Experimental manipulations
We will present the results week by week, in order to report the progressive advancement of our project as faithfully as possible. When possible, we will use the structure of the $\small WetLab$ $\small\&$ $\small Methods$ section in order to clarify the link between each result and the part of the project it applies.
I. 30/06 – 06/07
• All the preliminary steps (brainstorming, organisation of the project,etc.) have already been done officiously during the year with the help of our supervisors.
• 1$^{st}$ meeting in order to organize the search of the sponsors, the creation of the wiki, and the completion of the safety form.
• A sponsoring folder has been written in French by the sponsoring team, and a first list of biotech companies and public services susceptible of offering us sponsorship has been made.
II. 07/07 – 13/07
• 2$^{nd}$ meeting to make a briefing of the coming manipulations and to find some ideas of Human Practice. Research of sponsorship is intensified.
• Familiarization with the lab and the first basic manipulations (preparation of media, dyalisis, electroporation) as well as the presentation of the safe lab practices.
• Extraction of the biobricks we will use in our manipulation for cloning.
• The electrocompetent bacteria used for the cloning are MC1061 $\EColi$.
• Spectrophotometric verification of the well-functioning of the RFP and GFP. The chosen biobrick of GFP had no promoter, we have to fuse it with a promoter.
• Conservation of the transformed bacteria at -80°C (in glycerol).
• Miniprep of the RFP and GFP biobricks, midiprep of the vector AraC-pbad33 (which we will use to make our constructions).
III. 14/07-20/07
• 3$^{rd}$ meeting in order to report our respective advances in our quest of sponsorship, to discuss the popularization event of the Brussels Game festival, to elect delegates for the Website and to select the tracks in which we compete.
• Linearization and PCR amplification for the PSB1(A/C/K/T) 3 plasmids.
• Preparation of the ccdB and Kid biobricks in the Standard 10 by PCR amplification. Cloning into bacteria and screening. We will discover next week that the cloning has failed.
• PCR amplification of phoA and proline::phoA (proline added via primer) from MG1655 $\EColi$, cloning into bacteria and screening. We will discover next week that the cloning has failed.
• Design and ordering of the sequences needed for the first constructions.
• Some technical problem have required the addition of intermediary steps that are kept silent in this summary (gel purification,…).
IV. 22/07-27/07
• The week begins with the negative results of the screening of the phoA and proline::phoA bacteria. Furthermore, the control group (electropored with linearized plasmids) has numerous colonies, meaning that we may have forgotten to inhibit the circularization of the plasmid by incubating with the alkaline phosphatase.
o We thus begin the digestion of the plasmid and the inserts (phoA and phoA::proline) again. We proceed to the electroporation, goth overnight and screening of the obtained clones, but the results are all negative, once again. (25/07) After investigation, we discover that we used the wrong antibiotic (Ampicilin instead of Chloramphenicol) and that we forgot to add glucose to the medium in order to inhibit phoA overexpression and promote the bacterial growth.
• Construction of RFP :: phoA, RFP :: p2A :: phoA and RFP :: ccdB by PCR amplification.
o The templates for phoA, RFP and ccdB have respectively been found in MG1065 $\EColi$, MC1061 $\EColi$ infected by a RFP biobrick from the iGEM, and in a sample coming directly from IDT DNA technologies.
o We failed the experiment completely, restarted, and this time we managed to amplify and successfully purify all the segments except phoA2 and RFP2.
• Screening of the Kid and ccdB clones for the making of biobricks. (23/07) KID has not been integrated into the colonies; on the other hand, was successfully cloned in several bacteria.
o We restart the amplification of KID from the beginning, and make an overnight culture of the colonies containing ccdB. We then store these cultures at -80°C in glycerol and, in parallel; we make a mini-prep out of it in order to send it to sequencing.
o The second try of the amplification of KID is also negative (25/07/14), even after considering more colonies (28/07/14).
• The synthetic sequences ordered by our supervisors for the constructions in $\SCere$ have arrived. We thus proceed to the PCR amplification to build our constructions. The manipulation has completely failed (24/07/14), we will have to start again.
V. 28/07-03/08
• After having observed the failure of the screening of phoA, proline::phoA and Kid, we decided to abandon the fabrication of the Kid biobrick to focus on $\MyColi$ (results not shown). We restarted the phoA and proline::phoA manipulation several times from intermediary steps, and we eventually restarted it one last time from the very beginning, by amplifying the gene phoA from MG1655 $\EColi$ (28/07/14).
o We later discovered that we did not use the proper annealing temperature during the screening, nor did we adjust the duration of the elongation phase of the PCR to the length of phoA. It means that we might have grown phoA and phoA+P bacteria without noticing it.
o This time we managed to find 1 colony possessing phoA but still none with proline::phoA (05/08).
• We restarted the PCR amplification for the constructions in $\SCere$ (RFP :: p2A :: GFP, RFP :: p2A :: GFP, Kid) and the amplification of the RFP(RFP2) gene for the construction RFP :: p2A :: phoA in $\EColi$. This time, the result was positive (29/07/14).
• We lost the RFP and ccdB segments for the construction RFP::ccdB, or forgot that we had amplified them. We amplified them a 2nd time (30/07/14)
• We digested pbad33 with the restriction enzyme sal1 in order to begin our constructions using the In-Fusion kit. The concentration was weak but the digestion seems to have functioned. We also digested pGAl1 with Hind3 for the same reason (31/07/14).
• Purification of the RFP:ccdB and ccdB-pbad segment for their fusion in RFP-CCDB-pBAD33 using the kit In-Fusion (31/07/14).
• Making of the pbad33 ::RFP ::phoA and pbad33 ::RFP ::CCDB constructions using the In-Fusion kit. Electroporation and overnight culture (01/08/14). We later screened the colonies to observe that the manipulation had failed (04/08/14).
July summary
July was marked by excitement, precipitation, confusion and failure. At the end of the 1$^{st}$ month, we do not have any construction yet (but hope that our overnight cultures have two), and we have around half of the PCR segments needed to complete the project.
Things could have gone better, but since we are now finally familiarized with the basic lab manipulations and since we have seemingly already made all the mistakes that can possibly be made, we are optimistic that things will go faster in August.
We can point out some difficulty to produce the constructions (phoA and proline::phoA) needed for the control experiment of the positive screening of the 2A peptides, and the giving up of the construction of the Kid biobrick.
VI. 04/08-10/08
• We begin the week by the screening of the proline::phoA, RFP ::phoA and RFP ::ccdb bacteria. The screening is negative, we have to begin all over again. We think that the ligation did not work because the vector was able to re-circularize itself. When possible, we will thus digest it with 2 restriction enzymes in order to be absolutely sure it cannot do it again. We will restart the proline::phoA manipulation from the step of the ligation.
• The digestion of pGAL has failed, we have to restart from the step of the mini-prep.
• Due to miscommunication and bad labelling, we had to remake most of the segment that had been previously made but somehow lost.
• The whole week has been spent carefully digesting pbad33 and pGAL1, regrowing proline::phoA colonies, re-amplificating the segment we had lost or depleted and purifying them (either on gel or on column). This took time since several of these steps have failed, so we had to restart so often that we were not more advanced at the end of the week.
o For example, when the digestion worked properly, something went wrong during the purification and everything was lost. We changed our protocols in small ways several time by listening to the advices of the PhD students in the lab.
o At the end of the week, we had sent 2 phoA plasmids and 1 proline::phoA plasmid to the sequencing, but not much more.
VII. 11/08/14- 17/08/14
• We continued this week what we had begun the week before: all our time has been consumed by digestions that did not want to work anymore (although we never had digestion problem in July) and purification that removed the DNA as well as the impurities.
o At the end of the week, we had launched a In-Fusion construction of RFP::p2A::GFP in pGAL, and RFP::phoA and RFP::ccdB were purified and available for the In-Fusion kit but the pbad33 plasmids were not ready.
o We tested our restriction enzyme, but apparently they were functional, so we decided that the pbad33 plasmid we were using were somehow damaged.
• We had our Human Practice event at the Brussels Game Festival, which was a success.
VIII. 18/08/14-24/08/14
The screening of the colonies of bacteria electropored by the In-Fusion product was not satisfying. It is our second negative result with the In-Fusion kit, thus we transform by lithium acetate and try to exploit the homolog recombining property (in vivo, this is equivalent as with In-Fusion kit) of the yeast to make our constructions. See protocol: “Yeast transformation by lithium acetate». Our results were not satisfactory.
The pbad33::phoA & pbad33::phoA::proline sequences are now available.
In chromogenic zn, test fn ok $\rightarrow$ phoA works, but one really better than the other; we try to verify if phoA::proline doesn't works (mutation vs proline).
IX. 25/08/14-31/08/14
New pbad33::phoA::proline construction $\rightarrow$ 4 colonies were survived.
Extra functional test: the presence of proline deosn't stop the activity oh phoA.
We also discussed the problem of purification of plasmid pbad33 digested. We hypothesized that the digestion by the enzyme Sal1 results in a secondary structure of the plasmid such that it remains localized at the column. To prove this, we determined that a sample of digested pBAD33 was well concentrated in plasmid. We then performed the normal purification, except that at each step rather than discard the supernatant we have preserved to ascertain the presence of the plasmid. We also conducted several elutions. We have found the presence of the plasmid in any of these fractions and only at low concentration in elution fractions. This result supports our hypothesis and allows to explain the difficulties we have met.
August summary
During this month, despite a best skill, we encountered many difficulties that have hampered the progress of our project. Simple manipulations, yet well mastered, proved unsuccessful to numerous occasions.
Although the situation was very frustrating, it has nevertheless allowed us to deal with the vagaries of the research profession and to learn to think where problems come from and also how resolve them.
We also had the pleasure to get positive results for our constructions, in this case the BioBrick ccdb and the control test pbad::phoA::proline. However, these results have rubbed shoulders the arrival of September inducting the halting of the lab manipulations.
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- Université Libre de Bruxelles -
