María

Prepare 10mL of antibiotic stocks of both antibiotics that are going to be used during the project: Chloramphenicol (Cm) and Ampicillin (Amp). Make 500µL and 1mL aliquots.

Prepare 1L of LB medium and 3 400mL bottles of LB Agar. Send to autoclave.

María

Add antibiotics to the LB Agar bottles prepared the previous day and pour plates to have them ready for when we start transforming.

Lycka

Digestion gBlocks mVenus and mKate with EcoRI and PstI.

Made liquid culture of strain containing pSB4A5 bakcbone with RFP.

María

Digestion of pSB1C3 linearized backbone from the distribution kit with EcoRI and PstI.

María

Dissolution of necessary parts from distribution kit: GFP BioBrick BBa_E0840 and promoters BBa_J23100, BBa_J23105, BBa_J23108, BBa_J23113 and BBa_J23117.

Dissolution of K1149051 (a kind gift of Imperial College), which was sent dry on filter paper.

Lycka

Ligation of mKate into backbone pSB1C3.

Transformation of ligation product mKate and the BioBricks dissolved by María into Escherichia coli Top10 cells. These were plated on LB medium supplemented with Cm.

Lycka

Stocked primers VF2 and VR: storage stock (100µM) and working stock (10µM).

Colony PCR of transformants from yesterday with primers VF2 and VR followed by gel electrophoresis. Gel picture yielded no bands. This might be attributed to a fault in the transformation.

María

Transformation of same BioBricks as on 5^th July using a different transformation protocol since we believed that the negative result was due to a mistake during transformation.

María

Finally, the plasmids containing K1149051 and BBa_E0840 yielded a few colonies. However, the ones containing the promoters did not. Thus, we believe that the distribution kit plate containing the promoters is defective and the mKate ligation also featured some problems.

Lycka

To test whether the distribution kit might be the problem, transformation of BBa_J23113 in OneShot® TOP10 chemically competent cells (Invitrogen) to make sure the transformation process is not the cause of the negative results. Also, a plasmid containing TU Delft 2015 csgA Biobrick was used as a positive control.

For the mKate construct we will obtain the backbone from another construct instead of the linearized backbones from the iGem distribution kit.

Lycka

Measure DNA concentration of biobricks from registry by nanodrop.

Nanodrop

As can be seen from the table, there is DNA presence in the samples. Therefore, something else should be causing the transformations not to work. Since last year's distribution kit plates are still available in the lab and those plasmids were not taken we will try to get these parts from 2015 distribution kit.

María

Transfer colonies for K1149051 and BBa_E0840 to overnight LB+Cm culture. A colony containing CsgA was transfered as well to be able to isolate and use that plasmid as standard backbone for all synthesized parts.

María

Plasmid isolation of overnight cultures prepared yesterday and preparation of K1149051 and BBa_E0840 samples for sequencing.

Lycka

Cryostocks of K1149051 and BBa_E0840 overnight culures. Stored at -80 degrees.

Digestion of backbones pSB1C3, pSB4A5 and all gBlocks with EcorI and PstI.

Lycka

Gel purification of digested backbones. Dissolved in nuclease free water, stored at -20 degrees.

Ligation of synthesized fragments into backbones.

María

After all the problems transforming in the past weeks, we tested if we could use our homemade chemically competent cells for future transformations by transforming a couple of positive controls, which were taken from last year's plasmids. The transformations worked so these cells can still be used.

María

Transformation of Lycka's ligation products from yesterday and the BioBricks from the distribution kit were given one last chance. Transformed cells were plated after doubling their concentration.

Lycka

Dissolution of promoter BioBricks from 2015 iGEM distribution kit.

Lycka

Restriction of backbones pSB1C3 and pSB4A5 with EcorI and PstI. Gel electrophoresis of digested fragments. Gel picture yielded no bands.

Lycka

Transformation of BioBricks from the distribution kit of 2015, since the ones from 2016 yielded no colonies. Positive control: csgA. Negative control: water. Biobricks: J23100, J23108, J23105, J23117, J23113. Overnight cultivation yielded no result, we decide to drop this and add the promoters to the parts that need it by PCR.

María

Restriction of pSB1C3 and pSB4A5 backbones with EcoRI and PstI

Ligation of synthesized parts that could not be ligated on 14^th of July: INP_Sil_Sdom in pSB1C3 and BolA_ind and BolA_con in both pSB1C3 and pSB4A5.

Lycka and Tessa

Transformation of plasmids ligated by Maria (19^th of July) together with the ones ligated on 14^th of July. After overnight culture the following plates contained colonies. pSB1C3: INP_Sil_Sdom, OmpA_Sil_Taur, Sil_Sdom, SulA, BolA_ind, BolA_con, phaP. pSB4A5: BolA_con. The ones with a negative result were ligated again the next day.

María

Colony PCR of yesterday's transformation results from Lycka and Tessa. However, not all transformations yielded colonies.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.

Lycka

Ligation of mKate, mVenus, mCerulean, OmpA_Sil_Sdom and LacI into pSB1C3. Ligation of OmpA_Sil_Taur, OmpA_Sil_Sdom, Sil_Sdom, SulA and BolA_ind into pSB4A5. Left at room temperature overnight.

María

Continue with colony PCR of 20^th of July transformation results from Lycka and Tessa.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.

Lycka

Transformation of ligation products from yesterday. Cells containing backbones pSB1C3 or pSB4A5 were plated on plates supplemented with chloramphenicol or ampicilin, respectively. After overnight cultivation the following plates contained colonies: mKate, mVenus, mCerulean, OmpA_Sil_Sdom, Sil_Sdom, SulA, BolA_ind.

María

Continue with colony PCR of yesterday's transformation results from Lycka.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in selective LB overnight.

Pick all colonies selected by colony PCR on 21^st, 22^nd and 23^rd of July and transfer to selective LB according to the resistance expressed by each plasmid.

Lycka

Cryostock and plasmid isolation of overnight cultures prepared yesterday by Maria. Cryostocks stored at -80 degrees, isolated plasmids stored at -20 degrees.

Lycka

Nanodrop and prepare for sequencing plasmids isolated yesterday.

Sequencing resulted in the following. Correct sequence: Sil_Sdom (pSB1C3), OmpA_Sil_Taur (pSB1C3), Sil_Sdom (pSB4A5), SulA (pSB4A5). Sequence with mutations: SulA (pSB1C3), BolA_ind (pSB1C3), J23108 (pSB1C3), BolA_con (pSB4A5), BolA_ind (pSB4A5). Different sequence: BolA_con (pSB1C3), INP_sil_Sdom (pSB1C3), OmpA_Sil_Sdom (pSB4A5). Mutated sequences can be repaired by PCR and blunt end ligation.

María

Prepare new digested pSB1C3 and pSB4A5 backbones by restricting with EcoRI and PstI and gel isolating the restricted product.

New ligation of those synthesized fragments that have not yielded any positive results (transformation or sequence confirmation) in the past week in their respective backbones.

Lycka

Colony PCR of plates transformed yesterday.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated the same day in selective LB overnight.

María and Tessa

Make new chemically competent cells. They were tested for transformation capacity with a positive control plasmid (RFP in pSB4A5). Also, untransformed cells were plated in plates supplemented with Cm or Amp to test whether they had developed a resistance during the process.

María

Cryostock, plasmid isolation and preparation of samples for sequencing of the overnight cultures prepared yesterday by Lycka. Only INP_Sil_Sdom in pSB4A5 was sequenced confirmed with a mutation to be fixed by PCR. The other sequences aligned to a fragment of GFP unknown to us, we assumed it either came from a contamination of the old restriction enzymes we were using or from the pSB1C3 backbone that was taken from last year's team plasmid stock and probably was poorly labeled. Thus, we decided to use another backbone and the new enzymes sponsored to all iGEM teams by New England Biolabs.

Lycka and Célina

Repeat of the colony PCR of plates from the 26^th of July that yielded a negative result.

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.

Transformation of OmpA_Sil_Sdom in both different backbones.

Lycka

Colony PCR of OmpA_Sil_Sdom in both backbones from the 29^th of July. The PCR machine was filled with colonies from older plates which had not yielded any good colonies yet: mKate (26^th of July), mKate (mKate 30^th of June), INP_Sil_Sdom (20^th of July).

Picture on the left shows simulated agarose gel. Pictures in the middle and on the right show the actual agarose gel. Picked colonies marked with an arrow were cultivated in LB and the applicable antibiotic overnight.

Cryostocking and plasmid isolation of colonies picked by on 29^th of July. Cryostocks stored at -80 degrees, isolated plasmid stored at -20 degrees.

María

Cryostock and plasmid isolation of the overnight cultures prepared yesterday by Lycka and preparation of samples for sequencing of plasmids isolated yesterday and today. Sequence of mCerulean was confirmed (although it had a silent mutation). OmpA_Sil_Sdom in pSB4A5 and INP_Sil_Sdom in pSB1C3 had a deletion to be fixed by PCR. The rest either of sequences contained the random GFP sequenced mentioned before (since they are old ligation products) or multiple mutations that could not be fixed by PCR.

Tessa

Amplified E0840 (GFP) out of a pSB1C3-GFP plasmid using Phusion PCR. Four reactions of 50µl.

Nanodropped PCR products.

Nanodrop

Ran a 1% agarose gel of the PCR product.

Stored product 2 and 4 in the fridge for later use.

Tessa

Restricted PCR product of 3rd August, J23100 E0840, J23113 E0840 and J23117 E0840, with EcoRI-HF and PstI.

Purified restriction product.

Nanodropped purified product.

Nanodrop

Ligated purified product into pSB1C3.

Transformed ligation product into TOP10 strain. Using RFP as positive control and sterile MiliQ as negative control. Plated on plates with LB agar and CM.

Tessa

Colony PCR'd colonies from yesterdays transformation.

Ran a 1% agarose gel of the PCR product.

Transferred colony 14 (J23113 E0840 pSB1C3) and 21 (J23117 E0840 pSB1C3) into liquid LB.

Tessa

Miniprepped colony 14 and 21.

Nanodropped miniprep product.

Nanodrop

Cryostocked colony 14 and 21.

Send miniprepped product for sequencing. [Sequence Confirmed]

Lycka

PCR lineralization of vectors and inserts for gibson assembly pha operon (BBa_K1149051) to fluorophores (GFP, mVenus, mKate, mCerulean). Phusion polymerase. Annealing temperature 60 degrees, elongation time 2 minutes.

Lycka

Electrophoresis of PCR fragments yesterday.

Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. Gel did not yield bands at the right height. PCR is repeated at an annealing temperature of 57 degrees.

Second gel also did not yield the right bands. PCR repeated overnights by María at Tm 64 degrees and elongation time 3.5 minutes in the presence of DMSO.

Transfer from cryostock to plate BL21 strains with fluorophores expressed. On LB agar supplemented with chloramphenicol.

María

PCR with new primers of those parts that need to be expressed under the lac promoter. These primers will allow cloning on Addgene plasmids from the pBb series. The plasmids chosen (pBbS5a and pBbA5c) express lacI and the cloning site is placed downstream of a lac promoter. The parts to be amplified are:
1. BolA
2. INP_Sil_Sdom
3. OmpA_Sil_Sdom
4. OmpA_Sill_Taur
5. Sil_Sdom
6. SulA

Lycka

Electrophoresis of overnight PCR for lineralization.

Picture on the left shows simulated agarose gel. Picture on the right shows the actual agarose gel. 1 - 4: vectors for mCerulean, mKate, mVenus and GFP, respectively. 5 - 6 inserts for mVenus and GFP. For all lanes except lane 5, the right bands are present. Also many other bands were present so the product was gel isolated.

Transfered one colony of BL21 on each plate from yesterday in a 50mL falcon tube with 10mL filter sterilized M9 medium with glucose supplemented with chloramphenicol.