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Bio-bricks:

 

1) Constitutive Promoter (J23119) + Ribosomal Binding Site (B0034) + pvdQ Gene + Double Terminator (B0015)

 

This biobrick was constructed to test the expression of the pvdQ gene amplified by PCR from genomic DNA of Pseudomonas aeruginosa. It is an uncontrollable, standard biobrick that can later be used as a baseline reference to test whether our future biobricks result in an improvement in pvdQ expression level.  The biobrick is a composite part consisting of the already prevalent Constitutive Promoter [2006 Berkley], along with the newly inserted pvdQ gene. It is also composed of a strong RBS and a transcription double terminator. 

Constitutive Promoter (J23119): This promoter biobrick is the consensus sequence and therefore results in the idealized transcription scenario. Since it is a strong promoter and is constitutively expressed, it complies with our preliminary goal of testing transcription and translation of the pvdQ gene in an AHL-independent manner


2) LacI Promoter, RBS, LuxR Gene, Double Terminator, LuxR Promoter, & RBS (K137076) + pvdQ Gene + Double Terminator (B0015)

This biobrick was constructed to test the expression of the pvdQ gene in an environment containing the 3OC₁₂-HSL substrate. However, the synthesis of pvdQ is not exclusively dependent on AHL. This is because the LacI+pI promoter that initiates transcription of the LuxR gene can be effectively induced by IPTG. Moreover, the LuxR promoter is also expressed well in the absence of LuxR and AHL. The biobrick is composed part of the already existing K137076 [2008 Caltech] as well as the pvdQ gene and double terminator sequence. The first 1,096bp of the K237076 biobrick containing the LacI and LuxR promoters, two RBS, and the LuxR gene was amplified using the standard Prefix primer and a newly designed Suffix primer.

We chose the biobrick K137076 to ligate to the pvdQ gene for the following reasons:

•  K137076 consists of a strong, continually expressed promoter (R0011) that can, however, be further induced by IPTG. Such a promoter allows us to control the expression of the LuxR protein.

• The complex formed when two molecules of LuxR bind to two molecules of AHL, attaches to a palindromic site on the pLuxR to transcribe the pvdQ gene.This pLuxR promoter however causes leaky expression of pvdQ as it can be induced even in the absence of LuxR and AHL.

 
3). Constitutive Promoter (pLacI) + Ribosomal Binding Site + LuxR Gene + Double Terminator + LuxR Promoter (J09855) + Ribosomal Binding Site (B0034) + pvdQ Gene + Double Terminator (B0015)

This biobrick was constructed such that

 

4). LuxR Promoter (R1062) + Ribosomal Binding Site, LuxR Gene, Double Terminator, & Ribosomal Binding Site (amplified from K137076) + pvdQ Gene + Double Terminator (B0015)

This final biobrick is entirely an AHL-dependent system, as an identical promoter controls the transcription and translation of both the LuxR and the pvdQ gene. This LuxR promoter, unlike its Bba_R0062 counterpart, is only weakly expressed in the absence of LuxR and AHL. However, a desirable positive feedback mechanism is generated upon coupling of AHL with the LuxR protein.  The AHL-LuxR complex binds to p_LuxR and upregulates gene expression. As more LuxR is expressed, subsequent pairing of the molecules further activates the promoter. Thereby, a large yield of pvdQ is obtainable at low cell density. The outcome is advantageous as biofilm formation can be interrupted at an early stage.