Difference between revisions of "Team:IIT Madras/Notebook"

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<h2>Notebook</h2>
 
<h2>Notebook</h2>
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=====================DIARY OF EVENTS====================
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Week 1
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Cleaned fridge and transferred materials
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Circuit finalised toggle switch/ mutual depressor switch using TAL represoor.
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--------------------------------------------------------
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Week 2
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Inventory of all supplies is to be done.
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IISER Pune and IIT Delhi contacted us regarding meet up and characterisation of their projects
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Allisterin was chosen as the other protein APO has no defined structure. ZDOCK and online tools were used to generate a peptide that binds to hydrophobic surface. Possible peptides:
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IAGYAEEILEHVIAE (aka naly)
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--------------------------------------------------------
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Week 3
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-> GROMACS simulations started
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-> Made a catalog of all available materials
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-> Lacto Bacilus strains are available NZ9000 and MG1363
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-> Simulations finished the proteins were found to interact favourably
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-> Heidelberg 2008 Auto Inducer 2 produced by LuxS sensed by LuxQ
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-> Reference:Quorum Sensing AI@ in campylobacter Orla Cloak, Barbara Solow doi:10.1128
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Can we do the whole circuit using just Lux components??!!
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--------------------------------------------------------
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Week 4
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->Circuit will be composed of purely Lux components.
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-> MD Simulations were done with ionic solution of protein complex. MD Simultions showed that naly
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interacts favorably with Alytesterin forming a cavity of hydrophobic residues
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-> Sender is finalised to be E.Coli DH5Alpha with LuxP-PFS gene
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Receiver is L.lactis NZ9000/MG1316 with LUXPQOU coupled with Lux R through sRNA's qrr1-5 and HFQ
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--------------------------------------------------------
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Week 5
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-> Sequences were finalised for qr1-5 and HFQ.
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Email sent to iGEM HQ requesting extra parts for LuxS, LUXPQUO, Sigma 64, constitutive promoter and HFQ.
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MD Simulations on dis-oriented peptides job was submitted.
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Victoria provided us with L.lactis, restriction enzymes and buffers.
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Enzymes and buffers were stored at -20C yellow box labelled master mix.
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Strains(2 of each strain) were stored in Abrar's box at -80C.
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-> 11 Jun:Prepared SOC stock, stored in freezer for autoclaving the next day.
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-> 12 Jun:Made LB broth and LB agar. Plated one petri plate, made stock of 1M 45ml CaCl2.
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Alyteserin was found to have a localization signal attached to it in form of USP45. The same sequence was then attached to NAly.
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-> 13 Jun: We found some inconsistencies in the promoter sequences of LuxCDABE and expS and
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their role when bound to LuxR. It should work, but in vitro experiments carried out earlier
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showed that the promoters did not function as expected. Refer to this link for luxPQ which
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was found to have a signalling region at the beginning http://www.ncbi.nlm.nih.gov/nuccore/16082689?report=fasta.
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The stop codon for LuxP and the start codon for LuxQ overlap by one amino acid.
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For inoculation, first the bacteria on the plate were inoculated in a test tube of LB broth and
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kept overnight. Then bacteria from that LB broth are to be again taken and grown till OD of
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around 0.4 is reached. Negative control was also prepared, where the broth contained antibiotic.
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-------------------------------------------------------------
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Week 6
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15, 16 Jun: We performed competent cell preparation and transformation over two days. The first batch showed no colonies. This could be because after heat shock and addition of broth, the tubes were inoculated for only 40 miinutes. According to iGEM protocol they must be inoculated for atleast 2 hours. None of the transformed bacteria showed growth on 16th Jun. On 16th, this was attempted again following iGEM protocol.
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19 Jun: After analysing the results of our previous experiments, we decided that the competent cells prepared did not have hig transformation efficiency. We started preparation of new competent cells today afresh. Instead of SOC media, we used only LB broth throughout. SOC media might have degraded after autoclaving, so it is better to avoid it. Maybe next time we can use SOB media?
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21 Jun: Agar Stabs for 5 plasmids arrived. We streaked them on agar plates for plasmid isolation the next day. Also, RBS and promoter did not give good results. The DNA had dried up from the wells. Also, a new terminator that we cloned gave only 2 colonies. Adding salts to the competent cells before transformation helped in improving transformation efficiency.
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Week 7
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22 Jun: We performed plasmid isolation from bacteria received through agar stabs. We made one error in the procedure. GPS needs to be mixed well before it is used, because it is a suspension.
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23-27 Jun: We took a break from wet-lab work to focus on primer and sequence design for the plasmids we needed to order from IDT.
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Week 8
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29-30 Jun : We performed transformation for Biobricks [promoter, terminator], we failed as chemically competent cells were older than a week. We decides to make Ultra-competent cells.
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1 Jul : Qrr1-3 and Qrr4-5 were reported to have problems by IDT guys. We made changes and made them as Qrr1, Qrr2, Qrr3, Qrr4, Qrr5.
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2 Jul : Order for all gBlocks were confirmed and placed. Inoculation of a single colony to 5ml tubes [with antibiotic and without antibiotic].The bigger shaker in UG lab were occupied, so we couldn't go ahead with this. We booked the shaker for next two days.
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3 Jul : Agian, we incoulated the colony into two test tubes (with Antibiotic and without antibiotic), due to unknown error we did not get any growth.
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4 Jul : Agian, we incoulated the colony into three test tubes (with Antibiotic (Ampicillin)+ old LB broth and without antibiotic (old LB broth ans new LB broth)), no growth. Cells on plate are not viable. Streak a new E. coli DH5alpha plate.
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5 Jul - 25 Jul
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Plasmid isolation was performed 3 times(!).
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More or less no work was done.
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Work on the wiki has started.
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----------------------------------------------------------------
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10th August
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Plasmid Minipreps were run again, however the quantity of plasmid was found to be too low. We will run again taking larger quantity of cells in falcon tubes.
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IDT delivery delayed, should come by today.
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-----------------------------------------------------------------
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29-30 Aug
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We tried SDM with PQOU plasmid, unfortunately no amplification was observed. When checking the primers, we discovered that two primers were in low concentration. However, even with the other primers we obtained no amplification. We can only infer that the sequence for the part is wrong, as the primers should work otherwise.
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3A assembly was attempted for the first time with a sample of QRR1 and some PSB1K3.
  
 
<p> Document the dates you worked on your project.</p>
 
<p> Document the dates you worked on your project.</p>
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<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 
<li><a href="https://2014.igem.org/Team:Cornell/notebook">2014 Cornell</a></li>
 
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Revision as of 06:49, 1 September 2015