Team:OUC-China/Project/Thermosensitive Regulator

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Thermosensitive regulator

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 respond to sudden temperature changes measure 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. all, 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, only direct 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.

Fig.1. Genetic targeting of nanoparticles to specific cells and localized membrane heating. a–d, Microscopy images showing a group of HEK 293 cells, two of which are expressing Golgi-targeted GFP and the biotinylated membrane protein AP-CFP-TM (ref. 19). Differential interference contrast (DIC) image displaying all cells (a), green fluorescence image indicating the Golgi localized GFP (b), cyan fluorescence marking the membrane protein AP-CFP-TM (c), red fluorescence of the DyLight549 (d) on the nanoparticles, which are exclusively localized on the plasma membrane of the AP-CFP-TM expressing cells. Scale bar, 20 mm. e, During application of the RF magnetic field (t?30–45 s as indicated by the hatched box), the local temperature increased at the plasma membrane (red, measured by the change in DyLight549 fluorescence intensity), yet remained constant at the Golgi apparatus (green, measured by fluorescence intensity of Golgi-targeted GFP).[4]

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 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, DNA 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.

Fig.2. Scheme of 9 circuits: 3 promoters X 3 RNATs
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.).

Fig.3. Sketch map of the distribution of different group

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.).

Fig.4. All groups
Fig.5. The nine
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 calculated 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:

Fig.6. Fluorescence intensity for 9 circuits

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, and a RFP to indicate the combined effect.

Fig.8. Circuit for measure switch efficiency of FourU under heat stress

The following circuits are controls:

Fig.9. control for measure switch efficiency of FourU under heat stress

Fig.10. control for measure switch efficiency of FourU under heat stress

Fig.11. control for measure switch efficiency of FourU under heat stress

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.).

Fig.12. Schematic of thermosensitive T7 RNA polymerase

The mechanism of protein splicing[2]:

Fig.13. 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.).

Fig.14. extein sequences around the junctions affect the splicing activity significantly

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.).

Fig.15. Liang’s solution

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.).

Fig.16. Our solution

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.

Fig.17. Conditions for locus

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.).

Fig.18. Selected locus-front
Fig.19. Conditions for locus-right

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

Fig.20. Circuit of T7 platform

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].

Fig.21. SEG1: GFP under the control of PT7

Before construction, we measured efficiency of different PT7.
Here’s the result(Fig.22):

Fig.22. SEG2: PBAD with coding sequence of aracT7

(2) SEG2: BBa_I10500, PBAD with coding sequence of arac, which can tightly control expression of T7 RNA polymerase.(Fig.23.)

Fig.23. SEG2: PBAD with coding sequence of aracT7

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.

Fig.24. SEG3: Ribo J, RBS, UmuD degradation tag

(4) SEG4: BBa_I716103, T7 RNA polymerase(Fig.25.)

Fig.25. SEG4: T7 RNA polymerase

(5) SEG5: Terminator(Fig.26.)

Fig.26. SEG5: Terminator

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.

Fig.27. SEG5: Terminator

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.

Fig.28. schema of intein insertion

References

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