Team:UCAS-China/Composite Part
PART OVERVIEW
This year, we developed a collection of thermosensitive parts with high-performance, versatility and robustness.
Based on TCI transcription factor family and TlpA family, we collected some heat-inducible ON-switches, which can open the gene expression under high temperature. More importantly, we developed a series of cold-inducible ON-switches which are active under low temperature and inactive when temperature rises.
We fully characterized the performance of them in both Top10 strain and Nissle 1917, a probiotic with more than 100 years of medical application and most of them show robustness and high performance, some can even reach more than 100 fold-change under different temperatures.
We also constructed a double-status switch with two kinds of switches, which can turn on different genes‘ expression at different temperatures.
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 controlled.
BASIC PART
BEST BASIC PART CANDIDATE: TEVts18# , BBa_K2969005
TEVts18# , BBa_K2969005, is our best basic part this year which encodes a thermo-sensitive mutant of the TEV protease protein and serves as the important regulation part of our cold-inducible ON-switches to apply to the microbial therapy. It is created through an evolutionary strategy which use temperature as a screening condition. ‘ts’ means thermo-sensitive while 18# is the number to help differentiate it from other mutation types. When the temperature is between 35℃ and 40℃, the protease will gradually lose activity. When it is above 40℃, the activity will become less than the 1/100 of the activity below 35℃.
All basic parts are all listed in the table below.
| 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. |
| 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. |
| Doc | BBa_K2969023 | A toxin protein which can resulte in rapid cell growth arrest and marked inhibition of translation without significant perturbation of transcription or replication. |
| Phd | BBa_K2969024 | A protein which can inhibit the function of toxin protein and prevent host death. |
| PheOH | BBa_K2969025 | An key enzyme which catalyzes the hydroxylation of the aromatic side-chain of phenylalanine to generate tyrosine. |
| TyrOH | BBa_K2969026 | A rate-limiting enzyme which transforms Tyr into Dopa. |
| sfGFP-Tyr151 | BBa_K2969027 | sfGFP-Tyr151 is transformed from the reporting part sfGFP. The tyrosine residue at 151 site is mutated to TAG. |
| sfGFP-Asn39-Tyr151 | BBa_K2969028 | sfGFP-Asn39-Tyr151 is transformed from the reporting part sfGFP. The asparagine residue at 39 site and tyrosine residue at 151 site are both mutated to TAG. |
| sfGFP-Asn39-Tyr151-Tyr181 | BBa_K2969029 | sfGFP-Asn39-Tyr151-Tyr181 is transformed from the reporting part sfGFP. The asparagine residue at 39 site and tyrosine residue at 151 site and 181 site are all mutated to TAG. |
| sfGFP-pdt#4 | BBa_K2969030 | sfGFP-pdt4# is transformed from the reporting part sfGFP. Pdt4# is a degradation tag which can be recognized by protease mf-Lon from Mesoplasma florum bacteria. When there exists the protease mf-Lon, it will recognize sfGFP with tag pdt4# and degrade the protein. |
| 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. |
| mutTyrRS | BBa_K2969032 | MutTyrRS can encode aminoacyl tRNA synthase, which is used to insert the non-natural amino acid dopa into proteins. |
| 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. |
| pL(3) | BBa_K2969034 | The promoter of CI which consists of 3 operators(DNA binding sites) OL1, OL2, OL3. |
| pR(3) | BBa_K2969035 | The promoter of CI which consists of 3 operators(DNA binding sites) OR1,OR2,OR3. |
| PfnrS) | BBa_K2969036 | An anaerobic-inducible promoter. |
| CD47 nb | BBa_K2969037 | CD47 nb nanobody is the antibody of CD47. It can bind CD47 and inhibit the growth of the tumor cells. |
COMPOSITE PART
BEST COMPOSITE PART CANDIDATE: 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.
All composite parts are all listed in the table below.
| Parts Name | Number | Description |
|---|---|---|
| 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. |
| 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. |
| 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. |
| 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. |
| J23119-CI434ts-TEVsite-sfGFP | BBa_K2969050 | This part contains the coding gene of the thermo-sensitive transcription factor CI434ts-TEVsite under the constitutive promoter J23119 and a reporter gene sfGFP under the promoter of CI434ts. When the CI434ts is inactivated as the temperature changes, the inhibition of sfGFP expressing will be erased. |
