David Tulga Notebook

From 2007.igem.org

(Difference between revisions)
(RBS Library + T7rnap ATG, GTG (I716110, I716111) - Building (Re-Do Transformation))
m
 
(40 intermediate revisions not shown)
Line 2: Line 2:
[[Template:BerkiGEM2007_DavidConstructionFiles | My Construction Files]]<br>
[[Template:BerkiGEM2007_DavidConstructionFiles | My Construction Files]]<br>
[[Template:BerkiGEM2007_DavidSequencingFiles | My Sequencing Files]]<br>
[[Template:BerkiGEM2007_DavidSequencingFiles | My Sequencing Files]]<br>
-
Notebook Archives: [[David_Tulga_Notebook/Archive1 | May 30 - June 5]]<br>
+
Notebook Archives: [[David_Tulga_Notebook/Archive1 | May 30 - June 5]], [[David_Tulga_Notebook/ArchiveBasic1 | Finished Basic Parts 101-107]]<br>
===Progress Status Descriptions===
===Progress Status Descriptions===
Line 11: Line 11:
Finished : Correct clones are organized and saved<br>
Finished : Correct clones are organized and saved<br>
-
=Basic Part Construction=
 
-
==Low Copy ampR plasmid (pI716101) - Finished==
+
=Composite Part Construction=
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile101 | pI716101 (Low Copy ampR Plasmid - Produces RFP)]]
+
-
The midiprep of the plasmid pSB4A3-I7101 was successful, and I additionally digested and ligated the new 1144 Fragment into its EcoRI/XhoI insert to convert it into the Biobricks 2.0 Format.
+
==RBS Library + pir (I716108) - Finished==
 +
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile108 | I716108: RBS Library + pir]]
-
The colonies grown from this ligation/transformation were both red and white - the red ones being the Bca9145-1144 parent vector, while the white colonies I picked appear to be the correct product.
+
'''June 27:'''
 +
I digested the pir confirmed part (clone #4) with EcoRI and BglII, then ligated this to the digestion of pBca1122-Bca9235 with EcoRI and BamHI.  I then transformed these into some DH10B cells, and plated on AMP+KAN plates today, and will grow the culture tomorrow.
-
I then did a test digest with PstI and HindIII, and the results indicated that the red colonies were parent, and the large white colonies were the correct product.  Additionally, upon pelletization, the red colonies' cells appear distinctly red, while the white colonies appear slightly tinted red, which is the exact phenotype expected for the product, a low-copy plasmid with a RFP producing device.
+
'''June 29:'''
 +
Due to the incubator being turned off, I could not grow the culture yesterday, as the colonies were not visible, therefore, I scraped the plate in LB today, and grew in AMP+KAN LB Media (which I had to make, due to our being out of that particular mix), to then miniprep on Monday. (After being placed in the refrigerator on Saturday)
-
'''June 19 to June 21:'''
+
'''July 2:'''
-
A midiprep of clone #3 (large white colony) was then done, and the plasmid DNA was run, as well as a mapping digest, with 4 different enzyme pairs, was performed. The plasmid DNA gel matched the expected size perfectly, and the mapping digest appears correct. I can eliminate the possibility of original biobricks 1.0 vector, as the product is cut with BglII, which was not present in the original vector.  As well, the phenotype and size/map digest strongly support the indication that the product is correctTherefore, clone #3 is confirmed to be correct, and likely clone 4 is likely additionally right, but has not been extensively tested.
+
I have miniprep'd the library, and it appears correct, but I will not know whether the RBS + pir combination works until I test it with a promoter in the next pir composite part.
 +
 
 +
'''July 25:'''
 +
The sequencing result of some individual clones of 109 came back, indicating the RBS library must be correct.
 +
 
 +
'''July 27:'''
 +
I have now saved this library on the clone saver sheet.
 +
 
 +
==Salicylate Promoter + RBS Library + pir (I716109) - Finished==
 +
 
 +
'''July 2:'''
 +
I digested the RBS+pir (108) part with EcoRI and BglII, Gel Purifying the large fragment.  As well, I digested the Salicylate Promoter with EcoRI and BamHI, Gel Purifying the small fragment, for an insertionI then ligated the two fragments together, and transformed, and plated on an AMP plate, as the KAN resistance gene should have been excised.  However, the digest of the RBS+pir library looked very smeary, as there was likely incomplete digestion.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel002.jpg|100px]]
+
|[[Image:Berk-DTgel007.jpg|200px]]
-
|Gel Indicating the size of the resulting plasmid from the midiprep - lane 1 is the uncut plasmid, and lane 2 is the marker.  It shows up exactly as expected, as the plasmid size is at 4060, as both parent plasmids are 4276 and 2973.
+
|The Test Digest Gel.  Lane 1 is marker, lanes 2-9 are the T7rnap test Digests, the second to last is the RBS+pir library digest, and the last lane is the Salicylate Promoter digest.
-
|[[Image:Berk-DTgel004.jpg|200px]]
+
-
|This is the test digest gel. It is very  difficult to see the bands, but they seem to indicate that clone #3 is correct.
+
|}
|}
-
'''June 25:'''
+
'''July 3:'''
-
I performed another Test Digest, this time with BglII and BamHI, which was expected to give two bands at 3159 and 901 for the expected product, and to leave the parent uncut. I ran it with parent and product on the same gel and it gave the exact expected result.  Therefore, I saved clone #3 on the CloneSaver Card, as it is multiply confirmed.
+
I checked the plate, but no colonies grew, I think this is likely due to the incomplete digestion that I observed yesterday, and there being an insufficient amount of ligated product to transform with.
 +
 
 +
'''July 4:'''
 +
I again digested the RBS+pir (108) part with EcoRI and BglII, Gel Purifying the large fragment, with BglII and XhoI, Gel Purifying the small fragment, as well as digesting the Salicylate Promoter with BamHI and XhoI, Gel Purifying the large fragment, with all digests this time for two hours, to ensure complete digestion.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel005.jpg|100px]]
+
|[[Image:Berk-DTgel009.jpg|200px]]
-
|This gel is the second test digest, done with BglII and BamHI.  Lane 1 is the parent midiprep, lane 2 is the product midiprep of clone #3, and the rightmost lane is the marker. It gives the exact expected band pattern of two bands at 3159 and 901 for the product, and to leave the parent uncut.
+
|The Digest Gel.  Lane 1 is marker, lane 2 is the EcoRI/BglII digest of the RBS+pir library, lane 3 is the XhoI/BglII digest of the RBS+pir library, and the last lane is the BamHI/XhoI digest of the Salicylate Promoter.
|}
|}
-
==pir (I716102) - Finished==
+
'''July 5:'''
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile102 | pir I716102 (R6K Origin Inducer)]]
+
I have ligated the EcoRI fragements together as well as the XhoI fragments together, to hopefully ensure at least one insertion will work.  I then transformed and plated on AMP plates, as the KAN resistance should have been excised out.  I will then scrape and grow in AMP media tomorrow.
-
I attempted many PCR reactions for pir, many of which failed, or gave too dilute a product to transform with.  I additionally found that some of the incorrect colonies which were grown up from the first PCR product that appeared to succeed were actually primer dimers, and some were also mixed clones.
+
'''July 6:'''
 +
Neither of the plates looked sufficient, with only a few colonies.  I believe the ligase I was using might be bad, so I have attempted the same ligation/transformation/plating on AMP as yesterday, but this time with a different ligase.
-
'''June 21:'''
+
'''July 9:'''
-
I have re-tried PCR from the overlap A+B step, as both the A and B products did succeed.  The resulting transformation from that PCR has been grown up on an AMP plate.
+
Again, the plates looked very insufficient to no colonies, so I will have to re-do the transformations over again, possibly with a different procedure.
-
{|
+
'''July 10:'''
-
|-
+
I have re-done the same ligation / transformations with 4 times as much miniprep digest, and in TG1 cells, to hopefully to get more colonies.
-
|[[Image:Berk-DTgel003.jpg|200px]]
+
-
|The PCR gel, lane 1 is marker, 2 and 3 are failed T7 RNA Polymerases, ATG and GTG respectively, and lanes 3, 4, and 5 are the pir A, pir B, and combined overlap products, respectively.
+
-
|}
+
-
'''June 25:'''
+
'''July 11:'''
-
I have now cultured 4 white colonies from this transformation, and no obviously red colonies were spotted.
+
The plates looked a little better, but still very low yield.  I have tried the EcoRI digests, etc. again, as it was the most successful direction in the previous attempts.
-
'''June 26:'''
+
'''July 12:'''
-
I have miniprep'd the 4 cultures I made yesterday.  Additionally, a test digest was performed on the four minipreps, and all four came out exactly as expected - with two bands at 2652 and 344, when digested with HindIII, which cuts twice within the pir geneTherefore, I sent all four samples off for sequencing today.
+
The plate looked excellent, and so I scraped and cultured it in 5mL of LB+AMP Broth.
 +
 
 +
'''July 13:'''
 +
I have miniprep'd the library, and digested it and the 101 low copy plasmid with BglII and XhoI101's large fragment (backbone) was gel purified, but 109's bands were too close to differentiate, so I ran a ca998/G00101 PCR on it to amplify the part.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel006.jpg|200px]]
+
|[[Image:Berk-DTgel011.jpg|200px]]
-
|The Test Digest gel.  Lane 1 is marker, 2-5 are T7rnap PCRs, and the last 4 are the pir test digests.
+
|The Digest Gel.  Lane 1 is the 109 digest, lane 2 is the 101 digest, and the last lane is the Marker.
|}
|}
-
'''June 27:'''
+
'''July 16:'''
-
I received my sequencing results, and some mutations were found.  However, clone #4 had no additional mutations beyond what was present in all 4 samples. Therefore, I believe that those mutations were present in the template sequence, as they are somewhat conservative non-silent mutationsAdditionally, I sent off #4 for reverse sequencing, as the forward reads did not reach the end of the gene.
+
I gel-purified the PCR Product, and while there were multiple bands, the desired one was considerably brighter than the rest. I then digested the PCR Product with BglII and XhoI.  I then transformed the ligation of 109 in 101 into pBACr-AraGFP cells, and plated on an AMP+KAN+Ara+Sal plate.
 +
<!--Purify Gel Order: 3,3 ; 3,4 ; 4,1 ; 4,2-->
-
'''June 28:'''
+
'''July 17:'''
-
I now have the reverse read for clone #4 and it looked exactly correct, including the previously observed mutations, and so clone #4 is confirmed, and has been saved on the CloneSaver card.
+
The 109 in 101 ligation product had lots of colonies, but no obviously green ones, so I picked 96 and grew in 600 uL of AMP + KAN + Sal + Ara.
-
==T7 RNA Polymerase ATG and GTG starts (I716103, I716104) - Checking (Sequencing)==
+
'''July 18:'''
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile103 | T7rnap I716103, I716104 (T7 RNA Polymerase)]]
+
All 96 wells showed uniform uninduced activity, even with Salicylate in the media, so I miniprep'd 2 of them and did a PCR with ca998 / pir-R, for use in sequencing tomorrow.
-
I have attempted numerous PCR reactions for the T7 RNA Polymerases, and so far none have succeeded, either by returning no product, incorrect product due to mis-annealing, or genomic bleed through.
+
'''July 19:'''
-
 
+
I did another digest of 101 with BglII and XhoI, in case my previous digest was too low-quality.  Additionally, the PCR for the two wells failed, so I will have to sequence a high copy plasmid.  I also transfomed a new ligation of 109 in 101 on AMP+KAN+ARA+SAL, as well as directly transformed 109 on that kind of plate. I also transformed the ligation product of only the digest itself, to test the quality of the digests.
-
I will attempt the PCR reactions again, potentially from a different template, when we receive more Expand polymerase.
+
{|
{|
|-
|-
-
|[[Image:Berk-DTgel001.jpg|300px]]
+
|[[Image:Berk-DTgel012.jpg|200px]]
-
|This gel indicates the test digests of a variety of miniprepsThe lane order is low copy plasmid clones in order for lanes 1,2,3,4; then pir clones 3 and 4; the T7rnap ATG clones 1-6, then GTG  3-6.  None of the pir or T7rnap products are correct.
+
|The Digest GelLane 1 is Marker, 2 is the 101 digest, and lane 3 is a T7rnap EcoRI/BglII digest.
|}
|}
-
'''June 26:'''
+
'''July 20:'''
-
We now have more Expand polymerase, so I re-did the T7rnap PCRs with a new template Chris miniprep'd, and all 4 (2 ATG, 2 GTG) worked correctly.  I then cleaned up the PCR products, and will then proceed to digest, ligate, and transform tomorrow.
+
The first 101 digest looked clean, the second had many colonies, and the 109 digest had lots of colonies.  Additionally, the 109 direct and 109 in 101 plates showed no significant activityTherefore, I transformed into TG1 cells 1 uL of a 20x dilution of 109 onto AMP to then pick and analyze individual colonies.  I also test digested with HindIII the 109 library to see if its any good.  The band pattern looked approximately right, but somewhat fuzzy and uncertain.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel006.jpg|200px]]
+
|[[Image:Berk-DTgel013.jpg|200px]]
-
|The PCR gel.  Lane 1 is marker, lanes 2 and 3 are ATG Start PCRs, 4 and 5 are GTG Start PCRs, and the last 4 are the pir test digests.
+
|The Digest Gel.  Lane 1 is Marker, 2 is the 101 digest, lane 3 is the 109 test digest with HindIII, and the last lane is the PCR for the low/high plasmid Arthur is making.
|}
|}
-
'''June 27:'''
+
'''July 23:'''
-
I have digested, ligated, and transformed the four PCR products into DH10B cells today, using Austin's backbone digest, and will grow the colonies up tomorrow.
+
I picked 4 colonies of 109 to then miniprep and sequence, to determine if the construct is correct.
-
'''June 29:'''
+
'''July 24:'''
-
Due to the incubator being turned off, I could not grow any colonies yesterday, as they were not visible, therefore, I grew up 8 colonies today in culture, to then miniprep on Monday.  (After being placed in the refrigerator on Saturday)
+
I have miniprep'd the 4 109 colonies, and sent off for sequencing with ca1111F, which binds to the middle of the nahR gene in the Salicylate promoter part.
-
'''July 2:'''
+
'''July 25:'''
-
I have miniprep'd the 8 colonies, then performed a test Digest with AlwNI. (AlwNI cuts within the backbone, and the T7rnap gene). All clones except 103 #3 looked correct, so I sent of clones 103 #2, #4 and 104 #2, #3.
+
The 4 clones came back as correct, and so this library is correct, even though it does not appear to work, most likely due to the ineffectiveness of the Salicylate promoter.
 +
 
 +
'''July 27:'''
 +
I have now saved this library on the clone saver sheet.
 +
 
 +
==RBS Library + T7rnap ATG, GTG (I716110, I716111) - Finished==
 +
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile110 | I716110,I716111: RBS Library + T7rnap ATG, GTG]]
 +
 
 +
'''July 4:'''
 +
I digested the T7rnap ATG and GTG (103,104) parts with EcoRI and BglII, Gel Purifying the large fragment.  I performed this digest for 2 hours, as previous digests were somewhat smeary.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel007.jpg|200px]]
+
|[[Image:Berk-DTgel008.jpg|200px]]
-
|The Test Digest Gel.  Lane 1 is marker, lanes 2-5 are the ATG Start Digests, in clone order, 6-9 are the GTG Start Digests, also in clone order, and the last 2 are gel purification digests.
+
|The Digest Gel.  Lane 1 is marker, All digests are EcoRI and BglII, and the clone order is: 103 #2, 103 #4, 104 #2, 104 #3.
|}
|}
-
'''July 3:'''
+
'''July 5:'''
-
I received the sequencing results from Quintara, and in all clones were some template mutations, but otherwise they were all perfect.  However, as the T7rnap gene is rather long (2652bp), the reads did not reach the end, and I sent off all 4 samples for reverse sequencing.
+
I ligated the EcoRI/BglII digest with the EcoRI/BamHI digest of the RBS Library, and then transformed some DH10Bs and plated on AMP Plates, to then grow in AMP/KAN Media tomorrow.
-
'''July 4:'''
+
'''July 6:'''
-
I received the reverse sequencing results from Quintara. Again, the clones looked reasonable, with some template mutations.
+
None of the plates looked sufficient, with none to a few colonies. I believe the ligase I was using might be bad, so I have attempted the same ligation/transformation/plating on AMP+KAN as yesterday, but this time with a different ligase.
'''July 9:'''
'''July 9:'''
-
I have now designed and ordered the oligos for sequencing the middle of the gene, as it is over 2500 bp long, it requires two oligos in the middle of the gene to read it fully.
+
Again, the plates looked very insufficient to no colonies, so I will have to re-do the transformations over again, possibly with a different procedure.
-
==T7 Promoter Variants (I716105, I716106, I716107) - Finished==
+
'''July 16:'''
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile105 | T7 Promoters I716105, I716106, I716107]]
+
I have digested the new T7rnap parts with EcoRI and BglII, to then insert the RBS part.  I have ligated in the RBS part, and transformed into TG1 cells, and plated on AMP+CAM.
-
I designed and ordered oligos to create the T7 promoters synthetically, as their short length (29bp), and multiple variants (Wild Type, D, and E) mean that creating them from an extension of two annealing primers was the best method.
+
'''July 17:'''
 +
I have scraped and cultured the plates in AMP+CAM, as there were a ridiculous number of colonies.
-
It succeeded in producing products and colonies for all three variants.  Two clones for each were sent off for sequencing, and one of each variant came back perfect.  The other three were an unusual RFP parent recombination.  Therefore, clones: 1 for I716105, 1 for I716106, and 2 for I716107 are correct.
+
'''July 18:'''
 +
I have now miniprep'd the two libraries, as they appear to be correct.
-
'''June 25:'''
+
'''July 26:'''
-
I have now saved the correct clones on the CloneSaver Card, as indicated in the spreadsheet.
+
As the phenotypic analysis indicates definite functionality in both high and low copy plasmids, this library is confirmed to be correct.
 +
'''Aug 19:'''
 +
I have clonesaved clones 110,3 and 111,1.
-
=Composite Part Construction=
+
==Salicylate Promoter, Iron Promoter + RBS Library + T7rnap ATG, GTG<br>(Salicylate: I716112, I716113; Iron: I716114, I716115) - Finished==
-
==RBS Library + pir (I716108) - Checking (Waiting for Phenotypic Analysis)==
+
'''July 19:'''
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile108 | I716108: RBS Library + pir]]
+
I have digested the T7rnap ATG and GTG RBS Libraries (110,111) with EcoRI and BglII and purified the Large fragment. This was then ligated with an EcoRI and BamHI digest of the Salicylate Promoter and Iron Promoters, and transformed into some TG1 cells, and plated on AMP.
-
'''June 27:'''
+
'''July 20:'''
-
I digested the pir confirmed part (clone #4) with EcoRI and BglII, then ligated this to the digestion of pBca1122-Bca9235 with EcoRI and BamHI.  I then transformed these into some DH10B cells, and plated on AMP+KAN plates today, and will grow the culture tomorrow.
+
The T7 GTG Starts have a decent colony yield, but the ATGs don't have very many colonies.  This is likely due to the toxicity of the T7rnap, as there is some leaky expression even when uninduced.  Therefore, I have scraped and miniprep'd directly off the plate for the GTG starts.  I also re-did the ATG (110) Ligation / Transformation with more T7rnap material into TG1s and plated on AMP.
 +
<!--Gel Order: Marker, 101, 109, PCR-->
-
'''June 29:'''
+
'''July 23:'''
-
Due to the incubator being turned off, I could not grow the culture yesterday, as the colonies were not visible, therefore, I scraped the plate in LB today, and grew in AMP+KAN LB Media (which I had to make, due to our being out of that particular mix), to then miniprep on Monday. (After being placed in the refrigerator on Saturday)
+
The T7rnap ATG plates have a sufficient colony yield, but not too many, indicating high toxicity.  I then scraped and miniprep'd the Promoter/RBS/T7RA plates.  I have also digested the Promoter/RBS/T7RA,G plasmids with BglII and XhoI and ligated into a BglII/XhoI digest of 101, to make them low copy.  I then transformed into GH455G cells on AMP: The Ligations of 112-115 in 101, and the direct plasmids of 112-115, and 112-113 on AMP+SAL.
 +
<!--Gel Order: 1 I, 1 S ; 0 I, 0 S-->
-
'''July 2:'''
+
'''July 24:'''
-
I have miniprep'd the library, and it appears correct, but I will not know whether the RBS + pir combination works until I test it with a promoter in the next pir composite part.
+
These is confirmed activity with the Iron Promoter with the ATG and GTG Starts, and the low copy ATG starts have very few colonies.  I have then picked 96 colonies on a 96-well plate from the low copy GTG starts - columns 1-8 are 115 (Iron) in 101, columns 9-12 are 113 (Salicylate) in 101, and put this in the incubator to grow overnight, with TB+AMP+Glycerol.
-
==Salicylate Promoter + RBS Library + pir (I716109) - Building (Re-Do Transformation)==
+
'''July 25:'''
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile109 | I716109: Salicylate Promoter + RBS Library + pir]]
+
I grew up the 96 well plate (10 uL of culture from each well) in two 96 well blocks (600 uL culture in each well), one with LB+AMP only, and the other with LB+AMP+Iron or Salicylate, depending on the construct, to then do TECAN analysis when the cultures are at saturation.
-
'''July 2:'''
+
'''July 26:'''
-
I digested the RBS+pir (108) part with EcoRI and BglII, Gel Purifying the large fragmentAs well, I digested the Salicylate Promoter with EcoRI and BamHI, Gel Purifying the small fragment, for an insertion.  I then ligated the two fragments together, and transformed, and plated on an AMP plate, as the KAN resistance gene should have been excisedHowever, the digest of the RBS+pir library looked very smeary, as there was likely incomplete digestion.
+
I have done the TECAN analysis with 10uL of each well (on/off), with the parameters of bandwidth = 7.0, GFPmut2 (Ex: 481nm, Em: 507nm) as the spectrum, and a gain of 80.  The well D4 was a hit with a variance of 1850 (off: 1300, on: 3150), or 4.3 times a fluorescent, after removing the backgroundI have also miniprep'd the D4 well (off), and set up a ca998/T7-R PCR to amplify the part for sequencing.
 +
 
 +
'''July 27:'''
 +
The D4 PCR failed, so I ran an Iron-F/T7-R PCR on D4 to then sequence.
 +
 
 +
'''July 30:'''
 +
The D4 PCR was successful this time, so I gel purified it, and sent it off for sequencing with the ca570F oligo, which is a reverse internal T7 sequencing oligo.
 +
 
 +
'''July 31:'''
 +
The sequencing result came back correct, with the 1106-H RBS.
 +
 
 +
'''Aug 18:'''
 +
I have run a gel of D4, and the band is quite faint, indicating it is definitely low-copy.
 +
 
 +
'''Aug 20:'''
 +
I have clonesaved the D4 clone.
 +
 
 +
==Iron Promoter + RBS Library + pir (I716116) - Discontinued due to instability==
 +
 
 +
'''July 25:'''
 +
I have ligated the Iron Promoter digest with EcoRI and BamHI, Small Part Fragment, and the EcoRI/BglII, Large part fragment, digest of 108 (RBS+pir).  I then transformed in TG1s, and plated on AMP.
 +
 
 +
'''July 26:'''
 +
I have now scraped and cultured the 116 (Iron+108) library, as the colony yield was very good.
 +
 
 +
'''July 27:'''
 +
I have miniprep'd the 116 (Iron+108) library, and digested it with BglII and XhoI, as well as digested 101 with BglII and XhoI.  I then ligated the digests of 116 and 101, transformed in TG1, plated on AMP, to then use these for the EIPCR Quickchange oligos to create a larger library of RBS's.  I also transformed 116 directly in pBACr-AraGFP and plated on AMP+KAN+ARA, to test if there was any obvious activity in high copy.
 +
 
 +
'''July 30:'''
 +
There is no obvious activity in high copy, and the 116(101) plate had a reasonable colony yield, and so I picked 4 colonies to grow in AMP and sequence.
 +
 
 +
'''July 31:'''
 +
All the cultures were red in the pellet except for #2, indicating they were low-copy plasmid bleedthrough.  I then miniprep'd #2 and sent it off for sequencing.
 +
 
 +
'''Aug 1:'''
 +
The sequencing came back as a high-copy Truncation Recombination, and so it is incorrect. I have picked and grown in AMP 4 more 116(101) colonies, that do not appear red, to miniprep/test/sequence.  I have also tried an extra-long gel purify of the 116 and 101 digests, to clean up the bleedthrough.  I then ligated this and transformed into DH10Bs, and plated on AMP.  I also did a ligation digest check plating the 116 and 101 digests in DH10Bs on AMP.  I also transformed a dilution of the 116 library into DH10Bs on AMP to pick a few to then use a single clone to insert into the low-copy plasmid.  I also transformed some DH10Bs on AMP with 101 to then make more with a midiprep.
 +
 
 +
'''Aug 2:'''
 +
The 116(101) transformation failed, and there are very few/no colonies on the 101 and 116 digests.  I also miniprep'd the 116(101) cultures I picked yesterday from the first transformation, and ran 8ul on a gel.  All the bands are bright and are high-copy bleed-through.  I have also picked a few of the 116 colonies and grew in AMP, to then miniprep and sequence. Finally, I also picked a 101 colony to make more with a midiprep.
 +
 
 +
'''Aug 3:'''
 +
I performed a midiprep of the 101 plasmid, and the pellet appeared slightly red, indicating it is correct.  I also miniprep'd the 116 cultures, and sent off for sequencing.
 +
 
 +
'''Aug 4:'''
 +
The sequencing results came back all as some variation on a truncation recombination, indicating that the 116 library is unstable, and so I will try the EIPCR library instead.
 +
 
 +
==Bca1106-H RBS + T7rnap GTG (I716117) - Finished==
 +
 
 +
'''July 31:'''
 +
I have digested the 1106-H RBS with EcoRI and BglII (L), and the 1122 plasmid with EcoRI and BglII (S), to add the KAN resistance gene to the RBS.  I then ligated these two fragments and transformed into TG1s on AMP+KAN.
 +
 
 +
'''Aug 1:'''
 +
The colony yield for the KAN+RBS was excellent, so I picked 4 colonies and grew in AMP+KAN to miniprep.
 +
 
 +
'''Aug 2:'''
 +
I have miniprep'd the 4 KAN+RBS clones, and test digested them with EcoRI/BamHIAll clones look correct, and so I have cut out and gel purified the Small fragment (1047) of 2 of them.  I then ligated this fragment (1) with the T7RG (104,1) EcoRI/BglII Digest, and transformed into DH10Bs on AMP+KAN.
 +
 
 +
'''Aug 3:'''
 +
The colony yield for the KAN+RBS+T7RG was very good, so I picked 4 clones and grew up in AMP+KAN, to then miniprep and sequence.
 +
 
 +
'''Aug 6:'''
 +
I have miniprep'd the RBS+T7RG (117) clones, and sent off for sequencing. A quick test gel indicates that the sizes are good, indicating they are not RBS-only bleedthrough.
 +
 
 +
'''Aug 7:'''
 +
The sequencing came back, and all 4 clones are perfect.  #1 is the confirmed first clone that will be used in the further experiments.
 +
 
 +
'''Aug 19:'''
 +
I have clonesaved 117,1 on the clonesaver card.
 +
 
 +
==Bca1106-H RBS + T7rnap GTG + b0015 (Bca1092) Terminator (I716118) - Finished==
 +
 
 +
'''Aug 7:'''
 +
I have digested the terminator (Bca1092) with EcoRI/BglII, 117 #1 with EcoRI/BamHI, and ligated, transformed into DH10Bs on AMP+KAN.
 +
 
 +
'''Aug 8:'''
 +
I picked 4 118 colonies in AMP+KAN, as the plate's colony yield was good.
 +
 
 +
'''Aug 13:'''
 +
I have miniprep'd the 118's, and sent #1 and #2 off for sequencing with the reverse oligo.
 +
 
 +
'''Aug 14:'''
 +
The sequencing results came back with #1 perfect, and confirmed, with #2 looking correct, but the signal is too low to say for certain.
 +
 
 +
'''Aug 19:'''
 +
I have clonesaved 118,1 on the clonesaver card.
 +
 
 +
==Iron Promoter (014) + Pir (102) (I716119) - Confirmed==
 +
 
 +
'''Aug 6:'''
 +
I have ligate the EcoRI/BamHI digest of the Iron promoter with the EcoRI/BglII digest of pir, and Transformed into DH10Bs on AMP.
 +
 
 +
'''Aug 7:'''
 +
The colony yield was very good, so I picked & grew 4 colonies.
 +
 
 +
'''Aug 8:'''
 +
I have miniprep'd the colonies, and digested with BglII/XhoI.  However, all the digests appear to be single-digests, and so are likely to be a recombination failure or pir bleedthrough.
 +
 
 +
'''Aug 13:'''
 +
I set up colony-ish PCRs with ca998/G00101 on the four 119 (Iron+pir) minipreps.
 +
 
 +
'''Aug 14:'''
 +
The gel indicates that they are all some recombination failure, or pir-only bleed-through.  Therefore, I transformed the Iron Promoter into Lefty (Y), pir into Righty (G), and plated on AMP to try the 1-2-3 method.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel007.jpg|200px]]
+
|[[Image:Berk-DTgel015.jpg|200px]]
-
|The Test Digest Gel.  Lane 1 is marker, lanes 2-9 are the T7rnap test Digests, the second to last is the RBS+pir library digest, and the last lane is the Salicylate Promoter digest.
+
|The PCR Gel.  Lane 1 is marker, then the 119 clones, #1, #2, #3, and #4.
|}
|}
-
'''July 3:'''
+
'''Aug 15:'''
-
I checked the plate, but no colonies grew, I think this is likely due to the incomplete digestion that I observed yesterday, and there being an insufficient amount of ligated product to transform with.
+
I have picked a colony from each plate, and grown in AMP, to then miniprep.
-
'''July 4:'''
+
'''Aug 16:'''
-
I again digested the RBS+pir (108) part with EcoRI and BglII, Gel Purifying the large fragment, with BglII and XhoI, Gel Purifying the small fragment, as well as digesting the Salicylate Promoter with BamHI and XhoI, Gel Purifying the large fragment, with all digests this time for two hours, to ensure complete digestion.
+
I have miniprep'd the Iron and Pir plasmids in lefty and righty.  I then digested Iron with BamHI/XhoI (L-only), and Pir with BglII/XhoI (S), and gel purified the fragments.  I then ligated the iron+pir fragments, heat-killed the ligase at 65C for 10min, then I diluted to 50uL, while adding 5 NEB buffer 2, and digested this product with BglII/BamHI, and transformed into DH10Bs on AMP.
 +
 
 +
'''Aug 17:'''
 +
I have picked & grown 4 119 colonies in AMP, as the colony yield was excellent.
 +
 
 +
'''Aug 18:'''
 +
I have miniprep'd the 4 119 cultures, test digested with EcoRI/XhoI.  All appear to be correct.
{|
{|
|-
|-
-
|[[Image:Berk-DTgel009.jpg|200px]]
+
|[[Image:Berk-DTgel016.jpg|200px]]
-
|The Digest Gel.  Lane 1 is marker, lane 2 is the EcoRI/BglII digest of the RBS+pir library, lane 3 is the XhoI/BglII digest of the RBS+pir library, and the last lane is the BamHI/XhoI digest of the Salicylate Promoter.
+
|The Digest Gel.  Lane 1 is marker, then the 119 clones, #1, #2, #3, and #4.
|}
|}
-
'''July 5:'''
+
'''Aug 20:'''
-
I have ligated the EcoRI fragements together as well as the XhoI fragments together, to hopefully ensure at least one insertion will work.  I then transformed and plated on AMP plates, as the KAN resistance should have been excised out.  I will then scrape and grow in AMP media tomorrow.
+
I sent 119 #1 off for sequencing with ca998.
-
'''July 6:'''
+
'''Aug 21:'''
-
Neither of the plates looked sufficient, with only a few coloniesI believe the ligase I was using might be bad, so I have attempted the same ligation/transformation/plating on AMP as yesterday, but this time with a different ligase.
+
The sequencing results came back correctTherefore, clone #1 is confirmed to be correct.
-
'''July 9:'''
+
==Iron Promoter (014) + PCR RBS Library + Pir (102) (I716120)==
-
Again, the plates looked very insufficient to no colonies, so I will have to re-do the transformations over again, possibly with a different procedure.
+
-
'''July 10:''' I have re-done the same ligation / transformations with 4 times as much miniprep digest, and in TG1 cells, to hopefully to get more colonies.
+
'''Aug 8:'''
 +
I have set up 2 PCRs, dt012-T7-seq2/dt014-IRP-R on D4, and dt013-IRP-F/G00101 on pir (102).
-
==RBS Library + T7rnap ATG, GTG (I716110, I716111) - Building (Waiting for New T7rnap)==
+
'''Aug 10:'''
-
'''Construction File:''' [[BerkiGEM2007-DavidConstructionFile110 | I716110,I716111: RBS Library + T7rnap ATG, GTG]]
+
The PCRs were successful, with bands at the expected sizes.  I have gel-purified the PCR Products in preparation for digestion.
-
'''July 4:'''
+
'''Aug 11:'''
-
I digested the T7rnap ATG and GTG (103,104) parts with EcoRI and BglII, Gel Purifying the large fragment.  I performed this digest for 2 hours, as previous digests were somewhat smeary.
+
I have digested the PCRs with SpeI/XhoI/DpnI, column purified, Ligated, and Transformed into DH10Bs on AMP.
-
{|
+
'''Aug 12:'''
-
|-
+
I have picked & grown in AMP 4 Iron+RBS/PCR+Pir(101) = 120(101) coloniesThere were not enough colonies to scrape and grow the library, however.
-
|[[Image:Berk-DTgel008.jpg|200px]]
+
-
|The Digest GelLane 1 is marker, All digests are EcoRI and BglII, and the clone order is: 103 #2, 103 #4, 104 #2, 104 #3.
+
-
|}
+
-
'''July 5:'''
+
'''Aug 13:'''
-
I ligated the EcoRI/BglII digest with the EcoRI/BamHI digest of the RBS Library, and then transformed some DH10Bs and plated on AMP Plates, to then grow in AMP/KAN Media tomorrow.
+
I have Miniprep'd the 120(101) cultures, but all are High-Copy - they are most likely pir-only bleedthrough.
-
'''July 6:'''
+
'''Aug 18:'''
-
None of the plates looked sufficient, with none to a few colonies. I believe the ligase I was using might be bad, so I have attempted the same ligation/transformation/plating on AMP+KAN as yesterday, but this time with a different ligase.
+
I have performed the EIPCR reaction with dt013-IRP-F/dt014-IRP-R on 119 #1.
-
'''July 9:'''
+
'''Aug 19:'''
-
Again, the plates looked very insufficient to no colonies, so I will have to re-do the transformations over again, possibly with a different procedure.
+
The EIPCR was successful, so I digested the EIPCR product with SpeI, Ligated, Transformed the EIPCR products into DH10Bs.
 +
 
 +
'''Aug 20:'''
 +
The 119->120 EIPCR plates have very few/no colonies, indicating that the digestion may not have succeeded.
=Strain and Competent Cell Production=
=Strain and Competent Cell Production=
Line 242: Line 378:
'''July 10:'''
'''July 10:'''
The plate I grew yesterday has hundreds of colonies, and glows slightly green under UV light, indicating that the cells are indeed both competent and functional.
The plate I grew yesterday has hundreds of colonies, and glows slightly green under UV light, indicating that the cells are indeed both competent and functional.
 +
 +
'''July 17:'''
 +
I am testing the cells' sensitivity to AMP, by plating directly some competent cells on an AMP plate.
 +
 +
'''July 18:'''
 +
The cells are indeed AMP-sensitive, as the AMP-only plate appears clean, with no colonies whatsoever.

Latest revision as of 23:51, 24 October 2007

<< Back to UC Berkeley iGEM 2007
My Construction Files
My Sequencing Files
Notebook Archives: May 30 - June 5, Finished Basic Parts 101-107

Contents

Progress Status Descriptions

Designing : Making Construction Files and Ordering Oligos
Building : PCR, Transforming, Culturing
Checking : Analytical Digesting and Sequencing
Confirmed : Correct clones are confirmed and mini/midiprep'd
Finished : Correct clones are organized and saved


Composite Part Construction

RBS Library + pir (I716108) - Finished

Construction File: I716108: RBS Library + pir

June 27: I digested the pir confirmed part (clone #4) with EcoRI and BglII, then ligated this to the digestion of pBca1122-Bca9235 with EcoRI and BamHI. I then transformed these into some DH10B cells, and plated on AMP+KAN plates today, and will grow the culture tomorrow.

June 29: Due to the incubator being turned off, I could not grow the culture yesterday, as the colonies were not visible, therefore, I scraped the plate in LB today, and grew in AMP+KAN LB Media (which I had to make, due to our being out of that particular mix), to then miniprep on Monday. (After being placed in the refrigerator on Saturday)

July 2: I have miniprep'd the library, and it appears correct, but I will not know whether the RBS + pir combination works until I test it with a promoter in the next pir composite part.

July 25: The sequencing result of some individual clones of 109 came back, indicating the RBS library must be correct.

July 27: I have now saved this library on the clone saver sheet.

Salicylate Promoter + RBS Library + pir (I716109) - Finished

July 2: I digested the RBS+pir (108) part with EcoRI and BglII, Gel Purifying the large fragment. As well, I digested the Salicylate Promoter with EcoRI and BamHI, Gel Purifying the small fragment, for an insertion. I then ligated the two fragments together, and transformed, and plated on an AMP plate, as the KAN resistance gene should have been excised. However, the digest of the RBS+pir library looked very smeary, as there was likely incomplete digestion.

Berk-DTgel007.jpg The Test Digest Gel. Lane 1 is marker, lanes 2-9 are the T7rnap test Digests, the second to last is the RBS+pir library digest, and the last lane is the Salicylate Promoter digest.

July 3: I checked the plate, but no colonies grew, I think this is likely due to the incomplete digestion that I observed yesterday, and there being an insufficient amount of ligated product to transform with.

July 4: I again digested the RBS+pir (108) part with EcoRI and BglII, Gel Purifying the large fragment, with BglII and XhoI, Gel Purifying the small fragment, as well as digesting the Salicylate Promoter with BamHI and XhoI, Gel Purifying the large fragment, with all digests this time for two hours, to ensure complete digestion.

Berk-DTgel009.jpg The Digest Gel. Lane 1 is marker, lane 2 is the EcoRI/BglII digest of the RBS+pir library, lane 3 is the XhoI/BglII digest of the RBS+pir library, and the last lane is the BamHI/XhoI digest of the Salicylate Promoter.

July 5: I have ligated the EcoRI fragements together as well as the XhoI fragments together, to hopefully ensure at least one insertion will work. I then transformed and plated on AMP plates, as the KAN resistance should have been excised out. I will then scrape and grow in AMP media tomorrow.

July 6: Neither of the plates looked sufficient, with only a few colonies. I believe the ligase I was using might be bad, so I have attempted the same ligation/transformation/plating on AMP as yesterday, but this time with a different ligase.

July 9: Again, the plates looked very insufficient to no colonies, so I will have to re-do the transformations over again, possibly with a different procedure.

July 10: I have re-done the same ligation / transformations with 4 times as much miniprep digest, and in TG1 cells, to hopefully to get more colonies.

July 11: The plates looked a little better, but still very low yield. I have tried the EcoRI digests, etc. again, as it was the most successful direction in the previous attempts.

July 12: The plate looked excellent, and so I scraped and cultured it in 5mL of LB+AMP Broth.

July 13: I have miniprep'd the library, and digested it and the 101 low copy plasmid with BglII and XhoI. 101's large fragment (backbone) was gel purified, but 109's bands were too close to differentiate, so I ran a ca998/G00101 PCR on it to amplify the part.

Berk-DTgel011.jpg The Digest Gel. Lane 1 is the 109 digest, lane 2 is the 101 digest, and the last lane is the Marker.

July 16: I gel-purified the PCR Product, and while there were multiple bands, the desired one was considerably brighter than the rest. I then digested the PCR Product with BglII and XhoI. I then transformed the ligation of 109 in 101 into pBACr-AraGFP cells, and plated on an AMP+KAN+Ara+Sal plate.

July 17: The 109 in 101 ligation product had lots of colonies, but no obviously green ones, so I picked 96 and grew in 600 uL of AMP + KAN + Sal + Ara.

July 18: All 96 wells showed uniform uninduced activity, even with Salicylate in the media, so I miniprep'd 2 of them and did a PCR with ca998 / pir-R, for use in sequencing tomorrow.

July 19: I did another digest of 101 with BglII and XhoI, in case my previous digest was too low-quality. Additionally, the PCR for the two wells failed, so I will have to sequence a high copy plasmid. I also transfomed a new ligation of 109 in 101 on AMP+KAN+ARA+SAL, as well as directly transformed 109 on that kind of plate. I also transformed the ligation product of only the digest itself, to test the quality of the digests.

Berk-DTgel012.jpg The Digest Gel. Lane 1 is Marker, 2 is the 101 digest, and lane 3 is a T7rnap EcoRI/BglII digest.

July 20: The first 101 digest looked clean, the second had many colonies, and the 109 digest had lots of colonies. Additionally, the 109 direct and 109 in 101 plates showed no significant activity. Therefore, I transformed into TG1 cells 1 uL of a 20x dilution of 109 onto AMP to then pick and analyze individual colonies. I also test digested with HindIII the 109 library to see if its any good. The band pattern looked approximately right, but somewhat fuzzy and uncertain.

Berk-DTgel013.jpg The Digest Gel. Lane 1 is Marker, 2 is the 101 digest, lane 3 is the 109 test digest with HindIII, and the last lane is the PCR for the low/high plasmid Arthur is making.

July 23: I picked 4 colonies of 109 to then miniprep and sequence, to determine if the construct is correct.

July 24: I have miniprep'd the 4 109 colonies, and sent off for sequencing with ca1111F, which binds to the middle of the nahR gene in the Salicylate promoter part.

July 25: The 4 clones came back as correct, and so this library is correct, even though it does not appear to work, most likely due to the ineffectiveness of the Salicylate promoter.

July 27: I have now saved this library on the clone saver sheet.

RBS Library + T7rnap ATG, GTG (I716110, I716111) - Finished

Construction File: I716110,I716111: RBS Library + T7rnap ATG, GTG

July 4: I digested the T7rnap ATG and GTG (103,104) parts with EcoRI and BglII, Gel Purifying the large fragment. I performed this digest for 2 hours, as previous digests were somewhat smeary.

Berk-DTgel008.jpg The Digest Gel. Lane 1 is marker, All digests are EcoRI and BglII, and the clone order is: 103 #2, 103 #4, 104 #2, 104 #3.

July 5: I ligated the EcoRI/BglII digest with the EcoRI/BamHI digest of the RBS Library, and then transformed some DH10Bs and plated on AMP Plates, to then grow in AMP/KAN Media tomorrow.

July 6: None of the plates looked sufficient, with none to a few colonies. I believe the ligase I was using might be bad, so I have attempted the same ligation/transformation/plating on AMP+KAN as yesterday, but this time with a different ligase.

July 9: Again, the plates looked very insufficient to no colonies, so I will have to re-do the transformations over again, possibly with a different procedure.

July 16: I have digested the new T7rnap parts with EcoRI and BglII, to then insert the RBS part. I have ligated in the RBS part, and transformed into TG1 cells, and plated on AMP+CAM.

July 17: I have scraped and cultured the plates in AMP+CAM, as there were a ridiculous number of colonies.

July 18: I have now miniprep'd the two libraries, as they appear to be correct.

July 26: As the phenotypic analysis indicates definite functionality in both high and low copy plasmids, this library is confirmed to be correct.

Aug 19: I have clonesaved clones 110,3 and 111,1.

Salicylate Promoter, Iron Promoter + RBS Library + T7rnap ATG, GTG
(Salicylate: I716112, I716113; Iron: I716114, I716115) - Finished

July 19: I have digested the T7rnap ATG and GTG RBS Libraries (110,111) with EcoRI and BglII and purified the Large fragment. This was then ligated with an EcoRI and BamHI digest of the Salicylate Promoter and Iron Promoters, and transformed into some TG1 cells, and plated on AMP.

July 20: The T7 GTG Starts have a decent colony yield, but the ATGs don't have very many colonies. This is likely due to the toxicity of the T7rnap, as there is some leaky expression even when uninduced. Therefore, I have scraped and miniprep'd directly off the plate for the GTG starts. I also re-did the ATG (110) Ligation / Transformation with more T7rnap material into TG1s and plated on AMP.

July 23: The T7rnap ATG plates have a sufficient colony yield, but not too many, indicating high toxicity. I then scraped and miniprep'd the Promoter/RBS/T7RA plates. I have also digested the Promoter/RBS/T7RA,G plasmids with BglII and XhoI and ligated into a BglII/XhoI digest of 101, to make them low copy. I then transformed into GH455G cells on AMP: The Ligations of 112-115 in 101, and the direct plasmids of 112-115, and 112-113 on AMP+SAL.

July 24: These is confirmed activity with the Iron Promoter with the ATG and GTG Starts, and the low copy ATG starts have very few colonies. I have then picked 96 colonies on a 96-well plate from the low copy GTG starts - columns 1-8 are 115 (Iron) in 101, columns 9-12 are 113 (Salicylate) in 101, and put this in the incubator to grow overnight, with TB+AMP+Glycerol.

July 25: I grew up the 96 well plate (10 uL of culture from each well) in two 96 well blocks (600 uL culture in each well), one with LB+AMP only, and the other with LB+AMP+Iron or Salicylate, depending on the construct, to then do TECAN analysis when the cultures are at saturation.

July 26: I have done the TECAN analysis with 10uL of each well (on/off), with the parameters of bandwidth = 7.0, GFPmut2 (Ex: 481nm, Em: 507nm) as the spectrum, and a gain of 80. The well D4 was a hit with a variance of 1850 (off: 1300, on: 3150), or 4.3 times a fluorescent, after removing the background. I have also miniprep'd the D4 well (off), and set up a ca998/T7-R PCR to amplify the part for sequencing.

July 27: The D4 PCR failed, so I ran an Iron-F/T7-R PCR on D4 to then sequence.

July 30: The D4 PCR was successful this time, so I gel purified it, and sent it off for sequencing with the ca570F oligo, which is a reverse internal T7 sequencing oligo.

July 31: The sequencing result came back correct, with the 1106-H RBS.

Aug 18: I have run a gel of D4, and the band is quite faint, indicating it is definitely low-copy.

Aug 20: I have clonesaved the D4 clone.

Iron Promoter + RBS Library + pir (I716116) - Discontinued due to instability

July 25: I have ligated the Iron Promoter digest with EcoRI and BamHI, Small Part Fragment, and the EcoRI/BglII, Large part fragment, digest of 108 (RBS+pir). I then transformed in TG1s, and plated on AMP.

July 26: I have now scraped and cultured the 116 (Iron+108) library, as the colony yield was very good.

July 27: I have miniprep'd the 116 (Iron+108) library, and digested it with BglII and XhoI, as well as digested 101 with BglII and XhoI. I then ligated the digests of 116 and 101, transformed in TG1, plated on AMP, to then use these for the EIPCR Quickchange oligos to create a larger library of RBS's. I also transformed 116 directly in pBACr-AraGFP and plated on AMP+KAN+ARA, to test if there was any obvious activity in high copy.

July 30: There is no obvious activity in high copy, and the 116(101) plate had a reasonable colony yield, and so I picked 4 colonies to grow in AMP and sequence.

July 31: All the cultures were red in the pellet except for #2, indicating they were low-copy plasmid bleedthrough. I then miniprep'd #2 and sent it off for sequencing.

Aug 1: The sequencing came back as a high-copy Truncation Recombination, and so it is incorrect. I have picked and grown in AMP 4 more 116(101) colonies, that do not appear red, to miniprep/test/sequence. I have also tried an extra-long gel purify of the 116 and 101 digests, to clean up the bleedthrough. I then ligated this and transformed into DH10Bs, and plated on AMP. I also did a ligation digest check plating the 116 and 101 digests in DH10Bs on AMP. I also transformed a dilution of the 116 library into DH10Bs on AMP to pick a few to then use a single clone to insert into the low-copy plasmid. I also transformed some DH10Bs on AMP with 101 to then make more with a midiprep.

Aug 2: The 116(101) transformation failed, and there are very few/no colonies on the 101 and 116 digests. I also miniprep'd the 116(101) cultures I picked yesterday from the first transformation, and ran 8ul on a gel. All the bands are bright and are high-copy bleed-through. I have also picked a few of the 116 colonies and grew in AMP, to then miniprep and sequence. Finally, I also picked a 101 colony to make more with a midiprep.

Aug 3: I performed a midiprep of the 101 plasmid, and the pellet appeared slightly red, indicating it is correct. I also miniprep'd the 116 cultures, and sent off for sequencing.

Aug 4: The sequencing results came back all as some variation on a truncation recombination, indicating that the 116 library is unstable, and so I will try the EIPCR library instead.

Bca1106-H RBS + T7rnap GTG (I716117) - Finished

July 31: I have digested the 1106-H RBS with EcoRI and BglII (L), and the 1122 plasmid with EcoRI and BglII (S), to add the KAN resistance gene to the RBS. I then ligated these two fragments and transformed into TG1s on AMP+KAN.

Aug 1: The colony yield for the KAN+RBS was excellent, so I picked 4 colonies and grew in AMP+KAN to miniprep.

Aug 2: I have miniprep'd the 4 KAN+RBS clones, and test digested them with EcoRI/BamHI. All clones look correct, and so I have cut out and gel purified the Small fragment (1047) of 2 of them. I then ligated this fragment (1) with the T7RG (104,1) EcoRI/BglII Digest, and transformed into DH10Bs on AMP+KAN.

Aug 3: The colony yield for the KAN+RBS+T7RG was very good, so I picked 4 clones and grew up in AMP+KAN, to then miniprep and sequence.

Aug 6: I have miniprep'd the RBS+T7RG (117) clones, and sent off for sequencing. A quick test gel indicates that the sizes are good, indicating they are not RBS-only bleedthrough.

Aug 7: The sequencing came back, and all 4 clones are perfect. #1 is the confirmed first clone that will be used in the further experiments.

Aug 19: I have clonesaved 117,1 on the clonesaver card.

Bca1106-H RBS + T7rnap GTG + b0015 (Bca1092) Terminator (I716118) - Finished

Aug 7: I have digested the terminator (Bca1092) with EcoRI/BglII, 117 #1 with EcoRI/BamHI, and ligated, transformed into DH10Bs on AMP+KAN.

Aug 8: I picked 4 118 colonies in AMP+KAN, as the plate's colony yield was good.

Aug 13: I have miniprep'd the 118's, and sent #1 and #2 off for sequencing with the reverse oligo.

Aug 14: The sequencing results came back with #1 perfect, and confirmed, with #2 looking correct, but the signal is too low to say for certain.

Aug 19: I have clonesaved 118,1 on the clonesaver card.

Iron Promoter (014) + Pir (102) (I716119) - Confirmed

Aug 6: I have ligate the EcoRI/BamHI digest of the Iron promoter with the EcoRI/BglII digest of pir, and Transformed into DH10Bs on AMP.

Aug 7: The colony yield was very good, so I picked & grew 4 colonies.

Aug 8: I have miniprep'd the colonies, and digested with BglII/XhoI. However, all the digests appear to be single-digests, and so are likely to be a recombination failure or pir bleedthrough.

Aug 13: I set up colony-ish PCRs with ca998/G00101 on the four 119 (Iron+pir) minipreps.

Aug 14: The gel indicates that they are all some recombination failure, or pir-only bleed-through. Therefore, I transformed the Iron Promoter into Lefty (Y), pir into Righty (G), and plated on AMP to try the 1-2-3 method.

Berk-DTgel015.jpg The PCR Gel. Lane 1 is marker, then the 119 clones, #1, #2, #3, and #4.

Aug 15: I have picked a colony from each plate, and grown in AMP, to then miniprep.

Aug 16: I have miniprep'd the Iron and Pir plasmids in lefty and righty. I then digested Iron with BamHI/XhoI (L-only), and Pir with BglII/XhoI (S), and gel purified the fragments. I then ligated the iron+pir fragments, heat-killed the ligase at 65C for 10min, then I diluted to 50uL, while adding 5 NEB buffer 2, and digested this product with BglII/BamHI, and transformed into DH10Bs on AMP.

Aug 17: I have picked & grown 4 119 colonies in AMP, as the colony yield was excellent.

Aug 18: I have miniprep'd the 4 119 cultures, test digested with EcoRI/XhoI. All appear to be correct.

Berk-DTgel016.jpg The Digest Gel. Lane 1 is marker, then the 119 clones, #1, #2, #3, and #4.

Aug 20: I sent 119 #1 off for sequencing with ca998.

Aug 21: The sequencing results came back correct. Therefore, clone #1 is confirmed to be correct.

Iron Promoter (014) + PCR RBS Library + Pir (102) (I716120)

Aug 8: I have set up 2 PCRs, dt012-T7-seq2/dt014-IRP-R on D4, and dt013-IRP-F/G00101 on pir (102).

Aug 10: The PCRs were successful, with bands at the expected sizes. I have gel-purified the PCR Products in preparation for digestion.

Aug 11: I have digested the PCRs with SpeI/XhoI/DpnI, column purified, Ligated, and Transformed into DH10Bs on AMP.

Aug 12: I have picked & grown in AMP 4 Iron+RBS/PCR+Pir(101) = 120(101) colonies. There were not enough colonies to scrape and grow the library, however.

Aug 13: I have Miniprep'd the 120(101) cultures, but all are High-Copy - they are most likely pir-only bleedthrough.

Aug 18: I have performed the EIPCR reaction with dt013-IRP-F/dt014-IRP-R on 119 #1.

Aug 19: The EIPCR was successful, so I digested the EIPCR product with SpeI, Ligated, Transformed the EIPCR products into DH10Bs.

Aug 20: The 119->120 EIPCR plates have very few/no colonies, indicating that the digestion may not have succeeded.

Strain and Competent Cell Production

T7GFP Strain - GH455G

July 2: Chris took the -80 of this strain out of the freezer, and plated it on an LB-Only plate.

July 3: I picked a colony from the plate, and grew it up in 5mL of LB-Only Broth.

July 4: I took the culture out of the incubator, and put it in the refrigerator, to enable simultaneous competent cell production with the pBACr-AraGFP Strain.

July 5: I made competent cells from the culture grown on the 3rd, for a total of approximately 25 200uL aliquots. I then froze them using liquid nitrogen, and placed them in a box in the top shelf of the -80 freezer.

July 6: I then transformed some of the competent cells with pBAC-T7621B, as this plasmid produces the T7rnap constitutively, and should indicate whether the cells are indeed competent and functional. These were then plated on a KAN-only plate.

July 9: The transformation with pBAC-T7621B worked very well, and the transformed cells do, in fact, glow green under UV light, indicating that the cells are indeed competent, and functional.

pBACr-AraGFP (in DH10B) Competent Cells

July 2: I have transformed some DH10B cells with pBACr-AraGFP, and plated on a KAN-only plate.

July 3: Yesterday's transformation failed, as there were no colonies. Therefore, I have tried transforming some DH10B cells with pBACr-AraGFP again, this time with a longer rescue (>1 hour, in 2yT), and plated them on a KAN-only plate.

July 4: The plate appeared to have very few colonies, but they did have the resistance gene, so I picked a colony from the plate, and grew it up in 5mL of LB+KAN Broth.

July 5: I made competent cells from the culture grown yesterday, for a total of approximately 25 200uL aliquots. I then froze them using liquid nitrogen, and placed them in a box in the top shelf of the -80 freezer.

July 9: I transformed some of these competent cells with I716107 to give them AMP resistance, and have plated on an AMP + Arabinose plate, to test their competency and functionality, as they should grow slightly green upon induction with Arabinose.

July 10: The plate I grew yesterday has hundreds of colonies, and glows slightly green under UV light, indicating that the cells are indeed both competent and functional.

July 17: I am testing the cells' sensitivity to AMP, by plating directly some competent cells on an AMP plate.

July 18: The cells are indeed AMP-sensitive, as the AMP-only plate appears clean, with no colonies whatsoever.