Introduction

As 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:

• If it was possible to trigger positive feedback mechanism of the N protein by variying promters strength, that would result in an increased read through of the terminator.
• If the N-mechanisms can be triggered by induction of N from a seperate inducible plasmid

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.

The 5 test plasmids where transformed into e. coli Xlblue, with the synthetic promoter library in front of the constructs to give a large range of expressions. This first series of test strains are called serie A. Isolated colonies from this transformation (serie A) where again transformed with the inducible pAT01 plasmid containing to give the test serie N. For an overview of the test strains constructed se the table below.

Figure 1: pAT12: Insert´s organisation in plasmid pAT12 with pSB2K3 as a backbone. As the figure shows, there is an RBS followed by FACS optimised GFP (from pAT03) and lambda natural nutR (from pAT05). Next, there is the N gene followed by RBS and RFP (from pAT02) downst ream of the nutR site.

Figure 2: pAT13, 14, 15: Insert´s organisation in plasmids pAT13-15 with pSB2K3 as a backbone. These plasmids contain RBS followed by FACS optimised GFP (from pAT03). Next there is a nutR followed by a terminator site, which is different for each of the plasmids. pAT14 and pAT15 contain a single terminator site, namely BBa_B0011 (from pAT09) and BBa_B1003 (from pAT10) respectively, while pAT13 contain both (pAT11). Downstream of the terminator site, there is the N gene followed by RBS and RFP (from pAT02) present in each of the three constructs.

Figure 3: pAT16: pAT16 has been constructed by inserting pAT10 and BBa_I13507 into pSB2K3 backbone. The insert organisation of pAT16 is shown in figure X3 below. Insert´s organisation in plasmid pAT12 with pSB2K3 as a backbone. First there is an RBS and FACS optimised GFP (from pAT03), followed by nutR and terminator site BBa_B1003 (from pAT07). Downstream of the terminator there is another RBS followed by the RFP and a double terminator site BBa_B0010 and BBa_B0012 (from BBa_I13507). N should be removed from the figure

Figure 4: pAT01:pAT01 contain the arabinose inducible promoter pBAD, and the N plasmid.

Figure 1: pAT12: Insert´s organisation in plasmid pAT12 with pSB2K3 as a backbone. As the figure shows, there is an RBS followed by FACS optimised GFP (from pAT03) and lambda natural nutR (from pAT05). Next, there is the N gene followed by RBS and RFP (from pAT02) downst ream of the nutR site.

Construction of parts

The 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:

• The lambda N protein from e. Coli EMG2.
• The lambda NutR site, from e. Coli EMG2.
• A GFP – derived from GFP, called GFPmut2.

Characterization plasmids and strains

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.

The 5 test plasmids where transformed into e. coli Xlblue, with the synthetic promoter library in front of the constructs to give a large range of expressions. This first series of test strains are called serie A. Isolated colonies from this transformation (serie A) where again transformed with the inducible pAT01 plasmid containing to give the test serie N. For an overview of the test strains constructed se the table below.

Figure 1: pAT12: Insert´s organisation in plasmid pAT12 with pSB2K3 as a backbone. As the figure shows, there is an RBS followed by FACS optimised GFP (from pAT03) and lambda natural nutR (from pAT05). Next, there is the N gene followed by RBS and RFP (from pAT02) downst ream of the nutR site.

Figure 2: pAT13, 14, 15: Insert´s organisation in plasmids pAT13-15 with pSB2K3 as a backbone. These plasmids contain RBS followed by FACS optimised GFP (from pAT03). Next there is a nutR followed by a terminator site, which is different for each of the plasmids. pAT14 and pAT15 contain a single terminator site, namely BBa_B0011 (from pAT09) and BBa_B1003 (from pAT10) respectively, while pAT13 contain both (pAT11). Downstream of the terminator site, there is the N gene followed by RBS and RFP (from pAT02) present in each of the three constructs.

Figure 3: pAT16: pAT16 has been constructed by inserting pAT10 and BBa_I13507 into pSB2K3 backbone. The insert organisation of pAT16 is shown in figure X3 below. Insert´s organisation in plasmid pAT12 with pSB2K3 as a backbone. First there is an RBS and FACS optimised GFP (from pAT03), followed by nutR and terminator site BBa_B1003 (from pAT07). Downstream of the terminator there is another RBS followed by the RFP and a double terminator site BBa_B0010 and BBa_B0012 (from BBa_I13507). N should be removed from the figure

Figure 4: pAT01:pAT01 contain the arabinose inducible promoter pBAD, and the N plasmid.

Constructing our Biobricks and Parts

In 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
In order to construct our biobricks, we used a set of forward and reverse primers to amplify a region of interest by using PCR. The fragments created by PCR amplification were purified using a PCR clean-up kit. The amplicon and the linearized backbone plasmid pSB1C3 (containing a chroramphenicol resistance marker) were digested, resulting in sticky ends. This was achieved either by the standard assembly or the three-way ligation approach (3A assembly). Both the digested PCR product and the digested plasmid were run on a gel in order to estimate DNA concentration. T4 ligase was used for ligation with a 5:1 ratio of insert to backbone. After the ligation, the plasmid was transformed into electrocompetent DH5_α E. coli cells. After an hour of recovery in LB medium at 37 °C, the cells were plated on LB plates containing chloramphenicol (Cam) and left overnight. Several colonies from the plates were then selected and restreaked on LB+Cam plates in order to assure pure colonies. Overnight cultures of the transformants were made by taking one colony from each restreak and inoculating it in LB+Cam at 37 °C over night. Minipreps were made from the overnight cultures and a verification PCR was run on these in order to make sure that the plasmid had the expected insert.

construction details

Below is listed our different constructs, what they contain and information on construction.

pATN, pAT05
Lambda natural N-gene (encoding the N protein) and nutR have been inserted into the backbone plasmid pSB1C3 to construct pATN and pAT05 respectively. These parts have been submitted to the parts registry.

pAT02
pAT02 containing RFP reporter, has been constructed by inserting K374011 (lambda N-gene with its natural RBS), followed by BBa_B0034 (RBS) and BBa_E1010 (RFP) into pSB1C3. This part has also been submitted to the parts registry.

pAT03
pAT03 has been constructed by inserting RBS site BBa_B0034 and K374012 (FACS optimised GFP) into the recipient plasmid pSB1C3.

pAT04, pAT06, pAT07
pAT05 with the lambda nutR insert has been used to construct pAT06, pAT07 and pAT04, also containing different terminator sites. Plasmids pAT06 and pAT07 contain a single terminator site, but with different terminator strengths. Thus, pAT06 contains BBa_B0011 with medium terminator strength, while pAT07 contains BBa_B1003, which is a strong terminator site. Plasmid pAT04 contains both BBa_B0011 and BBa_B1003. These plasmids have been constructed in order to test termination efficiency and have also been submitted to the parts registry.

pAT08, pAT09, pAT10 and pAT11
plasmids pAT05, pAT06, pAR07 and pAT04 have been used along with pAT03 to construct pAT08, pAT09, pAT10 and pAT11 respectively. pAT08-pAT11 contain a GFP reporter that is important for the construction of pAT12-15.

pAT12, pAT13, pAT15
pAT08 has been used to construct pAT12, pAT09 to construct pAT014, pAT10 to construct pAT15 and pAT11 has been used to construct pAT13. All of these new constructs also contain pAT02. The organisation of inserts in plasmid pAT12 is shown in figure 1, while the organisation of inserts in plasmids pAT13-15 is presented in figure 2.

pAT16
pAT16 has been constructed by inserting pAT10 and BBa_I13507 into pSB2K3 backbone. The insert organisation of pAT16 is shown in figure 3.

Construction of BioBrick K374005

This 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 K374006

This 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 K374007

This 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 K374013

This 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 K37015

The 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 K374016

This 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

Characterization

Strategy

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

Analysis

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.

Results The results are summarized in the table below.

Table 1: INSERT TEXT.

Construct	Green filter	Red Filter	Description
A: Strong SPL No Terminator			An example of a colony with a strong promoter from the SPL. The colony have both strong GFP and RFP signal.
A: Weak SPL No Terminator			An example of a weak SPL promoter. The colony have weak expression of both GFP and RFP. It can be seen that it is weak due to the background color intensity compared to the stong promoter above
B: Strong SPL Strong Terminator			The construct with the strong terminator B0015. The Colony have strong expression of GFP and only very weak expression of RFP proving the high efficiency of the terminator
B: Multiple SPL Strong Terminator			On this picture is seen three colonies with weak, medium and strong promoter. The medium promoter in the bottom right corner cannot read through the strong terminator and express the RFP. The Strong promoter in the middle seems to have triggered the positive feed back mechanism and expresses RFP.
E: No N protein Weak Terminator B1003			The control construct E, without the N protein. Again three different strengths of promoters strong, medium and weak. It is seen from this and other E-colonies that the B1003 terminator have a very weak effect. B1003 cannot create a visible difference in florescence, or the anti-terminator effect is triggered.