Purified PCR products were treated with the wrong restriction enzymes, resulting in a worthless ligation. PCR was done again to repeat the experiment using correct enzymes.
Performed colony PCR on P. putida with the following primer combinations:
Reaction 1: P01_RFC10_pp1262_fwd + P03_RFC10_FAdP_rev
Reaction 2: P01_RFC10_pp1262_fwd + p04_RFC10_FAdP_Delta-RBS_rev
Reaction 3: P01_RFC10_pp1262_fwd + p05_RFC10_FAdP_Delta-RBS2_rev
Reaction 4: P01_RFC10_pp1262_fwd + p06_RFC10_FAdP_Delta-RBS3_rev
All PCR reactions resulted in a positive band. The product was isolated with a DNA purification kit, using 50 ul of elution buffer.
Concentrations according to the nanodrop:
J01: 25ng/ul
J02: 15ng/ul
J03: 15ng/ul
J04: 15ng/ul
Products are restriction digested:
J01: 500/25 = 20 ul
J02: 500/15 = 33 ul
J03: 500/15 = 33 ul
J04: 500/15 = 33 ul
pSB1C3: 500/80 = 6.25 ul
Added 1 ul EcoRI, 1 ul SpeI, 5 ul buffer and MQ to a total of 50 ul.
Digested for 60 minutes at 37°C.
Denatured for 20 minutes at 80°C.s
Put all the parts (digested and undigested) on gel. In J01-J04, no difference in band size was detectable, because the cut off part is only a few bp. The vector showed 1 band when uncut and 2 bands when cut.
Added J01-J04 to the cut vector pSB1C3 and ligated:
10 minutes at room temperature
20 minutes denature at 80°C
The ligated parts now contain either mRFP or J01-J04.
These vectors are used for chemical transformation, using a heatshock of 42°C on chemically competent E. coli cells. After 2 hours of growing on SOC medium, 35 ul and 175 ul is poured onto plates containing Cm antibiotics, then incubated at 37°C.
Picked white colonies from all plates, dipped them into PCR mix to check for inserts (same primers as before, to detect the insert itself, p01-P06). Also, every picked colony is grown over night in LB containing Cm antibiotics in the 37°C climate room, 160 rpm rotating machine.
PCR showed only inserts in 3 of the J01 colonies and 1 of the J03 colonies. Both strains are grown overnight an additional time, to make stock.
The J03 insert seemed to be mRFP, since it was completely red in the morning. J01 had grown normally and was made into a glycerol stock.
Picked different colonies for J02-J04 from the same plates and tried to find the right insert again. Strains grown in LB medium again. PCR gave unclear results, so repeated the PCR in the morning, with only the non-red strains: Only 1 strain of J04 had the insert.
Used miniprep to isolate the plasmids of the successful J01 and J04 strains. After nanodropping, they had a concentration of 110ng/ul and 85ng/ul, respectively. Both samples are sent off for sequencing.
PCR is performed on putida again, to obtain J02 and J03 once more and try to isolate those as well for transformation purposes.
Reaction 1: P01_RFC10_pp1262_fwd + p04_RFC10_FAdP_Delta-RBS_rev
Reaction 2: P01_RFC10_pp1262_fwd + p05_RFC10_FAdP_Delta-RBS2_rev
Results will be put on gel
Inoculated 5ml + 5ul Cm with the successful J04 strain to make glycerol stock. Put in the 37°C climate room at 160 RPM.
J02 and J03 were still vague bands. The products were ligated and transformed into E. coli. However, no transformations took place.
The sequences of BBa_J01 and BBa_J04 were returned. J01 was indeed the right insert of the pp1262 and intergenic region (including RBS). J04 was as well, yet there was a mistake in the promoter region, probably leading from a mistake by the company in the primer design.
Cut the isolated BBa_J01 and BBa_J04 vectors with EcoRI and SpeI. Also added these restriction enzymes to BBa_J23100, a reporter biobrick, with its active promoter between E & S. By inserting the J01 or J04 promoter, the reporter should in theory only work if the added promoter is active. By using alkaline phosphatase, the ability to self-ligate became much smaller.
After using the ligated products for transformation, many white colonies were present. 4 of each is picked, PCR’ed (with VF2 and VR primers) and grown overnight in 5ml LB medium + Amp. The PCR showed inserts in 1 of each colony (1.2 and 4.1). Also made stocks of these two transformed strains.
Performed the following PCR
p01 + p04, Q5, J01+J23100
VF2 + VR, Q5, J01+J23100
p01 + p04, Q5, J04+J23100
VF2 + VR, Q5, J04+J23100
p01 + p04, Q5, BBa_J01 (positive control)
VF2 + VR, Q5, BBa_J01 (positive control)
p01 + p04, Q5 (negative control)
VF2 + VR, Q5 (negative control)
Also performed a gradient PCR for J02 and J03, ranging from Ta 56 to 66
The band of the J01 part has the same size as the band of the J01 part + mRFP part from the J23100 biobrick. This indicates something is wrong. To find out what happened, restriction ligations are performed on J01, BBa_J23100 and J01R (the J01 part in the J23100 reporter plasmid) with restriction enzymes E-S, S-P and E-S. The results of J01 and BBa_J23100 are as expected. However, the J01R gives questionable results. To gain further insight into this vector, PCR is run with all combinations of the primers VF2, VR, p01forward and p03reverse. All experiments give a band. To be sure what happened, both J01R and J04R are sent for sequencing with primers VF2 and VR.
A new approach is started, using the BBa_I13507 biobrick (RBS, mRFP and 2 terminators in a pSB1A2 plasmid). J01 and J04 are cut with E&S, I13507 with E&X. Both parts are ligated together.
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