Part:BBa_K5427000
RBS 1_12000
The Ribosome Binding Site (RBS) is a crucial sequence in mRNA that facilitates the initiation of translation in prokaryotes. RBS3 is a specific short nucleotide sequence located upstream of the start codon, typically including a Shine-Dalgarno sequence. This Shine-Dalgarno sequence is complementary to a region at the 3' end of the 16S rRNA, enabling the ribosome to align correctly with the start codon and ensuring efficient initiation of protein synthesis (Ryu & Gomelsky, 2014).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Background Information
RBS 1, 2, 3, 4 (alongside T7 inducible promoter) were previously tested for their efficiency to control and regulate synthetic operon containing bphS_bphO_yhjH genes (diguanylate cyclase (DGC), heme oxygenase (BphO) and phosphodiesterase (PDE), respectively.
Part Assembly
ptac + RBS1_12000 Constructs
Construct 1: pTac_RBS1_sfGFP in pJUMP24
One of the primary goals of the ReneWool 2024 project was to produce spider silk using a synthetic plasmid construct expressed in bacteria. To achieve this, we designed and constructed four plasmid variants, designated as pJUMP24-pTac-RBS(1-4)-sfGFP. These plasmids were created to investigate how different ribosome binding sites (RBS) affect the proliferation of prokaryotic species in LB media. To begin, the vector plasmid pJUMP24-sfGFP was PCR-linearized to incorporate Gibson assembly-compatible overlapping sequences. This was achieved using forward primer #BBa_K5427045 and reverse primer #BBa_K5427046, aiming to generate RBS1 and RBS2-compatible linearized plasmids. The promoter pTac_RBS1 and pTac_RBS2 fragments were PCR-amplified using forward primer #BBa_K5427055 and reverse primer #BBa_K5427047. For the pTac-RBS3 construct, forward primer #BBa_K5427055 and reverse primer #BBa_K5427050 were employed, while the pJUMP24 vector was linearized with primers #BBa_K5427049 and #BBa_K5427046 to ensure compatibility with the RBS3 fragment. Similarly, the pTac-RBS4 amplification used forward primer #BBa_K5427055 and reverse primer #BBa_K5427052. The pJUMP24 vector was linearized with primers #BBa_K5427051 and #BBa_K5427046 to facilitate Gibson assembly with the RBS4 fragment. All constructs were assembled using the designated RBS fragments and their corresponding linearized vectors through standardized Gibson assembly, employing the NEBuilder HiFi DNA Assembly Master Mix (Lot #10238675) following the established protocol, as described in the construction of pET22b(+)-T7-Krt31.(#BBa_K5427070)
Construct 2: pTac_RBS1_SpiderSilk_sfGLP in pJUMP24
We tested the four previously constructed RBS-containing synthetic plasmid sequences, and the preliminary results indicated that RBS1 exhibited the highest growth potential for E. coli in LB media. Based on these findings, we selected RBS1 as the most suitable candidate for prokaryotic production of spider silk. Accordingly, we constructed the synthetic plasmid pJUMP24-pTac-RBS1-SpiderSilk-sfGFP using the following strategies.
We linearized the vector pJUMP24-pTac-RBS1-sfGFP through PCR amplification to introduce Gibson assembly-compatible overlapping tail sequences, using forward and reverse primers #BBa_K5427034 and #BBa_K5427035, respectively. The forward primer was specifically designed to introduce a spacer between the sfGFP gene and the spider silk gene to provide the necessary structural flexibility. The spider silk gene block was ordered from IDT and amplified using primers #BBa_K5427036 (forward) and #BBa_K5427037 (reverse).
J23119 + RBS1_12000 Constructs
Construct 3: J23119_RBS1 in pJUMP28
We constructed a new plasmid, pJUMP24-J23119-RBS1, to perform a comparative growth experiment between the IPTG-inducible promoter pTac and the constitutive promoter J23119, assessing their effects on bacterial growth in both solid and liquid LB media. The pJUMP24 vector and the J23119_RBS1 upstream regulatory sequence were double-digested with EcoRI and PstI restriction enzymes. Agarose gel electrophoresis confirmed the success of the digestion. The cleaved nucleotide fragments were ligated using NEB T4 DNA Ligase following the standardized protocol. Recombinant plasmid formation was further confirmed by agarose gel electrophoresis. The synthesized pJUMP24-J23119-RBS1 plasmid was then transformed into E. coli. The growth of the transformed bacteria was analyzed and quantitatively compared to those transformed as described in pJUMP24-pTac-RBS1-SpiderSilk-sfGFP plasmid (BBa_K5427075).
Construct 4: J23119_RBS1_KerDZ in pJUMP28
We constructed a synthetic plasmid to express the keratin hydrolase, KerDZ. The KerDZ gene, originally derived from Actinomadura viridilutea strain DZ50, was synthesized and ordered from IDT. The pJUMP28-J23119-RBS1 vector backbone underwent sequential double digestion using the restriction enzymes SpeI and PstI. To introduce compatible restriction sites for pJUMP28, the KerDZ gene block was amplified via PCR using the forward primer P_pre10 (iGEM 2023 Designation #BBa_K4755025) and the reverse primer P_suf10 (iGEM 2023 Designation #BBa_K4755027). Both primers were sourced from the UAlberta iGEM 2023 project’s primer collection. Successful amplification was confirmed by agarose gel electrophoresis. The PCR product was subsequently double-digested with XbaI and PstI and ligated into the vector using NEB T4 DNA Ligase according to standard protocols.
Characterization of RBS 1_12000
pTac_RBS1_sfGFP in pJUMP24 Results
This experiment was used to compare the four different RBS’ by measuring both the growth of each strain of bacteria, and the amount of fluorescence generated by sfGFP in each sample. The best RBS would theoretically produce the strongest sfGFP signal/appearance and have minimal detrimental effects to the growth of the bacteria that it was transformed into. This experiment was repeated in two different growing conditions (30℃ and 37℃) to observe how temperature affected the plasmid containing bacteria. The conditions at 30℃ slowed the growth of each bacteria but it is unlikely that the transformed plasmids had any effect. There was no appreciable change in growth or generation of sfGFP for each RBS tested. While there was no statistical significance between the different RBS’ tested it seems that RBS 4 routinely resulted in less overall growth of each bacterial strain. On the opposite end of the spectrum RBS 1_12000 seemed to have the most positive effect on bacterial growth overall
Figure 1 | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta Gami. These strains were cloned with our construct and measured for growth and 30C using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
Figure 2 | Growth curve of pTac_RBS1/2/3/4_sfGFP_pJUMP24 vector in 4 different E. coli bacterial strains; A) DH5alpha, B) BL21, C) K12, and D) Rosetta Gami. These strains were cloned with our construct and measured for growth and 37C using optical density of 600 nm to measure the growth of each strain. OD measurements were taken every 2 hours for a total of 10 hours for each culture while growing in liquid LB.
To determine overall production of sfGFP we performed a fluorometric characterization experiment. E. coli strain DH5alpha was induced with 3mM IPTG for 3 hours and subsequently lysed via French Press. Fluorescence was then measured for ribosome binding sites 1,2,3 and 4. Our results indicated that RBS1 showed a relatively strong fluorescence signal of 10.33, compared to RBS’s 2,3 and 4 signals of -0.66, -4.66, and -2.66, respectively. We then concluded that RBS1 produces sfGFP at a 1665.15% higher rate compared to RBS2. This result further confirms that RBS1 is the best ribosome binding site for our system when considering both growth and overall fluorescence production.
Figure 3 | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after 3 hour induction with 3 mM IPTG.
Figure 4 | Fluorescence (with blank subtracted) data for sfGFP isolated from DH5a containing the pTac_RBS 1/2/3/4_sfGFP_pJUMP 24 plasmid after overnight induction with 1 mM IPTG.
These pictures are all of the growth cultures of p(RBS 1,2,3,4_sfGFP_pJump24) in BL21, K12, DH5a, and R.Gammi. The fluorescence was measured after the 10 hour growth curve was performed. Notably, the only fluorescence was in two of the positive no-insert controls for the pJUMP24 plasmid, no other samples generated enough sfGFP to be detected by eye. The two vials containing the visible sfGFP left to right are DH5a and R.Gammi.
Figure 5 | Samples of RBS 1,2,3,4_sfGFP_pJump24 for BL21, K12, DH5a, and R.Gammi being tested for fluorescence.
pTac_RBS1_SpiderSilk_sfGLP in pJUMP24 Results
The results from our growth curve indicated that there was a significant difference between the two variables (empty vector vrs construct). Each culture was grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm. The data showed that although both strains grew at a controlled rate, the empty vector seemed to outperform our construct. We saw that our construct variable’s exponential phase of growth lasted until around the 6 hour mark where we can see a beginning of plateauing into stationary phase. When we compared that with the empty vector growth, we saw a continuation of the stationary phase until the end of our experiment. Specifically at the 10 hour mark we observed a 48.03% reduction in growth of our vector in DH5a. We concluded that there must be some metabolic strain that our construct placed on the bacteria. Other literature has stated that in E. coli during silk protein synthesis upregulates stress response proteins, and therefore hinder growth significantly. Since we still observed growth, just significantly diminished, we determined that spider silk production would still be possible, but at a lower output than desired and that its growth rate does not affect biomass accumulation.
Figure 6 | Growth Curve for pTac_RBS 1_Spider silk_sfGFP_pJUMP 24 in E. coli strain DH5a and a DH5a empty vector control at 37 degrees grown in regular LB media. Each culture was grown for 10 hours and measurements of Optical Density (OD) at 600nm was taken every 2 hours. Each culture had 3 replicates grown and measured for OD, the average values were taken and plotted on the growth curve chart for analysis.
J23119_RBS1 in pJUMP28 Results
Testing our J23119 constitutional promoter constructs in Rosetta Gami, DH5alpha, and BL21 strains allowed us to see if there was any metabolic difference between the growth of these strains when compared to each other. As seen in Figure 7, our constructs under perform when compared to the empty vector controls within the same strain. BL21 transformed vector was the only strain to outcompete its empty vector control, but was the worst at overall growth when compared to transformed DH5a and Rosetta Gami. Although DH5alpha and Rosetta Gami grew the best of the transformed construct strains, with an OD value of 0.414 and 0.338, respectively at hour 10, its value was lower than the non-transformed controls. This informed us that our construct placed some metabolic burden on DH5aplha and Rosetta Gami, but since growth was seen, biomass production is still of value. As there was not a significant difference between the strains, we continued to use DH5alpha to determine if salt concentrations had any metabolic effect on our strains transformed with our plasmid. Figure 8 highlights the effect of salt concentrations on our E. coli growth factors at 37C. Our experiment shows 2 conditions, the first our cultures were grown with regular LB, the second with Lennox LB which has 50% lower sodium contents. We used DH5alpha as determined in figure 7 to be an optimal strain to grow with the plasmid (J23119_RBS1_pJUMP28). We concluded from this growth curve that low sodium positively promotes the growth of transformed DH5alpha when compared to normal salt conditions. Salt concentration therefore seems to have a slight effect on the metabolic processes within the cell, but although we see an increase in growth for lower concentrations, there is no real significant difference between the conditions. Overall these results indicated that our construct could successfully be transformed into these strains tested, without inhibiting growth all together. Therefore we conclude that our constitutional promoter tested here will result in promoting effective keratin degradation within our system.
Figure 7 | Growth curve for J23119_RBS1_pJUMP28 in E. coli strains DH5alpha, Rosetta gami, and BL21. Cultures were grown for 10 hours at 37C in regular LB media, and measured for Optical Density (OD) every 2 hours at 600nm. Each culture was repeated 3 times and its averages were plotted on a growth curve for analysis.
Figure 8 | Growth curve for J23119_RBS1_pJUMP28 in E. coli strains DH5alpha grown in regular LB or Lennox LB (low salt condition) at 37C. Each culture was grown for 6 hours, and Optical Density (OD) was measured every 1.5 hours at 600nm.
J23119_RBS1_KerDZ in pJUMP28 Results
Once our construct was created with our keratin degradation enzyme, we transformed this plasmid into DH5alpha, BL21, and Rosetta Gami, and performed a growth curve analysis. Salt stress may have a small effect on the cell metabolism, therefore we grew each strain in regular and low salt LB. Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
The growth curves of each E. coli strain in regular LB and low salt LB categorized the metabolic effect of salt between each strain, as well as highlighting the overall growth rate. Figure 9 showed the effects of each strain of E. coli when growing in regular LB which allowed us to examine the effect our plasmid had on each strain. We then concluded that our KerDZ construct grows most effectively in DH5alpha and BL21, notably at 10 hours we saw a 51.85% and 23.67% increase in growth, respectively, in comparison to Rosetta Gami at the same time mark. Although we observed adequate growth from both BL21 and DH5alpha, when compared to the empty vector controls DH5alpha under performed, whereas BL21 grew more than its control. These results are consistent with what we observed previously where BL21 grew better than its control and DH5alpha the opposite. Therefore we determined that either BL21 or DH5alpha would be suited for transformation of our vector. We did not exhibit any significantly large inhibitions of growth between any strain. We then performed this experiment again with cells incubating in Lennox LB (low sodium). Figure 10 indicates the same growth factors as seen in figure 9. Even in low salt concentrations KerDZ grew the most in DH5alpha, and then BL21, where Rosetta Gami performed the worst. Consistent with our previous growth curve, BL21 grew better than its empty vector control, and DH5alpha grew less than its control. These tests confirm previous results and allow us to conclude that either BL21 or DH5alpha would give us adequate growth with our constructs. We then compared figure 9 and figure 10 to determine if there was any significant difference in salt concentrations on growth. Comparing the OD measurements at 10 hours for each strain between low salt conditions and high, we determined that these cells grew better in regular LB. Rosetta Gami at 10 hours of growth had an average OD measurement of 0.38 in regular LB but a measurement of 0.269 in Lennox LB, meaning we saw a 29% increase in growth for regular LB. Similarly, BL21 showed a 16.6% increase in growth in regular LB at the 10th hour. Contradictorily, Rosetta Gami when comparing both regular and Lennox LB saw a 0.25% reduction in growth in the regular LB at hour 10. These results indicate that there is very little if any salt stress happening in the cells for regular LB compared to Lennox. We then compared this result to earlier phases, figure 10 growth curve which tests a similar construct but without KerDZ, where we saw an increase in growth during the low salt condition. Since in figure 9 we saw the opposite results, where lower salt inhibited the growth of our organism, we determined that the KerDZ construct must have a higher tolerance to salt concentrations within the media, which allowed the plasmids to grow better in higher salt than seen in previous experiments. We can also conclude that there is very little effect that salt concentration has on the overall growth of the E. coli strains, and there was no real significant difference between the variables themselves.
Figure 9 | Growth Curve for J23119_RBS 1_KerDZ_pJUMP 28 in DH5a, R. Gami and BL21 at 37 degrees in Regular LB. Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
Figure 10 | Growth Curve for J23119_RBS 1_KerDZ_pJUMP 28 in DH5a, R. Gami and BL21 at 37 degrees in Lennox LB Cultures were grown for 10 hours and Optical Density (OD) was taken every 2 hours at 600nm.
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