Team:DTU-Denmark/AntiTermination Section
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IntroductionAs described in the design of the switch, the regulatory systems utilized in our switch can be divided into two major parts. In this section we focus on the lambda phage nut-site N protein termination system. In lambda bacteriophage, gene expression is regulated by the suppression of transcription termination which is mediated by the lambda N protein that interacts with the nut site. We have constructed several test plasmids with different terminator strentgh and references, these plasmids are presented the step-wise construction. The aim of the characterization experiments have been to test:
Characterization
Strategy
In order to test the funcionality of the antiterminator system the following 6 plasmids where constructed. the test plasmids pAT12 to 16 and the induction plasmid pAT01. They are presented in the figures below.
As described in the Stepwise Construction, several promoters of different strengths have been introduced into the final constructs pAT12-pAT16, all containing GFP and RFP reporters. Together these promoters constitute Synthetic Promoter Library (SPL). Transcription through the terminator site results in the expression of the N antiterminator. If enough N protein is produced, the antitermination is mediated. Our system enables us to measure the promoter strength by measuring the amount of GFP produced. At the same time the amount of transcription past the promoter can be estimated by measuring the amount of RFP produced. This is one of the strategies for the characterization of our final constructs. For a more detailed explanation see the section on SPL. Strains containing pAT12-16 have also been transformed with pAT01, containing pBAD and ARAC promoters upstream of lambda N-gene with its natural RBS. This has been done in order to induce expression of N protein. Supplement of bacterial cultures containing these plasmids with arabinose enhances the expression of the lambda N-gene and therefore increases the production of the N protein. As a consequence, the N produced in pAT12-16 can be measured and compared. Based on that, we have also tried to characterize our system by testing whether or not it is possible to trigger N-mediated antitermination by coupling the N protein to the pBAD promoter. The characterization experiments have been performed on the strains presented in the table X3 below. Results
In the following sections we present the analysis and results done on test strains constructed and presented in the table above. Florescence microscope
Flourescence microscope was used to investigate the success rate and verify the preformed transformations. We looked at the first transformations done with the test constructs and the SPL, and selected 10 colonies from each construct for further analysis.
Construction of BiobricksIn the following section we describe more in detail the method and strategy used for construction of our parts and Biobricks.
General sssemply standard and methods Construction of BioBrick K374005This part contains the lambda nutR site, inserted into the backbone plasmid pSB1C3. The lambda nutR site was sythesized by Integrated DNA Technology. In order to construct this part, the standard assembly ligation approach was used. In doing so, the nutR site was digested with restriction enzymes EcoRI and Pst1 and thereafter ligated into pSB1C3. The nutR site was verified by PCR using primers IG201 (VF2 forward primer) and IG004 (lambda nutR reverse primer). The following parts were taken into consideration when calculating the size of BioBrick K374005: IG201 + nutR + IG004 tail = 140 + 118 + 26 = 284 base pairs. Construction of BioBrick K374006This part contains the lambda N-gene that is responsible for the suppression of transcription termination downstream of part BBa_K374005. The lambda N-gene was synthesized by Integrated DNA Technology. As with the construction of K374005, the standard assembly ligation approach was also used in the construction of this part. For size verification, the lambda N-gene was amplified by PCR with primers IG201 and IG006 (lambda N-gene reverse primer). The size of K374006 is therefore: IG201 + IG006 tail + lambda N gene = 140 + 26 + 402 = 568 base pairs. Construction of BioBrick K374007This construct contains the lambda nutR site (BBa_K374005) and the downstream terminator BBa_B0015 (composed of two terminator parts, namely BBa_B0010 and BBa_B0012). The 3A assembly was used in the construction of this part. The nutR site has been digested with the restriction enzymes EcoRI and SpeI. The terminator part BBa_B0015 was, however, digested with Xbal and Pstl. NutR and BBa_B0015 were then ligated into the linearized plasmid pSB1C3 that had been restricted with EcoRl and Pstl. The size of K374007 has been verified by PCR with primers IG201 and IG202 (VR reverse primer) to be the following: IG201 + IG202 + nutR = 140 + 176 + 255 = 571 base pairs.
Construction of BioBrick K374013This part contains lambda N-gene (K374007) with its natural RBS. 3A assembly was used to construct this part. The lambda N-gene was excised with the restriction enzymes EcoRl and Spel. BBa_I13507 (containing RBS and RFP) was cut with Xbal and Pstl. Both parts were then ligated into pSB1C3 that had been restricted with EcoRl and Pstl. Aftertransformation and selection of the transformed colonies, verification PCR with primers IG201 and IG006 was carried out. The estimated size of this part: IG201 + IG006 tail + N-gene with RBS =140 + 26 + 420 = 586 base pairs. Construction of BioBricks K37014 and K37015The 3A assembly approach was used to construct these two parts. The lambda nutR site was exercised with EcoRl and Spel, while recipient vector pSB1C3 has been cut with EcoRl and Pstl. The BioBrick terminator, BBa_B1003 was restricted with Xbal and Pstl and ligated, along with the nutR site, into pSB1C3 to construct K37014. K37015 was constructed by restricting the BioBrick terminator, BBa_B0011, with Xbal and Pstl and ligated, along with the nutR site into pSB1C3. In order to ensure that the plasmid contained the desirable inserts, verification PCRs with primers IG201 and IG004 was carried out. The estimated sizes of the inserts are shown below: IG201 + IG004 tail + nutR = 284 base pairs.
Construction of BioBrick K374016This construct contains lambda’s natural RBS site (BBa_B0034), followed by a FACS optimized mutant of the Green Fluorescent Protein (BBa_K374012) and nutR site (BBa_K374005). Again, the 3A assembly approach has been used. The RBS-GFP was excised with EcoRl and Spel, while the nutR site with Xbal and Pstl. RBS-GFP and nutR were then ligated into pSB1C3 (with EcoRl and Pstl sticky ends). After transformation, the verification PCR with primers IG201 and IG004 was performed. The estimated size of this part includes the sizes of the following parts: IG201 + GFP + nutR + IG004 tail + RBS + biobrick scar = 140+717+118+2618+6=1025 base pairs
Construction of partsThe step-wise construction of the test plasmids and intermediate constructs are presented in this section. Some of these parts have been submitted to the parts registry as BioBricks. All of the parts constructed in our terminator system are presented in table 1. A more detailed description of the parts submitted as BioBricks and the experimental procedure of setting up these parts is presented in the Construction of BioBricks section below.
The plasmids and parts have been constructed of existing biobricks and we have submitted new genes not in the parts registry, these genes are:
construction detailsBelow is listed our different constructs, what they contain and information on construction. pATN, pAT05 pAT02 pAT03 pAT04, pAT06, pAT07 pAT08, pAT09, pAT10 and pAT11 pAT12, pAT13, pAT15 pAT16 |