 degrees Celsi­us increase in tem­per­at­ure, there’s about a doub­ling in the cell cycle speed,” Ben­nett said. Among bio­lo­gic­al pro­cesses, there’s a not­able excep­tion: cir­ca­di­an clocks that keep a steady beat des­pite the tem­per­at­ure. ​“We have genet­ic­ally con­trolled clocks that help us determ­ine the time of day and coordin­ate our response to the day-night cycle, chan­ging hor­mone levels and our alert­ness. And we’re not the only organ­isms that have them,” he said. ​“Plants and fungi and even some bac­teria that do not have intern­al tem­per­at­ure reg­u­la­tion also have cir­ca­di­an clocks. For those organ­isms, it’s very import­ant that the peri­od of their cir­ca­di­an clocks remains the same regard­less of tem­per­at­ure changes. Your crops, no mat­ter wheth­er it’s hot or cold, always keep to the same day-night cycle.” But cir­ca­di­an clocks are also bio­chem­ic­al. ​”As it gets colder, cir­ca­di­an clocks should slow down, and as it gets warm­er, speed up, but they do not,” he said. ​“It’s been a mys­tery as to why that doesn’t hap­pen.” Ben­nett sus­pec­ted the clocks take their cues from a com­bin­a­tion of cel­lu­lar feed­back loops and tem­per­at­ure-sens­it­ive pro­teins. ​“Instead of look­ing at cir­ca­di­an clocks in humans or plants, how­ever, we decided to build a sys­tem from the ground up,” he said. His research group star­ted with a syn­thet­ic gene oscil­lat­or that was built to run in E. coli. Then, by alter­ing a single amino acid of a key pro­tein – LacI, the lactose repressor – the research­ers made that pro­tein tem­per­at­ure-sens­it­ive and provided the syn­thet­ic clock a guide to com­pensate for chan­ging con­di­tions. Ben­nett noted in the paper that engin­eers have struggled with tem­per­at­ure com­pens­a­tion for a long time, per­haps most fam­ously in the search for a device to give sail­ors at sea their lon­git­ude. ​“Tem­per­at­ure com­pens­a­tion is a prob­lem with time­keep­ing in gen­er­al,” said the research­er, whose first paper as an under­gradu­ate also touched upon the lon­git­ude prob­lem. ​“Metals expand and con­tract in response to tem­per­at­ure changes, thus alter­ing the peri­od of mech­an­ic­al clocks. ​“This was a major obstacle for early nav­al chro­no­met­ers. The man who inven­ted those chro­no­met­ers, John Har­ris­on, had to com­pensate for tem­per­at­ure effects. It was a big prob­lem in engin­eer­ing back then, and we’re still find­ing it to be a prob­lem when we build gene cir­cuits in bac­teria today.” The research was sup­por­ted by the Nation­al Insti­tutes of Health through the joint Nation­al Sci­ence Foundation/​National Insti­tute of Gen­er­al Med­ic­al Sci­ences Math­em­at­ic­al Bio­logy Pro­gram, the Robert A. Welch Found­a­tion and the John S. Dunn Found­a­tion Col­lab­or­at­ive Research Award Pro­gram admin­istered by the Gulf Coast Con­sor­tia.

Ori­gin­al publication:

F. Hus­sain, C. Gupta, A. J. Hirn­ing, W. Ott, K. S. Mat­thews, K. Josic, M. R. Ben­nett. Engin­eered tem­per­at­ure com­pens­a­tion in a syn­thet­ic genet­ic clock. Pro­ceed­ings of the Nation­al Academy of Sci­ences, 2014; DOI: 10.1073/pnas.1316298111#sthash.cSNkoieN.dpuf Source: Rice University