| Line 135: | Line 135: | ||
<div id="week4puf" style="display:none"> | <div id="week4puf" style="display:none"> | ||
<h2>Week 4 Notes</h2><br/> | <h2>Week 4 Notes</h2><br/> | ||
| - | |||
<table border=1> | <table border=1> | ||
<tbody> | <tbody> | ||
Please select a week on the left.
| 5/29/2012 | - Designed 9 primers for LacI. LacZ. and eYFP. Prepared LB. - Inoculation of B0014.J23100.wtPUF. B0030.Q04121 - Transformed the psB1C3 biobrick into DH5a cells |
| 5/31/2012 | - Transformed C0012, I732005, E0030, I732017. Also transformed PSB1A3, PSB1AK3, PSB1K3. - Inoculated LB media with a psB1C3 colony from the plate grown the day before. - Met with Prof. Bhalerao to talk about PUF application and project direction (RNA scaffolding). |
| 6/1/2012 | - Short-term goal changed: make one testing construct, [rbs-puf-eYFP] cloned into P.protet plasmid with a ptet promoter and terminator - Began inoculation of C0012, I732005, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3. Was incubated for 9 hours, then put in 4 degree room - Inoculated again due to the round-bottom tube being misplaced. Mini-prepped the inoculation. |
| 6/2/2012 | - Cultured [PUF+PIN], E0030, and P. protet. designed check primers for [rbs-puf-eYFP] |
| 6/3/2012 | - Inoculated with a psB1C3 colony in order to prepare a glycerol stock of the strain. |
| 6/4/2012 | - Threw out old, incomplete inoculations from Friday. - Transformed I732005, R0010. - Miniprepped Venus and E0030 and put gDNA in the -20 temp box. - Did new inoculations on C0012, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3. - Made a glycerol stock of the psB1C3 strain. |
| 6/5/2012 | - Made glycerol stocks of C0012, E0030, I732017, PSB1A3, PSB1AK3, PSB1K3 and put them in the -80 freezer. - Transformation of I732005 failed, so replated with rest of culture. - Transformed J04500 and PSB3T5. - Minipreped PUF Amp and protet CM20 (spilled 1 tube of this) and put gDNA in the -20 degree in the temp box. - Inoculated R0010. |
| 6/7/2012 | - Set goal to create RNA scaffold by 6/15, begun design. |
| 6/11/2012 | - Innoculated K12 - Transformed split GFP constructs BBa_K157005 and BBa_K157006 into DH5a cells - Constructed the first RNA scaffold draft - Inoculated both parts of split GFP, as well as J04500 |
6/12/2012 |
- Miniprepped K12 cells - RNA scaffold design will inculde two scaffolds which need to be stored into one plasmid and then ligated into separate plasmids - Made glycerol stock of both split GFP parts, J04500, and miniprepped both split GFP |
| 6/13/2012 | - Transformed Pertobrick into K12 cells - Constructed the first draft of the scaffold, but it needs review. - Second, unique scaffold needs to be designed |
| 6/14/2012 | - Plated Petrobrick transformations; inoculated cell growth using CM resistance - Finalized an updated version of the scaffold based on new data of PUF binding. Final modifications need to be made before sent for synthesis. - Performed PCR: (2) 50 uL tubes of control (no PUF binding site) and (2) 50 uL tubes with the PUF binding site. Used WT PUF as a template and phusion as the polymerase. Ran gel and saw a result of all primer dimers. |
| 6/15/2012 | - Made plan to digest cells in order to see whether or not petrobrick was transformed into K12. - Final modifications made on the scaffold design. The construct is ready for synthesis. - Second try of PCR: Did a temperature gradient of 55-63-71 degrees Celsius. Used the WT PUF as template DNA. Gel results show only primer dimers, the faintest of faint lines at around 700 base pairs, but it's not a positive result. - Miniprepped pTET is in the "temporary" box of the -20 degree Celsius freezer. |
| 6/18/2012 | - Made our own NEB Buffer 2 - RNA scaffold ready for synthesis - PCR attempt 3: Using hotstart mix at 63 degrees, using protet as the template DNA and including a blank tube that contained no template DNA as a control. Gel reveals another failed PCR with all primer dimers. |
| 6/19/2012 | - Made digestions of Petrobrick and K12 cells using EcoRI as restriction enzyme; Ran a gel; brightest band was at 5 kb mark. - Made more 1x TAE buffer - Designed primers for BBa_K157006 restriction enzyme site (KpnI/XhoI) incorporation in order to put into pETDuet-1 along with BBa_K157005. - Created a plan to subclone WT PUF into pBAD (inducible by IPTG) and eYFP into protet. - Designed primers for the WT PUF with Angela, who submitted them for ordering. - Inoculated E0030 from glycerol stock for the PCR tomorrow as well as MC4100. |
| 6/20/2012 | - Primers and RNA Scaffold are ordered. - Will put in a pETDuet-1 subculture later today so miniprep of the plasmid and digestion/ligation can start tomorrow of BBa_K157005. - IDT responded saying the RNA scaffold can't be synthesized through gBlock synthesis. They suggested synthesis through Ultramer Oligos. - Miniprepped MC4100 and E0030 (stored both in the temp box of the -20). - Performed PCR with E0030 (eYFP) as the template to make 2 tubes of control (no PUF binding site) and 2 tubes with the PUF binding site. Used the hotstart mix and diluted the primers from 30 mM to 10 mM. Gel bands of 700-800 base pairs confirmed a successful PCR. PCR cleanup was performed, and the resulting DNA put into the tem pbox of the -20. |
| 6/21/2012 | - Miniprepped the pETDuet-1. - Digested both pETDuet-1 and BBa_K157005 with EcoRI and PstI in EcoRI buffer. - Emailed Dr. Silver about how they synthesized their scaffold and will base decision upon her reply. - Preliminary split-GFP/PUF tethering conceptualization done. - Digested the 2 minipreps of control eYFP, the 2 minipreps of PUF eYFP, pBAD, and ptet. - Ran digest and will view gel tomorrow. |
| 6/22/2012 | - The plan is to start growing cells in TB by Monday or Tuesday so that we can prepare M9 with Glucose. However, will need to test procedures with blank K12 and then with petrobrick-K12 so that we have a control. - Double digested BBa_K157005 and pETDuet-1 with EcoRI and PstI in NEB EcoRI buffer. Ran two samples on a gel. - Ran 2 gels of 1 kb ladder: pET duet and RID0015 and pBAD with ptet. The second gel was a 1 kb ladder with control eYFP 1, control eYFP 1, PUF eYFP 1, PUF eYFP 2. |
| 6/25/2012 | - Started making TB media. Added tryptone, yeast, glycerol to make media, autoclaved. Need to add a 100 mL filter of .17 M KH2PO4 and .72 M K2HPO4. After that, need to grow 5 mL of the cells (both normal and petrobrick-transformed) in the TB along with 1:1000 ratio of antibiotic (CM) and grow it overnight. - Extracted BBa_K157005 and pETDuet-1 from the gel. pETDuet-1 had a very faint band. - The wrong ladder was used for BBa_K157005 so the band was not located. - Digests were run again with NEB Buffer 3 and 2 uL of enzymes were used to cut the pETDuet-1 vector. - Scaffold sent for synthesis through IDT's miniGene option. - Ran gel to see if digests worked, all were successful, but not pET duet. - Ran digests through the nanodrop and got low numbers for all. - Went ahead and did ligation to make the following: pBAD+control eYFP, pBAD+PUF eYFP, ptet+control eYFP, ptet+PUF eYFP. - Transformed the ligations into both DH5a and BL21. |
| 6/26/2012 | - Finished making the TB media. - Prepared (2) 5 mL cultures of K12-Petrobrick in the TB. - An additional two innoculations of normal K12 were prepared to grow overnight. - Heard back from UW's team advisor from last year, Ingrid Swanson-Pultz, and she replied back with what strains of E. Coli they used. We will be fine using K12 cells because she said there wasn't much difference from the alkane production with the different cells. We will not need to order any more. - Need to order C15 Alkanes to inject in one of the production cultures to ensure alkane production. - Digestion of BBa_K157005 failed again with no presence of bands. - Digestion of pETDuet-1 worked so the BBa_K157005 plasmid was run on a gel to confirm viability. The plasmid was still viable so double digestion was done again with EcoRI-HF/PstI-HF in NEB Buffer 4 with BSA and Alkaline Phosphatase. - Transformations of DH5a and BL21 resulted in 3 lawns and no growth. A replate was necessary, and they were streaked out. - Performed additional transformation of all the following into DH5a: pBAD+control eYFP, pBAD+PUF eYFP, ptet+control eYFP, ptet+PUF eYFP. |
| 6/27/2012 | - Both the TB cultures were successful. However, one of the normal K12 innoculations did not grow. - Therefore, made two more TB cultures using just the K12 innoculation which worked. - In addition, prepared CM and Amp plates for future use. - Replates of pBAD with control/PUF eYFP in both DH5a and BL21 all had growth. However, BL21 cells looked translucent and flat. Inoculated all of the plates. No growth on transformations of the DH5a pBAD/ptet YFP constructs, so replated all of them. Checked resistance of ptet, and it's Cm, not Amp. - Started biobricking PUF WT. - Ran two PCR reactions with hotstart mix at 61 and 63 degrees C. - Ran gel of PUF PCR samples done in May, bands looked to be the correct size. Gel extracted all five samples. - Inoculated PSB1C3 overnight culture. |
| 6/28/2012 | - Made the M9 Media. - The TB cultures from the K12 innoculations were successful. - Currently, all cultures that we plan to use are in the 4 degree room. We will not take further action on the production protocol until we have received the C15 alkanes and Dr. Jin is back. - We have all the materials to start the production media culture, but we will need Dr. Jin's guidance on the extraction method as well as GCSM analysis as the last steps of the protocol happen rather immediately. We will wait until next week to finish it up. - Digestions of BBa_K157005 and pETDuet-1 seemed to have worked. However, the gel showed the split-gfp band at 300bp even though it was expected to be ~250bp. - Overnight inoculations: the DH5a inoculations of pBAD with control and PUF eYFP both grew. However, both of the BL21 inoculations didn't grow at all. - The replates of the YFP constructs didn't grow at all. So retransformed BL21 with WT PUF + control eYFP and WT PUF + PUF eYFP. - Also retransformed DH5a with pBAD that had both control and PUF eYFP as well as the same in ptet. - Digested the PSB1C3 culture made 6/27/12. - Digested the PSB1C3, then ran a gel of the 3 total PUF PCR samples and 2 PSB1C3 miniprepped sample. Both PSB1C3 showed two bands, which was not expected (one at about 2kb and one a 1kb). - Will have to run a control uncut PBSB1C3 with digested PSB1C3 to check what the problem is, could be that the strain used for inoculation had an insert in it. - The PUF PCR digests looked about right. - Gel extracted all 3 PUF PCR samples and the one band from the PSB1C3 that was at the right size. - Started an overnight culture of PSB1C3. |
6/29/2012 |
- Today more M9 media was made as we were unaware that it is not suppose to be autoclaved. - Did a miniprep of pSB1C3 - None of the eYFP constructs in DH5a grew. - Conclude that neither the control eYFP of the PUF eYFP constructs were properly ligated within either pBAD or ptet. - While the retransformations of BL21 grew some odd looking colonies, it is almost certain that they only contain the WT PUF plasmid and not a correct eYFP plasmid. - Attempt a second ligation, used same old digest (because the bands on that were correct). - Ran the ligation overnight. - Inoculated the successful but weird looking and dubious BL21 plates. |
"
