Background
To sense the heat produced by ferritin, we need thermosensitive regulator. There are many ways for bacteria to sense and respond to temperature change. However, most signal transduction system that responds to sudden temperature changes takes not the temperature itself but rather the consequences of temperature-induced damages[1].
The ferric oxohydroxide core in ferritin is structurally similar to the mineral ferrihydrite (γ-Fe2O3)[2], which is Superparamagnetic[3]. According to the work of Heng Huang et. al, Superparamagnetic ferrite nanoparticles can only generate local heat(Fig.1.), not the heat for whole cell[4]. So the consequences of temperature-induced damages are not obvious. Thus, direct only responses of regulator to temperature change could sense the heat produced by ferritin.
Taking all above into consideration, we tested RNA thermometers assembled with different promoters, and selected the best option. Also, we designed a Thermosensitive T7 RNA polymerase, based on thermosensitive intein.
RNA Thermometer
What is it?
RNA Thermometers(RNAT) are temperature-sensing RNA sequences in 5’UTR of their mRNAs. At low temperature, RNAT folds into the structure, blocking access of ribosome; at high temperature, RNAT switch from off to open conformation, increasing the efficiency of translation initiation[5].
Why choose it?
Unlike other thermosensitive regulators, especially those dependent on protein conformation change, RNA sequences of RNAT is short, conforming convenience. In addition, based on conformation changes, RNAT responses to temperature change in a more immediate manner[5].
How to?
Select the best assembly
TUDelft-2008 has modified 3 kinds of RNAT: ROSE(BBa_K115001), FourU(BBa_ K115002), PrfA(BBa_K115003). We constructed each RNAT under the control of 3 different constitutive promoters:BBa_J23101, BBa_J23106, BBa_J23119 and use RFP as a reporter(Fig.2.). After construction, the 9 circuits were transformed into DH5α.
Agar plate test
According to the results of TUDelft-2008, the temperature threshold is 37℃ for FourU and PfrA, 42℃ for ROSE. Thus, we set the culture temperature to 28℃, 37℃ and 42℃. For one temperature, we spotted one strain on 3 different plates, each plate containing 9 different strains(Fig.3.).
After 48 hours, as in the photo: only FourU significantly functioned, and FourU under the control of promoter J23119 worked best(Fig.4.&Fig.5.).
Liquid test
The 9 circuits were tested through plate reader as well. We set 5 temperature: 28℃, 35℃, 37℃, 40℃ and 42℃. After 36 h, when some replicates had changed color, we observed the Fluorescence(excitation wavelength-584 nm and emission wavelength-607 nm) and OD(600).
Unlike agar plate test, Four U(K115002) under the control of J23101 works best, and Four U(K115002) under the control of J23119 works far weaker than J23101+K115002. Here’s the results:
Measure RNAT under heat stress
Fluorescence of mCherry is a common indicator to indicate the switch efficiency of RNAT. However, most RNATs(ROSE for example) are derived from heat shock systems. Heat stress will cause protein unfolding, entanglement, and unspecific aggregation[24], which can interfere with protein expression. Thus, the fluorescence difference is the combined effect of RNAT and heat stress. Let’s use a formula to show these 2 factors and the indicator:
F stands for Fluorescence of mCherry
f1 stands for switch efficiency of RNAT
f2 stands for heat stress
We explored a new method to measure the switch efficiency: using 2 kinds of fluorescent proteins, a GFP to indicate the heat stress, while a RFP to indicate the combined effect.
The following circuits are controls:
Thermosensitive T7 RNA Polymerase
What is it?
Inspired by Liang’s work[6],we constructed the Thermosensitive T7 RNA Polymerase (TS T7 RNAP) by inserting a Thermosensitive intein(TS intein) into selected locus of T7 RNAP.
TS Intein is a polypeptide that can catalyze its own excision from the flanking polypeptides, or exteins, as well as ligation of the exteins at specific temperature[2](This process is also named “protein splicing”). In consequence, the interrupted T7 RNAP could only regain activity and transcribe gene under the control of T7 promoter at the permissive temperature(Fig.12.).
The mechanism of protein splicing[2]:
Step 1: The peptide bond linking the N-extein and intein is converted to a thioester or ester via nucleophilic attack by the N-terminal Cys or Ser of the intein.
Step 2: The N-extein is transferred from the side chain of the first intein residue to the side chain of the first C–extein residue (Cys, Ser or Thr) by transesterification, resulting in a branched ester intermediate .
Step 3: The branched ester is resolved by Asn cyclization coupled to peptide bond cleavage. This leaves the ligated exteins separated from the intein and linked by an ester bond, while the intein has a C-terminal aminosuccinimide.
Step 4: The ester bond connecting the ligated exteins is rapidly converted to the amide bond, and the C-terminal aminosuccinimide of the intein may be hydrolyzed .(Fig.13.)
Selected TS inteins
In Liang’s work, the TS intein from Saccharomyces cerevisiae vacuolar ATPase subunit (VMA) is active at low temperature(18℃), and dead at high temperature(37℃), which is contrary to our design. Consequently, we referred to literature to select TS inteins that active at high temperature and dead at low temperature, most of them from extreme thermophiles. The selected inteins are listed below:
Why select locus?
According to the mechanism of splicing for canonical inteins and many structural and mutational studies, extein sequences around the junctions affect the splicing activity significantly[8][10](Fig.14.).
Liang’s solution was to insert intein between Ala 491 and Cys 492, which is similar to VMA’s original extein context[6]. However, this solution can be limited when inserting other inteins into T7 RNAP: it’s difficult to find a locus similar to intein’s original extein context, while this locus happens to have significant influence on T7 RNAP’s activity(Fig.15.).
To offer a universal solution for inserting different kinds of inteins into T7 RNAP(which may become the foundation to construct T7 RNAP responses to different signals), we tried to insert inteins with several native residues of exteins[12] into selected locus of T7 RNAP(Fig.16.).
These locus must satisfy the following conditions(Fig.17.):
1. The inserted intein can function.
2. Before splicing, the inserted intein with residues could inactive T7 RNAP.
3. After splicing, the remaining residues of intein wouldn’t inactive T7 RNAP.
Selected locus
Since it’s difficult to predict intein’s function, and professor Liu told us that chances for inactive protein interrupted by 100~400 residues are pretty high, we concentrated more on condition 3. There are 2 main considerations for locus selection:
1. Avoid conserved sites and key sites for recognition, initiation and catalyzation.
2. Avoid α-helix or other important secondary structure, select loop region.
With the help of many papers[13~21,24] and PyMol, we selected 67N, 179K, 366D, 498E, 601N, 715T, 856S(Fig.18. Fig.19.).
How to ?
Construction of T7 expression platform
We designed a platform(Fig.20.) for T7 expression[21][22], which contains 2 circuits:
1. A T7 RNAP with UmuD tag under control of PBAD
2. GFP with LVA tag under control of PT7
We synthesized 3 segs and assemble them with BBa_I10500, BBa_I716103 using Gibson Assembly. Here are details for each seg:
(1) SEG1: Contains GFP with LVA tag under the control of PT7. (Fig.21.)
T7 expression is high level expression system, to exclude metabolic load[21], we employed GFP with LVA degradation tags, and a low-activity T7 promoter(according to literature, 21% activity[21]).
To avoid interference from upstream coding arac, we set a terminator BBa_B0015 before PT7, and a Ribo J after PT7[22]. RiboJ is a section of functional RNA which comprises the sTRSV- ribozyme which can excise its upstream fragment to reduce noise, and an additional 23-nt hairpin immediately downstream to help expose the RBS. It can be used to buffer synthetic circuits from genetic context[23].
Before construction, we measured efficiency of different PT7.
Here’s the result(Fig.22):
(2) SEG2: BBa_I10500, PBAD with coding sequence of arac, which can tightly control expression of T7 RNA polymerase.(Fig.23.)
Before construction, we tested the best induction concentration of PBAD.
(3) SEG3: Ribo J, RBS, UmuD degradation tag(Fig.24.)
This SEG is to fill the gap between PBAD and T7 RNA polymerase, and fuse a UmnD degradation tag.
(4) SEG4: BBa_I716103, T7 RNA polymerase(Fig.25.)
(5) SEG5: Terminator(Fig.26.)
Since we employ Gibson Assembly to assemble the above 5 parts, we synthesized SEG1, SEG3, SEG5 with 40bp sequences overlapped with their flanking segs(Fig.27.) and pSB1C3.
Insertion of intein
After the construction of T7 expression platform, we digested the platform using the native restriction site Nru I and Mfe I, with the remaining part of T7 RNAP overlapping sequences for Gibson Assembly. We amplified N-T7 and C-T7 divided from selected locus by PCR, and amplified intein with 20bp overlapping sequences with the flanking N-T7 and C-T7 by PCR.
References
[1] Klinkert B, Narberhaus F. Microbial thermosensors.[J]. Cellular & Molecular Life Sciences, 2009, 66(16):2661-2676.
[2] Arosio P, Ingrassia R, Cavadini P. Ferritins: A family of molecules for iron storage, antioxidation and more[J]. Biochimica et Biophysica Acta (BBA) - General Subjects, 2009, 1790(7):589–599.
[3] Gilles C, Bonville P, Rakoto H, et al. Magnetic hysteresis and superantiferromagnetism in ferritin nanoparticles[J]. Journal of Magnetism & Magnetic Materials, 2002, 241:430–440.
[4] Huang H, Delikanli S, Hao Z, et al. Remote control of ion channels and neurons through magnetic-field heating of nanoparticles.[J]. Nature Nanotechnology, 2010, 5(8):602-606.
[5] Kortmann J, Narberhaus F. Bacterial RNA thermometers: molecular zippers and switches[J]. Nature Reviews Microbiology, 2012, 10(4):255-265.
[6] Liang R, Liu X, Liu J, et al. A T7-expression system under temperature control could create temperature-sensitive phenotype of target gene in Escherichia coli.[J]. Journal of Microbiological Methods, 2007, 68(3):497-506.
[7] Topilina N I, Mills K V. Recent advances in in vivo applications of intein-mediated protein splicing.[J]. Mobile Dna, 2014, 5(1):804-804.
[8] Ellilä S, Jurvansuu J M, Iwaï H. Evaluation and comparison of protein splicing by exogenous inteins with foreign exteins in Escherichia coli.[J]. Febs Letters, 2011, 585(21):3471-3477.
[9] Mills K V, Manning J S, Garcia A M, et al. Protein splicing of a Pyrococcus abyssi intein with a C-terminal glutamine[J]. Journal of Biological Chemistry, 2004, 279(20): 20685-20691.
[10] Du Z, Liu J, Albracht C D, et al. Structural and mutational studies of a hyperthermophilic intein from DNA polymerase II of Pyrococcus abyssi[J]. Journal of Biological Chemistry, 2011, 286(44): 38638-38648.
[11] Xu M Q, Perler F B. The mechanism of protein splicing and its modulation by mutation[J]. The EMBO journal, 1996, 15(19): 5146.
[12] Mills K V, Connor K R, Dorval D M, et al. Protein purification via temperature-dependent, intein-mediated cleavage from an immobilized metal affinity resin.[J]. Analytical Biochemistry, 2006, 356(1):86-93.
[13] Sousa R, Chung Y J, Rose J P, et al. Crystal structure of bacteriophage T7 RNA polymerase at 3.3 Å resolution[J]. Nature, 1993, 364(6438): 593-599.
[14] Durniak K J, Bailey S, Steitz T A. The structure of a transcribing T7 RNA polymerase in transition from initiation to elongation[J]. Science, 2008, 322(5901): 553-557.
[15] Yin Y W, Steitz T A. The structural mechanism of translocation and helicase activity in T7 RNA polymerase[J]. Cell, 2004, 116(3): 393-404.
[16] Jeng S T, Gardner J F, Gumport R I. Transcription termination by bacteriophage T7 RNA polymerase at rho-independent terminators[J]. Journal of Biological Chemistry, 1990, 265(7): 3823-3830.
[17] Kinetic S, Middaughl C R, Woodyl R W, et al. Bacteriophage T7 RNA Polymerase and Its Active-site Mutants[J]. J. Mol. Biol, 1994, 231: 5-19.
[18] Sousa R. Fundamental aspects of T7 RNA polymerase structure and mechanism[M]//Mechanisms of Transcription. Springer Berlin Heidelberg, 1997: 1-14.
[19] Temiakov D, Patlan V, Anikin M, et al. Structural basis for substrate selection by T7 RNA polymerase[J]. Cell, 2004, 116(3): 381-391.
[20] Tunitskaya V L, Kochetkov S N. Structural–functional analysis of bacteriophage T7 RNA polymerase[J]. Biochemistry (Moscow), 2002, 67(10): 1124-1135.
[21] Schaerli Y, Munteanu A, Gili M, et al. A unified design space of synthetic stripe-forming networks[J]. Nature Communications, 2014, 5:4905-4905.
[22] Segall-Shapiro T H, Meyer A J, Ellington A D, et al. A‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase[J]. Molecular Systems Biology, 2014, 10(7):742-742.
[23] Lou C, Stanton B, Chen Y J, et al. Ribozyme-based insulator parts buffer synthetic circuits from genetic context[J]. Nature biotechnology, 2012, 30(11): 1137-1142.
[24] Richter K, Haslbeck M, Buchner J. The heat shock response: life on the verge of death[J]. Molecular cell, 2010, 40(2): 253-266.
