Team photo in the park at Frederiksberg Campus

Gel purification of bands from PCR amplification and subsequent nanodrop. Due to poor yield from this extraction gradient PCR was performed (56-68 degrees).



Traditional cloning of eGFP fragments into pDRIVE. Transformation of eFbFP-pEX-A (synthesized gene, Eurofins) and eGFP-pDRIVE.

cultures. Samples sent for sequencing.

Overnight cultures were made of colonies 1-4 (eFbFP-pEX-A). Miniprep of eFbFP-pEX-A cultures. Samples sent for sequencing. Colony PCR of eGFP-pDRIVE was repeated with the same outcome. Cloning and transformation was also repeated. CloneJET PCR cloning kit was used to create eGFP-pJET (pJET was chosen instead of pDRIVE). Not successful.

Gradient PCR of eGFP premade vector (56-65 degrees). Gel electrophoresis was carried out and the bands were purified from the gel.

An attempt to clone eGFP into pDRIVE was not successful. The cloning of eFbFP into expression vector pBBR1MCS-2 was successful instead.

Colony PCR of eGFP-pDRIVE and eFbFP-pBBR1MCS-2. Gel electrophoresis revealed only one colony to be successful (eFbFP-pBBR1MCS-2 no. 17).

Cloning and transformation of eGFP-pBBR1MCS-2 using plasmid no. 4 (mentioned just above). The transformation only gave rise to 2 colonies, thus suggesting the cloning to be unsuccessful. The cloning was repeated and transformation was carried out again. Colony PCR was performed alongside MamC-pJET (see gel below).
Liquid cultures were setup from eFbFP-pBBR1MCS-2 no. 2, 4, 5, 11, 17 for 2 hours in order to measure fluorescence. First, OD(600nm) was measured to ensure equal density of cells. The measurements did not show fluorescence (ex. 450 nm, em. 495 nm) from the cultures.
Cloning and transformation of MamC-pDRIVE. Colony PCR showed the cloning to be of cryptic success. Colony PCR was redone without success. Cloning was repeated using the CloneJET PCR cloning kit. MamC-pJET was transformed and plated. Colony PCR was carried out alongside eGFP-pBBR1MCS-2.
The gel electrophoresis indicated MamC-pJET to be successfully cloned. However, only a few of the eGFP-pBBR1MCS-2 colonies appeared to have contain the insert.

Assembly of constructs
Colonies 1, 2, 3 (MamC-pJET) and II, IV, VIII (eGFP-pBBR1MCS-2) were setup in liquid cultures (see colony PCR from previous week). Miniprep was carried out and plasmids were sent for sequencing. All MamC sequences were perfect while all eGFP sequences were wrong. The cloning of eGFP into pDRIVE was successfully redone (documentation lost). Fluorescence was also measured of eFbFP-pBBR1MCS-2 strains. No fluorescence detected as the sequencing explains. Inducing with IPTG (final conc. 1 mM) had no effect.

The CPH strain
Enrichment of magnetotactic bacteria was done according to the protocol. It was unclear whether the bacteria observed in the microscope were actually magnetotactic. They were compared to MS-1 and MSR-1 samples.

Six of each genotype were set up for liquid cultures overnight (colonies 1, 2, 6, 9, 13, 16 for eFbFP and 18, 19, 23, 25, 27, 31 for eGFP). Miniprep was done and samples were sent for sequencing. None of the eFbFP gave rise to good quality sequencing. Colonies 18, 19, 23 and 27 of eGFP contained the correct sequence.

Empty pBBR1MCS-2 vector was transformed into E. cloni in order to obtain more stock vector.

Culturing of MTBs
MSR-1 base medium culture was inoculated in new medium and put in 28 degrees shaker over the weekend. This was done to check whether the strain would grow aerobically as well.

Gateway BP reaction was performed to clone MamC-eGFP into pDONR207 using MamC-eGFP (GTW PCR product) no. 3 and 4 was used and eFbFP (GTW PCR product) no. 3 and 7. Colony PCR was carried out (see gel no. 3).
Colonies 2, 3, 5, 7, 9, 11, 13, 14 were selected to make liquid culture. Miniprep was done and samples were sent for sequencing.
BioBrick submission
PCR amplification of MamC15 (week 6), eFbFP-pEX-A, eGFP18 (week 6) and eGFP19 (week 6). Products were run on a gel and the bands were purified. Ligation was set up to clone the fragments into pSB1C3 and subsequently transformation was performed as well.
Culturing of MTBs
Microscopy of MSR-1 in order to check morphology is corresponding to what is reported in the literature (Jogler & Schüler, Annu. Rev. Microbiol. 2009. 63:501-21). We did not see any signs of bacterial growth in the liquid cultures set up in week 6.

The CPH strain
PCR amplification of 16S from our lake sample (enriched) and run on gel (see gel no. 4). The gel shows some amount of contamination in the negative control (no template).

on eFbFP-pEX-A was carried out. Gateway BP reaction was done to move the fragment into pDONR207. Afterwards the plasmid was transformed into E. coli.

The CPH strain
Cloning of 16S PCR products into pJET and subsequent transformation into E. coli. Liquid cultures were grown inoculating cells from 24 different colonies (12 from H.C. Ørsted Parken sample and 12 from Christiania sample). Miniprep was carried out and samples were sent for sequencing.

BioBrick submission
Colony PCR was performed. Colonies 1, 5, 12 and 15 were inoculated to make liquid cultures, that were grown overnight.

and eGFP(19)-pSB1C3 were correct while eFbFP-pSB1C3 held the wrong insert. We uncovered that this was due two "illegal" restriction sites in the FbFP sequence. We, therefore, ordered another synthetic gene from IDT with the restriction sites removed. To submit MamC-eGFP as a BioBrick we PCR amplified (1:20 min elongation time) the whole construct (MamC F primer and eGFP R primer). To do this we used 3 different templates. Afterwards, digestion and ligation into pSB1C3 was performed and the plasmid was transformed into E. coli.
Colony PCR was performed. Colonies 5 and 6 were inoculated into liquid culture and grown overnight. Miniprep was performed and samples sent for sequencing.

Culturing of MTBs
New base media was made according to Schüler et al. 1991 (see protocols). A variant was made with 0,2% agar as well. Both MS-1 and MSR-1 were inoculated and grown in 28 degrees in both media. 3 days after the tubes showed signs of bacteria growing.

Confocal imaging
Cultures set up for confocal imaging. Strains MamC-eGFP-pJAM1786 and eGFP-pBBR1MCS-2 were inoculated.

Confocal imaging
Confocal images were taken of MamC-eGFP-pJAM1786 and eGFP-pBBR1MCS-2 alongside MS-1 and MSR-1 cultures.
Hover the mouse over the picture to see!

 First photo: eGFP,    Second photo: MamC-eGFP

(3 control cultures, containing pBBR1MCS-2 without insert, were used as well). The measurements failed to show eFbFP-pJAM1786 to be significantly different from the control cultures. This was in contrast to eGFP-pJAM1786 that was shown be fluorescent.

Western blot was performed on the 3 strains above (eFbFP, eGFP and control), MamC-eGFP-pJAM1786 and a GFP control. Strangly, the MamC-eGFP-pJAM1786 cultures did not show fluorescence. This was highly unexpected since the cultures that was used for this glycerol stock earlier had been shown to be fluorescent. Therefore, we did not expect any bands in the MamC-eGFP lanes. The western blot somewhat failed since only one of the standard protein markers showed in the membrane. (See a visualization here). The GFP standard (band at the right side) seem to be highly overloaded. The lanes to left is eGFP-pJAM in different amounts.