Team:UCAS-China/Part Collection

PART COLLECTION

OVERVIEW

This year, our project collected a series of thermo-sensitive switches with high-performance, versatility and robustness.

Our switches are divided into two types: cold-inducible and heat-inducible ON-switches.

In synthetic biology, many heat-inducible switches have been well researched and described. We explore and choose TCI transcription repressor family and TlpA family whose activation is regulated by temperature. They are all temperature sensitive mutants. The structure of the corresponding transcription factors changes with the temperature’s change. We put our target gene under the promoter of TCI family and TlpA family. When it is above threshold temperature, transcriptional factors will be allosteric, losing their activity. Then the inhibition of target gene is relieved while the target gene is expressed.

Besides heat-inducible ON-switch, we also create a set of cold-inducible ON-switches which fill the vacancies of thermo-sensitive switches in synthetic biology. With the thermo-sensitive parts provided by our PI, we designed a circuit combining proteases with transcriptional factors to build a bistable switch. We put the coding region of protease under the promoter of the transcriptional factor while inserting the recognition site of the protease into the linking structuring of the transcriptional factor so that the two parts can inhibit each other. Therefore, once there is a small disturbance caused by change of temperature, the balance will lean to one side to act. To prove the function of our switches, we add reporter gene sfGFP downstream and fully characterized the performance of them through fluorescence expression in both TOP10 strain and Nissle 1917 which is a probiotic with more than 100 years of medical application. Most of them showed good robustness and high performance. Some can even reach more than 100-fold change under different temperatures.

What’s more, to expand the applications and function of thermo-sensitive switches, we integrated the cold- and heat-inducible circuit to build a double-status switch which can turn on different genes’ expression at different temperatures because these two kinds of switch can work independently. By choosing the different kinds of parts in our part collection, we can achieve the function of cold-inducible ON-switch, heat-inducible ON-switch, and double-status switch. Also, by choosing different parts of the same type, we can get thermo-sensitive switch of different transition temperature.

Our parts collection shows high performance and versatility, which ensure the potential for basic research, as well as industrial and biomedical applications, and truly makes engineered bacteria precisely under control.

PARTS OF HEAT-INDUCIBLE ON -SWITCHES

Parts Name Number Description
TCI BBa_K2969021 A temperature-sensitive variant of the bacteriophage λ repressor CI.
TCI38 BBa_K2969022 A temperature-sensitive variant of the bacteriophage λ repressor CI.
J23119-TCI-pL(3)-pR(3)-mRFP BBa_K2969041 This part contains the coding gene of the thermo-sensitive transcription factor TCI under the constitutive promoter J23119 and a reporter gene mRFP under the promoter of TCI. When the temperature is below 35℃, TCI is active to repress the transcription of mRFP so that there will be little fluorescence.
J23119-TCI42-pL(3)-pR(3)-mRFP BBa_K2969042 This part contains the coding gene of the thermo-sensitive transcription factor TCI42 under the constitutive promoter J23119 and a reporter gene mRFP under the promoter of TCI. When the temperature is below 37℃, TCI42 is active to repress the transcription of mRFP so that there will be little fluorescence.
J23119-TlpA36-pTlpA-mRFP BBa_K2969043 This part contains the coding gene of the thermo-sensitive transcription factor TlpA36 under the constitutive promoter J23119 and a reporter gene mRFP under the promoter of TlpA36 pTlpA. When the temperature is below the transition temperature of TlpA36, TlpA is active to repress the transcription of mRFP so that there will be little fluorescence.

PARTS OF COLD-INDUCIBLE ON-SWITCHES

Parts Name Number Description
TEV protease BBa_K2969000 A highly specific cysteine protease from Tobacco Etch Virus that recognizes the amino-acid sequence Glu-Asn-Leu-Tyr-Phe-Gln-(Gly/Ser) and cleaves between the Gln and Gly/Ser residues. It is often used for the removal of affinity purification tags such as maltose-binding protein (MBP) or poly-histidine from fusion proteins.
TEVts6# BBa_K2969001  A thermo-sensitive mutation type of TEV protease.
TEVts7# BBa_K2969002 A thermo-sensitive mutation type of TEV protease.
TEVts11# BBa_K2969003 A thermo-sensitive mutation type of TEV protease.
TEVts17# BBa_K2969004 A thermo-sensitive mutation type of TEV protease.
TEVts18# BBa_K2969005 A thermo-sensitive mutation type of TEV protease.
TVMV protease BBa_K2969006 A protease from Tobacco Vein Mottling Virus which can recognize a linear epitope of the general form T_V_R_F_Q / S with cleavage occurring between Q and S
SuM-MV protease BBa_K2969007 A protease from Sunflower Mild Mosaic Virus which can recognize special amino acids sequence EEIHLQ and cut the protein at special site.
HRV-3C protease BBa_K2969008 A protease from Human Rhinovirus 3C which can recognize a special amino acids sequence and cut the protein at special site.
CI-TEVsite BBa_K2969009 An engineered CI transcription factor which inserts the TEV protease recognition sequence and can be recognized and cut by TEV protease.
CI-TVMVsite BBa_K2969010 An engineered CI transcription factor which inserts the TVMV protease recognition sequence and can be recognized and cut by TVMV protease .
CI-SuMMVsite BBa_K2969011 An engineered CI transcription factor which inserts the SuMMV protease recognition sequence and can be recognized and cut by SuMMV protease.
CI-HRV3Csite BBa_K2969012 An engineered CI transcription factor which inserts the HRV3C protease recognition sequence and can be recognized and cut by HRV3C protease.
CI434-TEVsite BBa_K2969013 An engineered CI434 transcription factor which inserts the TEV protease recognition sequence and can be recognized and cut by TEV protease.
CI434-TVMVsite BBa_K2969014 An engineered CI434 transcription factor which inserts the TVMV protease recognition sequence and can be recognized and cut by TVMV protease.
CI434-SuMMVsite BBa_BBa_K2969015 An engineered CI434 transcription factor which inserts the SuMMV protease recognition sequence and can be recognized and cut by SuMMV protease.
CI434-HRV3Csite BBa_K2969016 An engineered CI434 transcription factor which inserts the HRV3C protease recognition sequence and can be recognized and cut by HRV3C protease.
HKCI BBa_K2969017 A transcription factor of CI family from the lambdoid phage HK022.
P22C2 BBa_K2969018 A transcription factor of CI family from the temperate lambdoid bacteriophage P22.
TP901 CI BBa_K2969019 A transcription factor of CI family from the temperate lambdoid phage 901-1.
CI434ts-TEVsite BBa_K2969020 CI434ts is a thermo-sensitive mutation type of CI434 transcription factor.This is an engineered CI434 transcription factor which inserts the TEV protease recognition sequence and can be recognized and cut by TEV protease.
mf-Lon BBa_K2969031 A protease from Mesoplasma florum bacteria which can recognize certain tags and degrade proteins which is similar to the E.coli lon protease.
pCI434 BBa_K2969033 The promoter of CI434 which can combine with it to prevent the expression of downstream genes when there exists the active transcription factor CI434.
pCI434-TEVts6# BBa_BBa_K2969040 This part contains the coding gene of TEVts6# which can code the thermo-sensitive protease TEVts6# under the promoter of CI434. When there exists the active transcription factor CI434, it will combine with the promoter of CI434 pCI434 to prevent enzymes combining with DNA, so the transcription of TEVts6# will be inhibited.
pTetO(wt)-mfLon BBa_K2969045 This part contains the coding gene of the protease mf-Lon under the promoter pTet. When there exists the transcription factor TetR, the transcription of mf-Lon will be inhibited. Mf-Lon is a protease from Mesoplasma florum bacteria (called mf-Lon for short). While it is evolutionarily related, and mechanistically similar to the E. coli Lon protease, mf-Lon is unable to recognize E. coli degradation tags (commonly known as ssRA tags), and vice versa. So if a protein contains mf-Lon degradation tag, mf-Lon will recognize the protein and degrade it.
pCI434-doc BBa_K2969046 This part contains the coding gene of toxin doc protein under the promoter of CI434 pCI434. When there exists the repressor CI434, the transcription of doc will be inhibited. But when the doc gene is expressed, the growth of the host will be repressed and a self-kill mechanism will be started.
pCI434-doc-J23119-CI434ts-TEVsite BBa_K2969047 This part combines the composite part pCI434-doc BBa_K2969046 with the transcriptional factor CI434ts-TEVsite BBa_K2969020.

PARTS OF DOUBLE-STATUS SWITCHES

Parts Name Number Description
pCI434-TEVts6#-TCI42-pL(3)-pR(3)-mRFP BBa_K2969044 This part contains TEVts6# and TCI42 which can encode the thermo-sensitive protease TEVts6# and thermo-sensitive transcription factor under the promoter pCI434 and pCI correspondingly. Therefore, this part can act as both a cold-inducible ON-switch(37℃) and a heat-inducible ON-switch(42℃). with this part, we can construct a double-status switch, which can turn on different genes‘ expression at different temperatures. This switch can respond to a narrow temperature range(<10℃) rapidly also with high fold-change(~100-fold). Its high performance and versatility ensure the potential for basic research, as well as industrial and biomedical applications.