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Team:Slovenia/Parts
From 2012.igem.org
Contribution to iGEM community
We have physicaly deposited 89 new BioBrick parts to the Registry. All of them were cloned into the standard pSB1C3 vector. We extensively documented performance of final composite parts into which the parts were implemented.
Most of our new parts work as expected. Experimental results showing the expected performance of those parts were entered in the "Main page" sections of the appropriate Registry entries.
We have improved the function of an existing BioBrick part from Registry, which was the NicTAL DNA binding domain (BBa_K323214) designed by iGEM team Slovenia 2010. This part was designed for binding to the Tet operator. The original part was not functional and could not efficiently bind to the expected DNA-binding site because at that time, more than two years ago, it had not been known which subdomains of TAL are required for the efficient binding to DNA. We identified the problem, added a missing subdomain and prepared a new part that was fully functional. We compared the performance of the old version of the NicTAL10 DNA binding domain (BBa_K323214) and new version of NicTAL12 DNA-binding domain (BBa_K782007). We entered this information to the registry entry of the improved part (BBa_K782007). The original NicTAL10 was also poorly characterized, so we additionally characterized the part already deposited in the Registry (BBa_K323214), preventing someone to use the nonfunctional part.
iGEM team Slovenia 2012 also improved the functionality of part "mouse guanylate kinase - thymidine kinase TK30 fusion protein" (BBa_K404113) for other teams. This part was previously deposited as a coding only sequence without mammalian promoter. Our team added the CMV promoter for application in mammalian cells. New part was deposited as BBa_K782063. New part was experimentally characterized in mammalian cell line HEK293. Results of experimental characterization are presented in the experience section of the old part (BBa_K404113:Experience) and on main page of new part (BBa_K782063).
Favourite parts
Natural
Interferon alpha2 (BBa_K782060) is a cytokine that is used for treatment of chronic hepatitis C. In our system we used interferon alpha2 as an effector protein produced by microencapsulated mammalian cells. We characterized the activity and measured the amount of interferon alpha2 produced in HEK293T cells. The experimental characterization is found on the part's main page (BBa_K782060). On our hepatitis C site you can find more about the way we incorporated interferon alpha2 into our project.
Synthetic
10x[TALA] operator_minimal promoter_TALA:NLS:VP16_t2a_BFP (BBa_K782085). This part is one of the four plasmids of the genetic bistable toggle switch with a positive feedback loop. It comprises an operator for the designed DNA binding protein TALA fused to the activator domain VP16. In frame with the designed activator the T2A sequence links the reprter blue fluorescent protein (BFP), which ensures the equimolar production of the activator and reporter protein. This part can also be used as the autoloop for the amplifier of signal in genetic circuits. Part is exhaustively described in the Registry (BBa_K782085) and on the page on the Positive feedback loop bistable switch.
Submitted parts ordered according their function
New TAL effector based DNA-binding domains
| TALA:NLS DNA binding domain | BBa_K782004 |
| TALD:NLS DNA binding domain | BBa_K782005 |
| TALB:NLS DNA binding domain | BBa_K782006 |
| NicTAL12:NLS DNA binding domain | BBa_K782007 |
New TAL effector based repressors
Normal
| TALA:NLS:KRAB | BBa_K782008 |
| TALD:NLS:KRAB | BBa_K782009 |
| TALB:NLS:KRAB | BBa_K782010 |
| NicTAL12:NLS:KRAB | BBa_K782011 |
Fast degradable
| TALA based fast degradable KRAB (CL1-PEST tag) | BBa_K782040 |
| TALD based fast degradable KRAB (CL1-PEST tag) | BBa_K782041 |
| TALB based fast degradable KRAB (CL1-PEST tag) | BBa_K782042 |
New TAL effector based activators
| TALD:NLS:VP16 | BBa_K782012 |
| TALB:NLS:VP16 | BBa_K782013 |
| TALA:NLS:VP16 | BBa_K782065 |
| NicTAL12:NLS:VP16 | BBa_K782066 |
TAL effector controlable reporters
| 7x[NicTAL]+7x[TALD] operator_CMV promoter_mCitrine | BBa_K782000 |
| 2x[NicTAL]+2x[TALD] operator_CMV promoter_mCitrine | BBa_K782001 |
| 4x[NicTAL]+4x[TALD] operator_CMV promoter_mCitrine | BBa_K782002 |
| 1x[NicTAL]+1x[TALD] operator_CMV promoter_mCitrine | BBa_K782003 |
| 12x[TALD] operator_CMV promoter_mCitrine | BBa_K782014 |
| 12x[NicTAL] operator_CMV promoter_mCitrine | BBa_K782015 |
| 10x[TALA+TALB] operator_CMV promoter_mCitrine | BBa_K782016 |
| 10x[TALA] operator_CMV promoter_mCitrine | BBa_K782017 |
| 10x[TALB] operator_CMV promoter_mCitrine | BBa_K782018 |
| 2x[NicTAL]+2x[TALD] operator_minimal promoter_mCitrine | BBa_K782024 |
| 4x[NicTAL]+4x[TALD] operator_minimal promoter_mCitrine | BBa_K782025 |
| 7x[TALA+TALB] operator_minimal promoter_mCitrine | BBa_K782026 |
| 7x[TALA] operator_minimal promoter_mCitrine | BBa_K782027 |
| 10x[TALA+TALB] operator_minimal promoter_mCitrine | BBa_K782028 |
| 10x[TALA] operator_minimal promoter_mCitrine | BBa_K782029 |
| 10x[TALB] operator_minimal promoter_mCitrine | BBa_K782030 |
| 12x[TALD] operator_minimal promoter_mCitrine | BBa_K782031 |
| 10x[TALA+TALB] operator_CMV promoter_fLuciferase | BBa_K782019 |
| 10x[TALA] operator_CMV promoter_fLuciferase | BBa_K782021 |
| 10x[TALB] operator_CMV promoter_fLuciferase | BBa_K782022 |
| 12x[NicTAL] operator_CMV promoter_fLuciferase | BBa_K782023 |
| 10x[TALA+TALB] operator_minimal promoter_fLuciferase | BBa_K782032 |
| 10x[TALA] operator_minimal promoter_fLuciferase | BBa_K782033 |
| 10x[TALB] operator_minimal promoter_fLuciferase | BBa_K782034 |
| 12x[TALD] operator_minimal promoter_fLuciferase | BBa_K782035 |
| 10x[TALA+TALB] operator_CMV promoter_BFP | BBa_K782073 |
| 10x[TALA] operator_CMV promoter_BFP | BBa_K782074 |
| 10x[TALB] operator_CMV promoter_BFP | BBa_K782075 |
| 12x[TALD] operator_CMV promoter_BFP | BBa_K782076 |
| 12x[NicTAL] operator_CMV promoter_BFP | BBa_K782077 |
| 10x[TALA+TALB] operator_CMV promoter_2xBFP:NLS | BBa_K782078 |
| 10x[TALA] operator_CMV promoter_2xBFP:NLS | BBa_K782079 |
| 10x[TALB] operator_CMV promoter_2xBFP:NLS | BBa_K782080 |
| 12x[TALD] operator_CMV promoter_2xBFP:NLS | BBa_K782081 |
| 12x[NicTAL] operator_CMV promoter_2xBFP:NLS | BBa_K782082 |
Parts for bistable Switch construction
| 10x[TALA+TALB] operator_CMV promoter_TALD:NLS:KRAB | BBa_K782036 |
| 10x[TALA] operator_CMV promoter_TALD:NLS:KRAB | BBa_K782037 |
| 10x[TALA] operator_CMV promoter_TALB:NLS:KRAB | BBa_K782038 |
| 10x[TALB] operator_CMV promoter_TALD:NLS:KRAB | BBa_K782039 |
| 12x[TALD] operator_CMV promoter_TALB:NLS:KRAB | BBa_K782064 |
| 2x[NicTAL]+2x[TALD] operator | BBa_K782067 |
| 4x[NicTAL]+4x[TALD] operator | BBa_K782068 |
| 10x[TALA+TALB] operator | BBa_K782069 |
| 10x[TALA] operator | BBa_K782070 |
| 10x[TALB] operator | BBa_K782071 |
| 12x[TALD] operator | BBa_K782072 |
| 10x[TALB] operator_CMV promoter_TALA:KRAB:NLS_t2a_mCitrine | BBa_K782083 |
| 10x[TALA] operator_CMV promoter_TALB:NLS:KRAB_t2a_mNeptune | BBa_K782084 |
| 10x[TALA] operator_minimal promoter_TALA:NLS:VP16_t2a_BFP | BBa_K782085 |
| 10x[TALA+TALB] operator_minimal promoter | BBa_K782086 |
| 10x[TALA] operator_minimal promoter | BBa_K782087 |
| 10x[TALB] operator_minimal promoter | BBa_K782088 |
Therapeutic effectors
| Interferon a2 | BBa_K782060 |
| VEGF-p2a-PDGF | BBa_K782061 |
Safety
| CMV promoter – mouse guanylate kinase_thymidine kinase 30 | BBa_K782063 |
| Alginate lyase | BBa_K782059 |
| dCBD Alginate lyase (mature variant) | BBa_K782062 |
System for subcloning of nonstandard genes into BioBrick vectors
We have designed a new system for cloning of genes with non-standard restriction sites at 5' and 3' ends into BioBrick vectors. Before our system was introduced cloning a gene from a non BioBrick vector with a different multi cloning site into a BioBrick vector with standardized multi cloning site was time consuming and expensive. If we wanted to clone a gene that was in a vector with noncompatible restriction sites into a BioBrick vector we previously had to perform a PCR reaction with which we would add overhangs with appropriate restriction sites. We would then have to digest our PCR product with restriction enzymes and ligate it into a BioBrick vector.
With the system we designed it is now possible to digest your gene from almost any plasmid with non-standard multi cloning site with several combinations of restriction enzymes and then insert it into a BioBrick vector using just ligation reaction. We can therefore skip the problematic, time consuming and nonreliable PCR reaction of your gene and thus avoid the possibility of new point mutations in your product.
The strategy is based on type II restriction enzyme BsaI. Target vectors are composed of standard BioBrick restriction sites and ccdB expression cassette. In the process of preparing target vectors ccdB was cloned into vector together with kanamycin resistance marker in order to simplify the cloning, but kanamycin resistance has no significant role in the final constructs. CcdB-KanR expression cassette is flanked with BsaI restriction sites. After BsaI restriction the target vector is digested into backbone with standard BioBrick restriction sites and any desired 4 nucleotide overhang. That means that any fragment can be cloned in-between standard BioBrick restriction sites. Both cutting sites of BsaI restriction enzyme are designed in a way that original restriction sites flanking the insert in original vector are never reconstituted. So someone can even use BioBrick restriction enzyme for subcloning of desired genes into BioBrick standard vector.
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| Figure 1. Scheme presenting new system for subcloning of non standard genes into BioBrick vector. Using series of vectors that we have prepaired and deposited to Registry, someone can subclone desired genes from almoust any available vector to BioBrick standard vector pSB1C3. |
We prepaired different standard vectors for subcloning of non standard genes into BioBrick format. With so far prepared vectors 16 different restriction enzyme combinations can be used to subclone genes into BioBrick format.
| EcoRI/BamHI target vector for insertion of non standardized parts | BBa_K782043 |
| EcoRI/XbaI target vector for insertion of non standardized parts | BBa_K782044 |
| EcoRI/SacI target vector for insertion of non standardized parts | BBa_K782045 |
| HindIII/BamHI target vector for insertion of non standardized parts | BBa_K782046 |
| HindIII/XbaI target vector for insertion of non standardized parts | BBa_K782047 |
| HindIII/XhoI target vector for insertion of non standardized parts | BBa_K782048 |
| HindIII/SacI target vector for insertion of non standardized parts | BBa_K782049 |
| BamHI/XbaI target vector for insertion of non standardized parts | BBa_K782050 |
| BamHI/XhoI target vector for insertion of non standardized parts | BBa_K782051 |
| BamHI/SacI target vector for insertion of non standardized parts | BBa_K782052 |
| XbaI/BamHI target vector for insertion of non standardized parts | BBa_K782053 |
| XbaI/XhoI target vector for insertion of non standardized parts | BBa_K782054 |
| XbaI/SacI target vector for insertion of non standardized parts | BBa_K782055 |
| SacI/BamHI target vector for insertion of non standardized parts | BBa_K782056 |
| SacI/SpeI target vector for insertion of non standardized parts | BBa_K782057 |
| SacI/XhoI target vector for insertion of non standardized parts | BBa_K782058 |
Nondeposited genes
We could not prepare parts for some effectors since they contained numerous restriction sites for the restrictases used for cloning according to the BioBrick standards such as MICA (3xPstI) and anakinra (1xXbaI and 1xSpeI).
We did not deposit the plasmids containing components of the erythromycin and pristinamycin induction systems since we obtained them under the MTA that prevents further distribution.
