Introduction

The Gifsy promoters with their respective repressors and anti-repressors are important regulatory elements in the switch and this is why we wanted to characterize them and submit them as BioBricks. We already know that this system is tight in its natural settings in Salmonella, but to our knowledge no one before us has tried to characterize the system in Escherichia coli. A good way to characterize it in E. coli is to test whether the repression of the pR promoters is tight, and if the antirepressors indeed do antirepress. Because of previous knowledge of the strength of the promoters in Salmonella (reference, spørg seb) all characterization had to be done in low copy number plasmids. We have characterized the following BioBricks:

• BBa_K374008
• BBa_K374009
• BBa_K374010

Construction of BioBricks

BioBricks BBa_K374008 and BBa_K374009

In wildtype Salmonella enterica Serovar Typhimurium (strain ATCC 14028) the Gifsy immunity region, consisting of the two divergent promoters pR and pRM and the repressor gogR/gtgR downstream of the pRM promoter, are present in the chromosome. We designed a primer pair to amplify the immunity region from Gifsy 1 and Gifsy 2, and by adding the standardized prefix and suffix as a tail, the amplicons were made Biobrick compatible. The amplicons were made by PCR. After the PCR the fragments were cleaned up using a PCR clean-up kit.

The amplicon as well as the linearized backbone plasmid pSB1C3 were digested with the restriction enzymes EcoRI and PstI leaving sticky ends. Both the digested PCR product and the digested plasmid were run on a gel in order to estimate DNA concentration.

The ligation was made using T4 ligase with a 5:1 ratio of insert to backbone. After the ligation the plasmid was transformed into electrocompetent DH5α E. coli cells. After an hour of recovery in LB media at 37 °C the cells were plated on LB plates containing chloramphenicol (Cam).

The next day five colonies on the plates were selected and restreaked on LB+Cam plates in order to assure pure colonies. Overnight cultures were made of the transformants 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 assure that the plasmid had the expected insert.

Figure 1 Left: GeneRuler DNA ladder mix from Fermentas. Right: Verification PCR of the Biobricks K374008, K374009, K374010. Lanes 1-5 show the expected amplicon of 650 bp from a PCR using the iGEM primer VR as reverse and a forward primer which anneals to the Gifsy2 immunity region. Lanes 6-10 show the expected amplicon of 672 bp from a PCR using the iGEM primer VR as reverse and a forward primer which anneals to the Gifsy2 immunity region. Lanes 11-13 show the expected amplicon of 450 bp from a PCR using the iGEM primer VR as reverse and a forward primer which anneals to the anti-repressor AntO. The Biobricks sent in are the ones corresponding to lanes 4, 9 and 11.

BioBrick BBa_K374010

The Gifsy1 anti-repressor AntO was amplified by PCR using a plasmid containing the anti-repressor as template (Sebastién Lemire). The primers had tails corresponding to the standardized prefix and suffix making the amplicon BioBrick compatible. Once we had the amplicon it was digested, ligated and transformed into DH5α E. coli cells following the same procedure as in the above mentioned BioBricks. The results from the verification PCR can be seen in figure 1.

Finally we decided to have the submitted BioBricks sequenced. To our joy, the results showed that our BioBricks do not contain any mutations.

Characterization

Strategy

In order to characterize the BioBricks we constructed three plasmids for each Gifsy phage using fluorescent proteins as reporter genes, see table 1. We used a BioLector microreactor system for our measurements. The BioLector is a microfermenting system that measures OD and fluorescence simultaneously, which is ideal to characterize our constructs.

In the first plasmids (pIGR01 and pIGR02) we cloned a GFP reporter downstream of the pR promoter, because this would give us the level of GFP expression from the pR promoter when the represser is not present, see figure 2.

Figure 2 Characterization plasmid pIGR01/02, containing promoters and GFP reporter. Expression of GFP shows that the pR promoters are working as expected.

The next plasmids (pIGR03 and pIGR04) contain GFP downstream of pR and the repressors (GogR or GtgR) downstream of pRM, see figure 3. These plasmids would allow us to see if the repressors repress the pR promoter – causing expression of GFP to cease. If no GFP is expressed, repressor system is tight.

Figure 3 Characterization plasmid pIGR03/04, containing promoters, repressor and GFP reporter. No expression of GFP shows that the repressor is working as expected.

The last plasmids, (pIGR05 and pIGR06) contains the arabinose inducible promoter pBAD with antirepressor antO/antT and RFP located downstream, see figure 4. This means that if there is expression of RFP, the antirepressor is also expressed assuming that both proteins are equally translated. pIGR05/06 are designed to have a different origin of replication than pIGR03/04 which allows transformation of both plasmids into the same E. coli strain. This enables us to see the influence of the anti-repressor on the repressor. We expect to see expression of both GFP and RFP, as the anti-repressor will hinder the effect of the repressor on the pR promoter.

Figure 4 Characterization plasmid pIGR05/6, containing pBAD promoter, antirepressor and RFP reporter. Expression of RFP shows that the antirepressor is expressed.

Results

Our first discovery was that when growing the E. coli transformants, carrying the pIGR01 or pIGR02 plasmid, we saw a wide variety not only in colony size, but also in level of GFP expression. Colonies with visible expression of GFP were significantly smaller in size indicating that the pR promoter expresses GFP to a level that becomes toxic to the cells,see figure 5.

Figure 5 Agar-plate with colonies of E. coli carrying pIGR01. We se small colonies expressing GFP.

The transformants carrying pIGR03 or pIGR04 did not show any expression of GFP – as expected.

Figure 6 Agar-plate with colonies of E. coli carrying pIGR02 and pIGR03. We se that colonies carrying plasmid pIGR02 are expressing GFP (left), and that colonies carrying pIGR03 are not expressing any GFP(right).

These results were very interesting and must be considered when making the final design for the switch. We might even have to consider the need for a different reporter in our final design of the switch.

Sequencing of characterization plasmids

Because of the differences in colony size and GFP expression that we saw on the transformation plates, see figure 5 and 6, we decided to have the plasmids pIGR01-04 sequenced. Results show that out of three chosen colonies two were mutated for strains carrying pIGR01 and one was mutated for strains carrying pIGR02. For pIGR03 and pIGR04 we chose two colonies carrying each plasmid, and we found one silent mutation in the repressor gogR, but none in the rest.

Biolector

We picked five different colonies from each construction and ran them on the BioLector.