Difference between revisions of "Part:BBa K4294091"

Latest revision as of 16:53, 13 October 2022

pTU1-A-lacZ destination Vector

Plasmid Vector from the Ecoflex MoClo Kit

Usage and Biology

We utilized pTU1-A-lacZ as our level 1 (as hinted by the “1” in its name) - Golden Gate acceptor vector for assembling the LuxI expressing transcriptional units with the different RBS in our senders. This vector has ampicillin (Amp) resistance gene and is a high copy plasmid; it is also approximately 2.5kbp in size. Since it would not be present in the senders to be induced, we did not have to select a version with a particular origin of replication (taking into consideration possible interactions with other plasmids transformed in the same cell or how copy number would affect expression dynamics). pTU1-A-lacZ contains the gene coding for LacZ, which allowed us to select recombined constructs through blue-white screening on agar plates spread with x-gal and IPTG (blue colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid). Another trait worth mentioning of pTU1-A-lacZ is that it is optimized to be used as a part-carrying vector should the transcriptional unit it contains be utilized as a fragment in level 2 assembly, as it also includes two BsmBI cut sites “outside” its level 1 restriction positions. The “A” in the vector’s name indicates that the transcriptional unit assembled inside this particular plasmid will be placed upstream of other transcriptional units (assembled in pTU1-A-lacZ “B”, “C”, etc. counterparts) should it be included in a level 2 - Golden Gate assembly. This detail was given particular attention, as many of our constructs contain repressor genes that are constitutively expressed. We designed our constructs placing the constitutively expressed genes downstream of all inducibly expressed CDS, to reduce leakiness from our constitutively translated transcriptional units [1,2].

Plasmid Map

Reference

[1] Addgene plasmid (#72935) https://www.addgene.org/72935/

[2] EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology. Moore SJ, Lai HE, Kelwick RJ, Chee SM, Bell DJ, Polizzi KM, Freemont PS. ACS Synth Biol. 2016 May 2. 10.1021/acssynbio.6b00031 PubMed 27096716

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2043
Illegal XbaI site found at 2058
Illegal XbaI site found at 2087
Illegal SpeI site found at 2477
Illegal PstI site found at 1
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2043
Illegal SpeI site found at 2477
Illegal PstI site found at 1
Illegal NotI site found at 2049
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2043
Illegal BamHI site found at 2483
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 2043
Plasmid lacks a suffix.
Illegal XbaI site found at 2087
Illegal SpeI site found at 2477
Illegal PstI site found at 1
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 2043
Plasmid lacks a suffix.
Illegal XbaI site found at 2058
Illegal XbaI site found at 2087
Illegal SpeI site found at 2477
Illegal PstI site found at 1
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal BsaI site found at 2495
Illegal BsaI.rc site found at 2081

@@ Line 7: / Line 7: @@
 ==Usage and Biology==
-We utilized pTU1-A-lacZ as our level 1 (as hinted by the “1” in its name) - Golden Gate acceptor vector for assembling the LuxI expressing transcriptional units with the different RBS in our senders. This vector has ampicillin (Amp) resistance gene and is a high copy plasmid; it is also approximately 2.5kbp in size. Since it would not be present in the senders to be induced, we did not have to select a version with a particular origin of replication (taking into consideration possible interactions with other plasmids transformed in the same cell or how copy number would affect expression dynamics). pTU1-A-lacZ contains the gene coding for LacZ, which allowed us to select recombined constructs through blue-white screening on agar plates spread with x-gal and IPTG (blue colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid). Another trait worth mentioning of pTU1-A-lacZ is that it is optimized to be used as a part-carrying vector should the transcriptional unit it contains be utilized as a fragment in level 2 assembly, as it also includes two BsmBI cut sites “outside” its level 1 restriction positions. The “A” in the vector’s name indicates that the transcriptional unit assembled inside this particular plasmid will be placed upstream of other transcriptional units (assembled in pTU1-A-lacZ “B”, “C”, etc. counterparts) should it be included in a level 2 - Golden Gate assembly. This detail was given particular attention, as many of our constructs contain repressor genes that are constitutively expressed. We designed our constructs placing the constitutively expressed genes downstream of all inducibly expressed CDS, to reduce leakiness from our constitutively translated transcriptional units.
+We utilized pTU1-A-lacZ as our level 1 (as hinted by the “1” in its name) - Golden Gate acceptor vector for assembling the LuxI expressing transcriptional units with the different RBS in our senders. This vector has ampicillin (Amp) resistance gene and is a high copy plasmid; it is also approximately 2.5kbp in size. Since it would not be present in the senders to be induced, we did not have to select a version with a particular origin of replication (taking into consideration possible interactions with other plasmids transformed in the same cell or how copy number would affect expression dynamics). pTU1-A-lacZ contains the gene coding for LacZ, which allowed us to select recombined constructs through blue-white screening on agar plates spread with x-gal and IPTG (blue colonies: transformed with the undesired parental plasmid, white colonies: transformed with a recombinant plasmid). Another trait worth mentioning of pTU1-A-lacZ is that it is optimized to be used as a part-carrying vector should the transcriptional unit it contains be utilized as a fragment in level 2 assembly, as it also includes two BsmBI cut sites “outside” its level 1 restriction positions. The “A” in the vector’s name indicates that the transcriptional unit assembled inside this particular plasmid will be placed upstream of other transcriptional units (assembled in pTU1-A-lacZ “B”, “C”, etc. counterparts) should it be included in a level 2 - Golden Gate assembly. This detail was given particular attention, as many of our constructs contain repressor genes that are constitutively expressed. We designed our constructs placing the constitutively expressed genes downstream of all inducibly expressed CDS, to reduce leakiness from our constitutively translated transcriptional units [1,2].
 [[File:Ptu1-a-lacz-map.png]]