Difference between revisions of "Part:BBa K2656200"

Revision as of 02:08, 18 October 2018

pBIOBRICKATOR

BBa_K2656200 (pBioBrickator backbone) is a modification of the pSB1C3 backbone, so being able to receive transcriptional units previously assembled in a [http://2018.igem.org/Team:Valencia_UPV/Design#Level1 GB alpha 1] vector.

To do so, it includes two divergently orientated BsmBI Type IIS recognition sites, whose cleavage leaves equivalent overhangs to those of the alpha 1 BsmBI digestion (resulting overhangs can be seen in Figure 1). Thus, this vector can be used to switch any transcriptional unit inserted in an alpha 1 GB vector into a BioBrick compatible one, so adapting the composite part into the BB grammar with an easy GB one-pot reaction.

To carry out the positive transformants screening method, common BBa_P1050 plasmid relies on the white-blue screening. It is based on the α-complementation, where the host strain must carry the ω-peptide while the vector includes the α-peptide sequence. Thus, if both subunits are expressed (when there is not insertion in the vector), functional beta-galactosidase is reconstituted. However, the blue/white colors depends of the subsequent addition of X-gal (D-lactose analog) and IPTG substances into the agar plates. Although the principle is simple, the method requires the strict use of lacZ mutant strains, and it is always necessary to add both chemical reagents into the medium.

In order to avoid these limitations, we have decided to rely on a different visual method. Thus, we have inserted an mRFP1 transcriptional unit [http://2018.igem.org/Team:Valencia_UPV/Design#LevGB BBa_BBa_K2656201] to act as the screening gene marker. With this approach, cloning is achieved in a more reliable, easy, low-cost way, so avoiding the necessity of adding chemical supplements and the possible non-function of these chemicals once on the solid medium. With this screening method, non-positive transformants will always express the a red fluorescent intense protein, easing the correct check.

As a proof of functioning, the pBioBrickator backbone has been used to adapt each of the composite parts from our [http://2018.igem.org/Team:Valencia_UPV/Part_Collection UPV iGEM 2018 Printeria Part Collection] into the BioBrick standard.

Figure 1. Transcriptional unit assembled in an alpha 1 GoldenBraid vector (KmR). Digestion with BsmBI Type IIS endonuclease leaves complementary overhangs (5'-GGAG-3' and 3'-CATG-5') to the pBioBrickator vector digest.

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found at 5
Plasmid lacks a suffix.
Illegal SpeI site found at 921
Illegal PstI site found at 935
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5
Illegal NheI site found at 44
Illegal NheI site found at 67
Illegal SpeI site found at 921
Illegal PstI site found at 935
Illegal NotI site found at 11
Illegal NotI site found at 928
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 5
Illegal XhoI site found at 1952
Illegal XhoI site found at 2844
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 5
Illegal suffix found at 921
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 5
Plasmid lacks a suffix.
Illegal XbaI site found at 20
Illegal SpeI site found at 921
Illegal PstI site found at 935
Illegal AgeI site found at 653
Illegal AgeI site found at 765
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.

@@ Line 7: / Line 7: @@
 To do so, it includes two divergently orientated BsmBI Type IIS recognition sites, whose cleavage leaves equivalent overhangs to those of the alpha 1 BsmBI digestion (resulting overhangs can be seen in Figure 1). Thus, this vector can be used to switch any transcriptional unit inserted in an alpha 1 GB vector into a BioBrick compatible one, so adapting the composite part into the BB grammar with an easy GB one-pot reaction.
-To carry out the positive transformants screening method, common BBa_P1050 plasmid relies on the white-blue screening method. It is based on the α-complementation, where the host strain must carry the ω-peptide, while the vector contains the α-peptide. Thus, if both subunits are expressed (when there is not insertion in the vector), functional beta-galactosidase is reconstituted. However, the blue/white colors depends of the subsequent addition of X-gal (D-lactose analog) and IPTG substances into the agar plates. Although the principle is simple, the method requires the strict use of lacZ mutant strains, and it is always necessary to add both chemical reagents into the medium.
+To carry out the positive transformants screening method, common BBa_P1050 plasmid relies on the white-blue screening. It is based on the α-complementation, where the host strain must carry the ω-peptide while the vector includes the α-peptide sequence. Thus, if both subunits are expressed (when there is not insertion in the vector), functional beta-galactosidase is reconstituted. However, the blue/white colors depends of the subsequent addition of X-gal (D-lactose analog) and IPTG substances into the agar plates. Although the principle is simple, the method requires the strict use of lacZ mutant strains, and it is always necessary to add both chemical reagents into the medium.
 In order to avoid these limitations, we have decided to rely on a different visual method. Thus, we have inserted an mRFP1 transcriptional unit [http://2018.igem.org/Team:Valencia_UPV/Design#LevGB BBa_BBa_K2656201] to act as the screening gene marker. With this approach,  cloning is achieved in a more reliable, easy, low-cost way, so avoiding the necessity of adding chemical supplements and the possible non-function of these chemicals once on the solid medium. With this screening method, non-positive transformants will always express the a red fluorescent intense protein, easing the correct check.