Part:BBa_K5427074
pTac + RBS 4_59 + sfGFP
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. In the beginning phase of our project we wanted to determine the strongest ribosome binding site within our construct, ensuring we should achieve optimal protein production. For this experiment we developed 4 key constructs as seen in the figures below.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 107
Illegal NheI site found at 130 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 918
Illegal BsaI.rc site found at 91
Illegal SapI.rc site found at 186
Assembly Information
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).
Characterization
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 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 30℃ 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 37℃ 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.
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