V09_10_1 Miniprep of pSB1C3-motA, pSB1C3-motB and pSB1C3-yhjH
Experiment: Plasmids were prepped using PeqGOLD MiniPrep Kit (Peqlab) according to the manual.
V09_10_2 Test digestion of pSB1C3-motA, pSB1C3-motB and pSB1C3-yhjH
Experiment: Test digestion of the three constructs was performed with the restriction enzymes EcoRI and PstI. Afterwards, the product was loaded in a 1% analytical gel in order to verify its success.
Observations & Results:
The digestion worked for all clones since fragments with the appropriate size were produced over all.
V09_10_3 Preparative double digestion of motA, motB, yhjH and three different promoter-constructs
Experiment:
In order to investigate our genes of interest under the control of different promoter strengths, we planned to clone our genes into three different plasmids with different promoters. The genes of interest, motA, motB and yhjH were digested with XbaI and PstI. The plasmids with the different promoters were treated with SpeI and PstI. The digestion was then controlled via gel-electrophoresis.
Observations & Results:
Since for all clones band of the expected size could be obtained these were isolated from the gel and purified using PeqGOLD Gelextraction Kit (Peqlab) according to the manual.
V09_10_4 Repetition of the Overlapping PCR of fliC
Experiment: This time the Overlapping PCR was performed using the designed primers and a new PfuTurbo polymerase (protocol). Also this time four short fragments were produced first and subsequently combined to a complete final DNA fragment.
Observations & Results:
Once again the Overlapping PCR failed for no PCR product was observed. Whether the primers, the enzyme or anything else cause the PCRs failure is not clear to us yet.
V09_11
V09_11_1 Ligation of motA, motB and yhjH into J61002
Experiment: motA, motB and yhjH were ligated into the plasmid J61002 with different promoters (20E, 20I and 18C) according to the protocol.
V09_11_2 Chemical transformation of pSB1C3-motA, pSB1C3-motB and pSB1C3-yhjH into E. coli DH10B
Experiment:
The ligation products were transformed into the E. coli DH10B as described in the standard protocol.
Observations & Results:
The transformation was successful since numerous colonies were grown on all plates except for the negative control.
V09_11_3 Repetition of Overlapping PCR of fliC (1st Part)
Experiment:
After consultation with our supervisor we decided to give the Overlapping PCR another try using Phusion polymerase this time. The first reaction round resulting in the production of the four short fragments was prepared according to the protocol and subsequently analyzed on gel.
Observations & Results:
This time the QC PCR was successful! The gel showed strong bands of the expected size. Obviously the PfuTurbo polymerase, which is known to be perfect for Overlapping PCR was the reason for the previous failures.
V09_12
V09_12_1 Repetition of the Overlapping PCR of fliC (2nd part)
Experiment:
Since the first round of amplifications was successful, the samples 1 and 2 as well as 3 and 4 were combined via overlapping PCR. Afterwards, the samples 1+2 and 3+4 were combined as well. For all reaction the new Phusion polymerase was used. The success of both reactions was investigated via agarose gel electrophoresis.
Observations & Results:
Both reactions were successful. We were able to receive fragments of a reasonable size.
V09_12_2 Preparative double digestion of J61002-Promoter-RFP constructs and pSB1C3
Experiment:
In order to bring the promoters of the BioBrick library we used for our experiments on the new BioBrick standard plasmid pSB1C3, the parts as well as pSB1C3 were digested with EcoRI and PstI as described in the protocol. The restriction products were subsequently loaded on a 1% agarose gel, cut out and purified using the PeqGOLD Gelextraction Kit (Peqlab).
Observations & Results:
The digestion was successful. We could observe bands of the correct size.
V09_12_3 Insertion of the promoter-RFP constructs into pSB1C3
Experiment:
The eight RFP-promoter constructs were ligated into the vector pSB1C3 according to the protocol.
V09_13
V09_13_1 Chemical transformation of the pSB1C3-promoter-RFP into E. coli (DH10B)
Experiment: The transformation was performed as described in the protocol.
Observations & Results:
The transformation was successful since numerous colonies were grown on all plates except for the negative control.
V09_13_2 Preparative double digestion of fliC
Experiment:
In order to clone fliC into pSB1C3 the product of the Overlapping PCR was digested with EcoRI and PstI according to the protocol. The restriction products were subsequently leaded on a 1% agarose gel, cut out and purified using the PeqGOLD MiniPrep Kit (Peqlab) according to the user manual.
Observations & Results:
The digestion was successful since fragments of the expected size could be obtained.
V09_13_3 Ligation of fliC into pSB1C3
Experiment: For the ligation of fliC into the vector pSB1C3 the protocol was followed.
V09_14
V09_14_1 Chemical transformation of the pSB1C3-fliC into E. coli (DH10B)
Experiment: The transformation was performed as described in the protocol.
Observations & Results:
The transformation was not successful since no colonies were grown on all plates except for the negative control.
V09_14_2 Preparation of over night cultures
Experiment:
Over night cultures of the pSB1C3-promoter-RFP constructs were prepared.
V09_14_3 Chemical transformation of pSB1C3-promoter-flhDC constructs into E. coli BL21 and MG1655
Experiment: The transformation was performed as described in the protocol.
V09_15
V09_15_1 Mini preps and test digestion of pSB1C3-promoter-RFP constructs
Experiment: Plasmids were prepped using PeqGOLD MiniPrep Kit (Peqlab) according to the manual. Test digestion was performed using XbaI and SpeI.
Observations & Results:
The digestion worked for all clones since fragments with the appropriate size were produced over all.
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