Team:Hamburg/Part Collection
Both trigger compositions contain strong constitutive promoters
(BBa_J23102) and terminators (BBa_B1002) along with the trigger
sequences. The gate composition contains a strong constitutive promoter
(BBa_J23100) and a terminator (BBa_B1002) as well. Downstream of the
gate sequence we implemented a chloramphenicol resistance gene to give
the bacteria ability to be selected. To use these parts, they should be
inserted into a backbone, where the antibiotic resistance for amplification can
be cut out.
One advantage of this collection is that users can add genes of interest to all
plasmids. The chloramphenicol resistance is translated only in bacteria that
contain all three plasmids, thus guaranteeing all genes of interest will
exclusively be expressed together. This works with one instead of three
different antibiotics, which means less stress to bacteria. Simultaneously it
shows the potential of RNA based gene regulation on translational level. Figure 1: A) Formation of trigger complex after translation. B) mRNA of gate sequence forms
secondary structures that hide the ribosome binding site and start codon. Figure 2: Opening of the gate due to annealing of trigger complex to gate.To use this system in
bacteria we implemented the gate sequence together with a gene for chloramphenicol
(BBa_K2970011), flanked by a constitutive promoter (BBa_J23100) and a strong terminator
(BBa_B1002) into pSB1A3 where the ampicillin resistance can be cut out.
After transformation of both trigger plasmids (BBa_K2970003 and
BBa_K2970004) and the gate plasmid in one bacterium all three mRNA
structures will be formed, the gate will be opened, and the translation of the
chloramphenicol resistance can take place. Bacteria that took all three
plasmids are able to survive on media with chloramphenicol. This collection
can be used together with both trigger compositions for triple transformation
in bacteria. Genes of interest that should be transformed together, can be put
on the three plasmids. Only if all three plasmids are taken by a bacterium the
chloramphenicol resistance is produced and the bacterium can survive on
medium with chloramphenicol. Thus chloramphenicol can be used to select
for bacteria that got all genes of interest. We tested the gate plasmid with our two trigger plasmids by performing
simultaneous transformations with these plasmids. After transformation we selected
the bacteria by plating on LB-agar plates with chloramphenicol. We compared the
results with positive and negative controls, using the empty pSB1C3 backbone and
pSB1A3 respectively.
The result of the transformation with all three plasmids after selection with
chloramphenicol is shown in figure 3. Figure 3: E. coli cells transformed with three plasmids, the plasmid carrying the
gate, the plasmid carrying trigger one and the plasmid carrying trigger two. The
cells were plated on LB-agar plates with chloramphenicol, bacterial growth was
observed. Colonies grew on chloramphenicol plates after transformation with all three plasmids. This
shows that our RNA based logic circuit allows the expression of the antibiotic resistance. No
colonies grew on the plates with the negative control. Colonies also grew on the plates with
the positive control.
To further test the trigger and the gate, we performed transformations with each plasmid
individually. The results are shown in figure 4. Figure 4: Cell plated out on chloramphenicol agar plates. Left: cells transformed
with the gate plasmid. Middle: cells transformed with the trigger 1 plasmid. Right: cells
transformed with the trigger 2 plasmid. Unfortunately colonies also grew on the plates with just the individual transformation of the
gate plasmid, though the number of colonies was much lower when compared to the
transformation with all three plasmids. We sequenced the gate plasmid to confirm correct
assembly. The results showed no mutation in the gate sequence. We concluded that the
gate is showing signs of leakage, expressing the antibiotic resistance even in the absence of
triggers. This basal expression was rather strong due to the strong promoter we used for our
experiments. To measure the gates leakage, we observed cells transformed with all three plasmids on
plates with different chloramphenicol concentrations and compared them to cells transformed
with only the gate plasmid. As a positive control we used the pSB1C3 backbone. The Excel
file used for the calibration can be viewed here. The number of colonies per plate after twelve hours of
incubation is shown in figure 5. Figure 5: Number of colonies per plate depending on the chloramphenicol
concentration. E. coli DH5α cells were transformed with the gate plasmid (blue), the
gate plasmid together with both trigger plasmids (red), or with the control backbone
pSB1C3 (green). The colonies were counted after 12 hours. The data clearly shows that the gate is leaking, but when compared to the triple
transformation (red) and the control (green) the number of colonies when transformed with
only the gate plasmid was significantly lower. Additionally the number of colonies when
transformed with all three plasmids was larger than when transformed with the positive
control.
We also used a plate reader to measure the growth rate of cells that were transformed with
three different plasmids (pSB1A3, pSB1C3, pSB1K3 or BBa_K2970003, BBa_K2970004,
BBa_K2970006) and compared it to cells without any plasmids. The results are shown in
figure 6. Figure 6: Plate reader growth curves of E. coli DH5α. The generation time was
measured and calculated for non competent cells (orange), for cells transformed with the
backbones pSB1A3, pSB1C3 and pSC1K3.M1 (blue), and for cells transformed with our two
trigger plasmids together with the gate plasmid (green). Error bars show the standard
deviation. For cells transformed with three plasmids an impaired growth rate can be observed.
We could not show that the transformation of our three plasmids was less harmful to
the bacteria than the transformation with three different antibiotics, instead it shows
comparable levels of stress.
Due to the linker between the gate and our gene of interest (chloramphenicol acetyl
transferase) additional bases were attached to the gene which might affect the
functionality of the protein as it contained an additional 19 amino acids. To test the
genes activity we inserted the sequence containing the additional bases into
pSB1A3, transformed it into bacteria and plated the cells on chloramphenicol plates.
The results of this experiment are shown in figure 7. Figure 7: Test transformation to check chloramphenicol resistance
gene. E. coli DH5α cells were transformed with a test plasmid containing the
modified chloramphenicol resistance gene (A). For negative control (B)
pSB1A3 was used. For positive control (C) pSB1C3 was used. Several colonies grew on the plate with bacteria transformed with the modified
chloramphenicol acetyl transferase. The negative control showed no cell growth
while on the plate with the positive control a cell turf has grown. This confirms that
the activity of the chloramphenicol acetyl transferase was not significantly impaired
by the additional amino acids. Collection
The RIBOT collection
BBa_K2970003 - Trigger 1 Composition
BBa_K2970004 - Trigger 2 Composition
BBa_K2970006 - Gate Composition
-Gate Composition
Results
