(Difference between revisions)

iGEM 2012

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 alpha-2a	BBa_K782060
VEGF-p2a-PDGF	BBa_K782061

Safety

CMV promoter – mouse guanylate kinase_thymidine kinase 30	BBa_K782063
Alginate lyase	BBa_K782059
ΔCBD 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.

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 equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782043
EcoRI/XbaI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782044
EcoRI/SacI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782045
HindIII/BamHI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782046
HindIII/XbaI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782047
HindIII/XhoI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782048
HindIII/SacI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782049
BamHI/XbaI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782050
BamHI/XhoI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782051
BamHI/SacI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782052
XbaI/BamHI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782053
XbaI/XhoI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782054
XbaI/SacI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782055
SacI/BamHI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782056
SacI/SpeI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	BBa_K782057
SacI/XhoI equivalent multicloning site for insertion of non standardized parts to BioBrick vectors	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.