Difference between revisions of "Part:pSB1C3"

Revision as of 22:39, 31 October 2017

High copy BioBrick assembly plasmid

pSB1C3 is a high copy number plasmid (RFC [10]) carrying chloramphenicol resistance.

The replication origin is a pUC19-derived pMB1 (copy number of 100-300 per cell).

pSB1C3 has terminators bracketing its MCS which are designed to prevent transcription from *inside* the MCS from reading out into the vector. The efficiency of these terminators is known to be < 100%. Ideally we would construct a future set of terminators for bracketing a MCS that were 100% efficient in terminating both into and out of the MCS region.

pSB1C3 is the designated Registry shipping plasmid backbone. All submissions for iGEM competitions must be submitted using pSB1C3.

Go to this page for more information on how to ship submissions to iGEM HQ.

Sample Location

The distribution kits contain many samples of pSB1C3 as it is the Registry's main backbone for shipping and maintaining samples. Our recommendation when cloning a part into pSB1C3 is that you can use either linearized plasmid backbone provided in the kits, or pSB1C3 with a reporter (BBa_J04450).

2017 Kit Plate 4 - Well 4B has a sample of BBa_J04450 in pSB1C3.

Usage and Biology

In Spring 2011, pSB1C3 was sequenced using several primers: Pre-R, Suff-F, VF2, VR, and internal primers for the origin and resistance. The reference sequence below matches the most recent sequencing verification of pSB1C3 (w/ BBa_J04450, located in SP 4000 Well 2A).

[http://2017.igem.org/Team:USP-Brazil Team USP-Brazil 2017] improved the function of part by showing that it can be used to sucessfully transform another chassis from the Enterobacteriaceae family, Pantoea agglomerans
(--lubdub 02:38, 30 October 2017 (UTC))

Improvement by Evry_Paris-Saclay 2017

The [http://2017.igem.org/Team:Evry_Paris-Saclay Evry_Paris-Saclay 2017 team] made two improvements of the pSB1C3 backbone BBa_K2448038 and BBa_K2448036.

BBa_K2448038: pSB1C3 is high copy number plasmid and the iGEM workhorse for gene expression. However, when it comes to the of use LacI regulated promoters, very frequently significant leakage is observe due to its important copy number, which leads to important regulation problems. This issue can be addressed either by including the LacI coding sequence into the construct or by using only LacI overexpressing cells. To facilitate further use of pSB1C3, we designed a pSB1C3 with a built-in LacI coding sequence under the control of a mutated version of its own natural promoter known as LacIq which leads to a 10-fold increase in LacI expression compared to the natural promoter. This improved backbone allow a fine regulation of LacI related parts.
BBa_K2448036: the pSB1C3 backbone vector contains a BsmBI cloning site within the chloramphenicol resistance gene. Its presence prevents from using the Golden Gate assembly technique with this backbone. To circumvent this issue, we performed a site-directed mutagenesis (G1385C) and created the pSB1C3 BsaI free backbone.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2049
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 2055
21
INCOMPATIBLE WITH RFC[21]
Plasmid lacks a prefix.
Plasmid lacks a suffix.
Illegal EcoRI site found at 2049
Illegal XhoI site found at 1033
Illegal XhoI site found at 1925
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found at 2049
Illegal suffix found at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found at 2049
Plasmid lacks a suffix.
Illegal XbaI site found at 2064
Illegal SpeI site found at 2
Illegal PstI site found at 16
1000
INCOMPATIBLE WITH RFC[1000]
Plasmid lacks a prefix.
Plasmid lacks a suffix.

@@ Line 29: / Line 29: @@
 ===Improvement by Evry_Paris-Saclay 2017===
-The [http://2017.igem.org/Team:Evry_Paris-Saclay Evry_Paris-Saclay 2017] made two improvements of the pSB1C3 backbone [[Part:BBa_K2448038|BBa_K2448038]] and [[Part:BBa_K2448036|BBa_K2448036]].
+The [http://2017.igem.org/Team:Evry_Paris-Saclay Evry_Paris-Saclay 2017 team] made two improvements of the pSB1C3 backbone [[Part:BBa_K2448038|BBa_K2448038]] and [[Part:BBa_K2448036|BBa_K2448036]].
 *[[Part:BBa_K2448038|BBa_K2448038]]: pSB1C3 is high copy number plasmid and the iGEM workhorse for gene expression. However, when it comes to the of use LacI regulated promoters, very frequently significant leakage is observe due to its important copy number, which leads to important regulation problems. This issue can be addressed either by including the LacI coding sequence into the construct or by using only LacI overexpressing cells. To facilitate further use of pSB1C3, we designed a pSB1C3 with a built-in LacI coding sequence under the control of a mutated version of its own natural promoter known as LacIq which leads to a 10-fold increase in LacI expression compared to the natural promoter. This improved backbone allow a fine regulation of LacI related parts.
 *[[Part:BBa_K2448036|BBa_K2448036]]: the pSB1C3 backbone vector contains a BsmBI cloning site within the chloramphenicol resistance gene. Its presence prevents from using the Golden Gate assembly technique with this backbone. To circumvent this issue, we performed a site-directed mutagenesis (G1385C) and created the pSB1C3 BsaI free backbone.