Difference between revisions of "Part:pSB1C3"

Latest revision as of 15:20, 12 August 2024

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 was the designated Registry shipping plasmid backbone. Sample submissions to the Registry or for the iGEM competitions are no longer required or accepted.

pSB1C3 is an extremely common plasmid backbone used in iGEM, and is one of the primary backbones used to propagate parts in the iGEM Distribution Kit.

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))

[http://2018.igem.org/Team:MichiganState MichiganState 2018] improved the function of part by using it to stably transform the grass endophytic bacteria Enterobacter ludwigii FCP2-01 (BBa_K2633006), of the Enterobacteriaceae family. (--liberjul 02:35, 17 October 2018 (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 allows 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 BsmBI free backbone.

Improvement by Newcastle iGEM 2018

• BBa_K2797013: In summer 2018, [http://2018.igem.org/Team:Newcastle/InterLab Newcastle iGEM] designed and introduced a constitutive, medium strength RFP expression construct into pSB1C3 to act as an internal standard (IS) for each test device. The device was cloned into the non-coding region between the chloramphenicol resistance gene and the Origin of Replication of pSB1C3 via Gibson Assembly. The goal was to use the IS to control for changes in plasmid copy number when comparing devices i.e. RFP signals should be consistent across plasmids allowing improved reporting of the activities of test devices. Results show that inclusion of the IS altered the behaviour of the test devices. The IS repressed expression of GFP from all devices and affected expression rates - particularly in devices with strong promoters. Further, the IS signal varied across plasmids though the only difference was the strength of the test device. The data generated using this approach indicates competition for cellular resources between the IS and test device. As a result, measurements and conclusions regarding the strength of strong reporters should be treated with caution.

Improvement by iGEM NCHU_Taichung 2018

• Part name:BBa_K2546004
• Group: NCHU_Taichung 2018
• Author: YI-CIAN CHEN
• Summary:To prove our gene work well, we use BBa_J364007, pBBR1, and pMP4657 to get BBa_K2546004. thus we replace the ori with the new ori and replication related proteins to let the part express in our endophyte and broaden its host range. We transform BBa_K2546004 in Burkholderia cenocepacia Strain 869T2, and test the efficient of green fluorescence protein. By interlab protocol: Cell growth, sampling, and assay, we can analyze the BBa_K2546004 work. We found that BBa_K2546004 work well in Burkholderia cenocepacia Strain 869T2.

Sequence and Features