The general stocks (IPTG, Kanamycin, LB medium and LB agar for the plates) were prepared.

The vector ordered at Genscript (pET28a(+)) with our insert dCas9-Sbit-Lbit was dissolved and transformed into BL21 and NovaBlue. The vector transformed into BL21 will be used for proteins expression of dCas9-Sbit. The vector transformed into NovaBlue will be used for restriction and ligation to form dCas9-Lbit. In the end the bacteria were plated and incubated overnight.

The plates were taken out of the incubator and were put in the fridge. Not that many colonies were formed, however there were enough colonies for the small cultures. In the afternoon the small cultures were prepared and incubated overnight. Furthermore, the media for the large cultures were prepared. We do not know yet which medium will be most optimal for protein expression, so LB, 2YT and TB medium were prepared.

Fig. 1: Culture plates.

The small cultures were taken out of the incubator. The two cultures with BL21(DE3) cells were put on ice for protein expression later in the morning. The plasmids from the other four cultures with NovaBlue cells were isolated using the QIAprep miniprep kit. The following DNA concentrations were obtained: 215.8 ng/μL, 225.0 ng/μL, 202.0 ng/μL, 215.2 ng/μL. The samples were put in the freezer (-20oC). Tomorrow these samples will be digested and ligated to form dCas9-Lbit.
After the two small cultures on ice were at room temperature, they were divided over the three large cultures (LB, 2YT, TB). When the OD600 had reached 0.6-0.7, protein expression was initiated by adding IPTG. The large cultures were incubated overnight.

The three large cultures (hopefully with the protein dCas9-Sbit inside the bacteria) were centrifuged in several rounds. The volumes were too big for just one round. After each round, the supernatant was discarded. In the end the pellets were taken out of the centrifuge bottles and put into three falcon tubes (pellets from TB, 2YT and LB medium). The pellets were snap freezed and will be purified after the weekend.
Furthermore, the DNA retrieved yesterday (miniprep) was digested to form the dCas9-Lbit. The digested DNA was put on an agarose gel to separate the digested part from the rest of the plasmid. The ladder had leaked into the samples, however we were still able to cut out the DNA of dCas9-Lbit. The DNA was retrieved from the agarose gel using the QIAquick Gel Extraction Kit. Due to lack of time, the sample will be ligated after the weekend.

The digested DNA (from 21-06) was ligated, transformed into BL21(DE3) bacteria (for protein expression) and plated. The plates are incubated overnight (dCas9-Lbit). Furthermore, the strep buffer W and IMAC columns were prepared for protein purification (of dCas9-Sbit), which will be done tomorrow. The Strep columns will be used from Eva van Aalen (PhD supervisor).

The pellets of dCas9-Sbit were taken out of the -80oC freezer and put on ice to thaw. Subsequently, the pellets were dissolved and lysed (chemically). When the lysing was finished, the proteins were purified using IMAC columns. The elution from the IMAC columns were then put on the strep columns. Amicon filters were used to concentrate the elution from the strep columns. NanoDrop was used to measure if there were any proteins in the solution. Unfortunately there were no proteins after the purification. The question is if the proteins were expressed. Samples were taken during the whole purification process, these will be put on an SDS-page gel tomorrow to check if the protein was lost during the purification. If this is not the case, the expression protocol needs to be further looked into to optimize it for this protein.

NcoI–His tag–GGS– SacI–dCas9–AgeI–(GGS)5–HindIII–SpeI–SBit–BamHI–(GGS)2–Strep tag–stop

Ten small cultures were also made for the dCas9-Lbit which was ligated and transformed in NovaBlue yesterday.

The small cultures were taken out of the incubator and the DNA for the dCas9-Lbit was isolated using the QIAprep miniprep kit. The final DNA concentrations are shown below. Furthermore, SDS-PAGE samples were made from the flow through samples from the purification proces; His bind, His wash, Strep lysate, Strep wash, Strep elution. The samples were loaded on the gel to evaluate the purification steps. To be able to evaluate the results, the gel was washed and stained with Coomassie for approximately 40 minutes. After this, the gel is washed in water overnight on the shaking table.

Since the first round of expression and purification of the dCas9-NL_Sbit protein was unfortunately unsuccessful, we adapted the expression and purification protocol to now test 4 different sets of expression conditions and 2 different lysis methods. To start the second trial round of protein expression of our sensor we again transformed our plasmid into E. coli BL21 (DE3).

For transformation, a 4x dilution of the miniprepped plasmid DNA was prepared, taking 1 μl from plasmid tube 1 and adding together with 3 μl in another tube. Final plasmid concentration ~50 ng/μl. 1 μl of this was added to the bacteria, the remainder was placed in the freezer. The bacteria were heat-shocked for 40 s. 100 μl of the final LB/bacteria solution was spread out over an agar plate and left to incubate at room temperature over the weekend.

Also, new IPTG stock solution was prepared. We added 6.7 ml MQ water to 1.6 g IPTG, totalling 8.0 ml solution (in hind sight the total solution should have been 6.7 ml to obtain 1M conc.), resulting in a 0.84M stock. Hence, for 1mM final concentration, add 1.2 ml stock IPTG to 1L medium. For 200nM final concentration, add 0.25 ml stock IPTG.

From the SDS-PAGE results we concluded that no dCas9-Sbit was formed. Therefore a new plan was made to test different combinations of expression conditions based on information we gathered. The plan is to test four different condition combinations.

The following conditions will be the same:

• Host: BL21(DE3)
• Medium: 750 mL LB
• OD600: 0.5
• Half of the culture will be lysed with ultrasound, the other half with Homogenizer