- 3rd: Once again, the transformation of E.
coli was attempted with the parts: promoter, RBS and terminator from
the 2023 plates.
The minipreps of the backbone and
terminator performed on June 30th were quantified using NanoDrop
2000.
Furthermore, inoculations of the
backbone and terminator were repeated in selective LB broth.
Additionally, an inoculation in selective LB broth was performed for
the RBS isolated through previous replating.
- 4th: Once again, no transformants were
obtained for either the promoter or terminator. For the RBS, fewer
than 5 colonies were obtained, displaying the same abnormal biofilm
growth beneath the colonies.
Using the inocula from July 3rd,
cryovials were prepared for the RBS, promoter, and backbone. With
the remaining inoculations, minipreps were conducted for the RBS,
promoter, and backbone.
Immediately after completing the
minipreps, quantification of all three minipreps was performed using
NanoDrop 2000.
To verify the sensitivity of the used
E. coli 5-alpha strain to the antibiotic chloramphenicol
(used for
selecting RBS transformants), non-transformed bacteria were replated
on an agar plate with chloramphenicol (25 µg/mL) and left to
incubate.
A Gram staining of the RBS was
performed to rule out the presence of any other strain resistant to
the antibiotic chloramphenicol. The result showed pink bacilli in
isolated arrangement, characteristic of E. coli.
Finally, electrophoresis was conducted
for the minipreps carried out on this day (backbone, terminator, and
RBS) and those from July 3rd (backbone and terminator).
- 5th: Upon reviewing the plating of
non-transformed E. coli 5-alpha seeded on a chloramphenicol
plate
(25 µg/mL), it was discovered that there was biofilm growth. Further
investigation on the specific strain from the supplier's page
revealed that the chloramphenicol concentration was not adequate,
but rather below the recommended level. The necessary concentration
was 33 µg/mL, not 25 µg/mL. This was attributed to the previous
results with the RBS. Additional media were prepared with the
adjusted chloramphenicol concentration.
5-alpha chemically competent cells
were prepared as the commercial chemically competent cells were
depleted. Subsequently, a transformation test was carried out with
the control plasmid pUC19.
- 6th:
The transformation result with the
control plasmid yielded 6 colonies. It was decided to repeat the
experiment with two main changes: the volumes were halved, and the
transformation protocol from the previous day was replaced with the
one recommended by the cell provider.
- 7th: The previous day's transformation
yielded 5 transformants. Given this equally unsatisfactory result,
it was decided to repeat the process early in the following week.
Competent cells were prepared to test
their effectiveness after being frozen at -80 °C. This experiment
would be conducted at the beginning of the following week.
- 10th: Due to the results of the
transformations with the parts from the 2023 plates, it was decided
to pursue an alternative approach. Previously, Twist Bioscience was
commissioned to synthesize complete transcriptional units (TUs) for
LysK and LysCSA13. The synthesized DNA fragment was then resuspended
for
subsequent assembly into the successful BBa_J435330 backbone
recovered from plate 2023. The resuspension of the synthesized pDNA
was carried out at a final stock concentration, and aliquots of each
TU were immediately prepared.
Furthermore, a pre-inoculum of E.
coli
5-alpha was prepared for subsequent use in producing chemically
competent cells.
- 11th: The chemically competent cell
production protocol was executed, with the modification of employing
a larger EM flask (250 mL) in an attempt to enhance transformation
efficiency by allowing better cell growth. The transformation was
carried out with the control plasmid pUC19 using the freshly
prepared cells and also with the cells thawed from July 7th. This
was done to assess transformation efficiency after undergoing deep
freezing.
- 12th: The transformation result from the
previous day yielded approximately 40 colonies, signifying a
significant improvement in transformation efficiency. This confirmed
that the use of larger flasks for the production of competent cells
was a better choice.
A pre-inoculum of E. coli
5-alpha was
prepared for subsequent use in competent cell production.
- 13th: It was decided to repeat the
production of chemically competent cells and the transformation,
this time varying the incubation time for cell recovery after the
heat shock. We tested with 1 and 2 hours.
A pre-inoculum of E. coli
5-alpha was
prepared for subsequent use in competent cell production.
- 14th: The transformation yielded between
40-50 colonies for the one-hour incubation, and between 80-90
colonies for the two-hour incubation. It was established that a
two-hour incubation period would be used for all future
transformations.
- 17th: A pre-inoculum of E. coli
5-alpha was
prepared for subsequent use in competent cell production.
- 18th: We performed the Golden Gate assembly
protocol to assemble the synthesized TUs of LysK and LysCSA13 into
the BBa_J435330 backbone.
Subsequently, we prepared chemically
competent E. coli 5-alpha cells and conducted the
transformation
protocol using the products of the Golden Gate assembly as genetic
material to transform.
A pre-inoculum of E. coli
5-alpha was
prepared for subsequent use in competent cell production.
- 19th: As a result of the transformation,
colonies grew on all plated dishes, including the negative controls.
Given this outcome and considering that the utilized backbone
typically possesses two antibiotic resistances, but only one when
ligated, re-plating was performed for screening with antibiotics.
This was done to determine whether any of the colonies had been
transformed with the cloned plasmid or if only colonies with the
unligated backbone were present./li>
- 20th: It was determined that two of the
re-plated quadrants, 16 and 18 of BBa_J435330-LysK, exhibited the
characteristic of having only one antibiotic resistance (kanamycin
and not ampicillin). This is typical of the ligated backbone, which
loses the ampicillin resistance gene after ligation. Consequently,
inoculations of these two quadrants were made in selective LB broth
for subsequent miniprep and analysis via electrophoresis.
No quadrant of BBa_J435330-LysCSA13
exhibited the same behavior.
- 21st: When retrieving the inoculations from
the previous day from quadrants 16 and 18 of BBa_J435330-LysK, we
noticed there was hardly any growth, as the turbidity of the broth
was nearly imperceptible.
Inoculations for quadrants 16 and 18
of BBa_J435330-LysK were repeated, taking special care to add the
appropriate amount of kanamycin antibiotic (50 µg/mL) to the LB
broth.
- 22nd: This time, the inoculations showed
growth, so minipreps were performed for both BBa_J435330-LysK
quadrants 16 and 18.
Subsequently, each miniprep was quantified on NanoDrop 2000 and
visualized on agarose gels through electrophoresis.
- 24th: Inoculations were repeated in
selective broth (kanamycin 50 µg/mL) for quadrants 16 and 18 of
BBa_J435330-LysK.
In an effort to retest the antibiotic
screening to identify colonies transformed with the
BBa_J435330-LysCSA13, 32 colonies solely from this transformation
were replated.
- 25th: This time, there were indeed quadrants
of BBa_J435330-LysCSA13 that displayed the expected behavior:
colonies resistant to kanamycin (50 µg/mL) but not to ampicillin
(100 µg/mL). Quadrants 9, 18, 20, and 26 stood out.
From the aforementioned four
quadrants, inoculations were made in selective LB broth (kanamycin
50 µg/mL).
Cryovials were prepared for quadrants
16 and 18 of BBa_J435330-LysK.
- 26th: Minipreps were conducted for quadrants
9, 18, 20, and 26 of BBa_J435330-LysCSA13. They were promptly
quantified using NanoDrop 2000 and visualized through agarose gel
electrophoresis.
Additionally, inoculations in
selective LB broth were repeated for these four quadrants.
- 27th: Cryovials were prepared for all four
quadrants of BBa_J435330-LysCSA13.
Enzymatic digestions were conducted
using the single-cut enzyme SmaI to verify the plasmids extracted
from the quadrants of both BBa_J435330-LysK and
BBa_J435330-LysCSA13. These digestions were visualized using agarose
gel electrophoresis.
- 28th: A transformation of the E. coli
Bl21
(DE3) strain was carried out with the BBa_J435330-LysK and
BBa_J435330-LysCSA13 obtained through Golden Gate assembly.
- 31st: Pre-inoculum of E. coli BL21
(DE3)
were conducted in selective LB broth (kanamycin 50 µg/mL) for
BBa_J435330-LysK and BBa_J435330-LysCSA13 transformants.
Additionally, a pre-inoculum for a negative control, untransformed
E. coli BL21 (DE3), was performed.
