Introduction

Construction of BioBricks

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 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

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 4: Prefix SPL primers that SHOULD be used depending on which BioBrick plamid backbone is selected for amplification is illustrated.

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.