Biobrick Primers for Site-Directed Insertion Mutagenesis

Ribosomal Binding Site Insertion

Purpose

The RBS primer is a standard primer that can be used for site directed mutagenesis in all linearized backbones available from the iGEM database. such and such and such and such and such and such and such

Background

Most BioBrick parts require a Ribosomal Binding Site to express the protein. This has proven problematic as the RBS is very small and hard to insert using 3A Assembly. We believe the creation of an oligo primer with this RBS may be easier to place the RBS in front of the protein that is needed to be expressed. An easy oligo primer that could be used in PCR extension and then placed into a vector backbone would save time and frustration as to whether the part had been inserted.such and such

Design

The format of this primer needs the BioBrick formatted prefix with an additional strong Ribosomal binding site within it. The prefix of the standard BioBrick was analyzed and the primer was found to be able to be inserted after the XbaI site and still have the scar intact between the part and the RBS site. The RBS sites chosen were selected from the Anderson RBS library degenerate sequence and a library was created with that degenerate sequence via the Salis Lab at Penn State University, optimized for E. coli 16s.

Promoter Sequence Insertion

Purpose

The Promoter primer is a standard primer that can be used for site directed mutagenesis in all linearized backbones available from the iGEM database and the Anderson RBS sites from the Anderson RBS library.

Background

Most BioBrick parts require a Promoter to express the protein. This has proven problematic as the promoter is very small and hard to insert using 3A Assembly. We believe the creation of an oligo primer with this promoter may be easier to place the promoter in front of the RBS site that is needed to be expressed. An easy oligo primer that could be used in PCR extension and then placed into a vector backbone would save time and frustration as to whether the part had been inserted.

Design

The format of this primer needs the BioBrick formatted prefix, the promoter and part of the RBS site. The prefix of the standard BioBrick was analyzed and the primer was found to be able to be inserted after the XbaI site and be able to insert the promoter just before the RBS site. The promoters were selected from the Anderson Constitutive Promoter Library in the iGEM parts registry.such and such and such and such and such and such and such and such and such and such and such and such and such and such and such and such and such and such

Characterization of Function

The flow cytometry data suggests successful insertion of both an RBS and a promoter via PCR with our specialized primers. The vertical lines transecting the histograms represent the “gate” or boundaries used to determine the presence of mCherry. When comparing the mean fluorescent molecules per cell, the high efficiency (HE) RBS and promoter combination is about 2,000 molecules per cell higher than the low efficiency (LE) RBS and promoter combination. This result was expected; however the degree to which the RBS or promoter individually played in the overall expression is yet to be determined. Insertion of the RBS and promoter crosses (e.g. RH2 with PL1 and RL1 with PH2) into pSB1C3_mCherry are underway. Theoretically, the flow cytometry data will yield plots that fit in between the current data.

Initial computer predicted results for these RBS and promoter efficiencies predicted that a much greater difference should have been seen between the high efficiency RBS and promoter pair and the low efficiency RBS and promoter pair. Predicted transcription rates for the RBS’s were: RH2= 0.914 and RL1=0.007. Theoretical translation rates for promoters were: PH2=0.86 and PL1=0.06. It is not yet determined why this discrepancy exists.