Team:Lethbridge/Notebook/Lab Work/Group2

Week 1 May 2 - 8 2011

Week 2 May 9 - 15 2011
Week 3 May 16 - 22 2011
Ryan vacationing in Whitefish. Having an awesome time.
Week 4 May 23 - 29 2011
First week Ryan begins working full time in the lab. Successfully used polymerase chain reaction (PCR) and specifically designed primers to alter prefix and suffix regions to to the Silver standard for the genes xylE and mms6. That way we can fuse signal sequences. On an agarose gel the DNA strands are of expected size for both xylE and mms6 with the fusion standard. After attempting to cut PCR products of xylE and mms6 and cloning into psb1c3 using red/white screening, no white colonies test positive for the product when the plasmids are restricted and resolved on a gel. Miscellaneous plasmids are taken out of the glycerol stock and grown in LB cultures.
Week 5 May 30 - June 5 2011
The problem with the ligation of PCR products into psb1c3 is diagnosed as a result of prefix and suffix restriction sites being too close to the ends of the PCR products. Instead of using red/white colony screening and psb1c3 the polymerase used in the PCR produces blunt ends and therefore blunt end ligation is used for cloning with puc19. Different conditions are used in the blunt end ligation. Since puc19 has to be cut bluntly half the samples have the blunt end restriction enzyme heat killed. Blue/white colony screening has to be used in for puc19 cloning which requires X-Gal and IPTG. Solutions of these are made for the plates. Attempts to assemble miscellaneous parts from synthesis vectors to psb1c3 results in positive testing white colonies on an agarose gel for k331025, an enhanced cyan fluorescent protein with a ribosomal binding site and a c-terminally fused arginine residue tail. One colony for the blunt end ligation cloning of an xylE PCR product produces a band at the expected size when the plasmid is restricted and run on a gel. Attempts to move the remaining miscellaneous parts into psb1c3 produce no positive testing white colonies.
Week 6 June 6 - 12
A glycerol stock of the one colony of xylE and k331025 were made after they were re-transformed and grown in liquid LB culture. After another attempt the mms6 PCR product still refuses to be cloned via blunt end ligation. The same can be said for the another assembly of the miscellaneous parts to psb1c3.
Week 7 June 13 - 19
While these other aspects continue to elude success a plasmid preparation of SO4261, the ten residue arginine tag with a double transcriptional terminator, is transformed and a glycerol stock of it is made is made.
Week 8 June 20 - 26
The xylE from puc19 is assembled with the arginine tag. The miscellaneous parts are also re-assembled into psb1c3. Both through PCR and restriction the gels do not show a positive result for the xylE-arg tag assembly. A blunt end ligation of mms6 into puc19 shows some white colonies.
Week 9 June 27 - July 3
The miscellaneous parts are restricted and run on a gel along with mini-preps of the mms6 colonies that were grown in liquid LB media and while all the remaining miscellaneous parts are consistent with expected results, the mms6 is blank on the gel. A PCR of the mms6 in puc19 also gives the same result on an agarose gel. The xylE and re-assembled, plated, possible colonies are picked and grown in liquid LB media and cultures are mini-prepped.
Week 10 July 4 - 10
After restricting and running on a gel all the xylE-arg tag assemblies came back negative. The lack of progress on xylE and mms6 being suspicious, new approachs are used. PCR products of both xylE and mms6 with the fusion standard are gel extracted using a Qiagen gel extraction kit. These gel extractions have DNA presence confirmed on a gel and are then used in a blunt end ligation with puc19 and a restriction free cloning attempt with psb1c3. Neither first attempt succeeds.
Week 11 July 11 - 17
Sequencing data from samples sent to genewiz come back showing k331025 and k331008, a signal sequence for localization to the outer membrane of E. coli is the correct sequence and confirmed suspicions regarding what was thought to be xylE in puc19. Six more attempts at restriction free cloning fail to produce results, including attempts to use non gel extracted PCR products that have had the original plasmid digested. K331008 is re-transformed, colonies picked, cultures grown, and a glycerol stock is made.
Week 12 July 18 - 24
Another attempt at restriction free cloning with a different polymerase fails. Running low on PCR product mms6 and out of PCR product xylE. Strangely, an attempt at repeating the PCR with the same conditions fails to reproduce the xylE when run on an agarose gel. Another attempt at gel extracting mms6 PCR product and blunt end ligation with puc19 comes to naught. Another assembly is done on the two remaining miscellaneous parts to get them into psb1c3.
Week 13 July 25 - 31
After hearing about a system for cloning PCR products, an attempt at reproducing the PCR products using a polymerase possessing template independent terminal transferase activity that produces adenine overhangs is done but, to no avail. A gel of the two reassembled parts is consistent with expected sizes. Three more attempts at PCR using different conditions to replace the PCR products either with or without adenine overhangs fail to produce results. The PCR cloning system from Promega is ordered along with a new polymerase.
Week 14 August 1 - 7
A test is done using different generic primers and two different polymerases on a confirmed plasmid. The polymerase that produces adenine overhangs successfully produces a band of the expected size. Using this polymerase, another PCR of xylE is attempted, the largest yet, with a slightly longer elongation time and a wide variety of annealing temperatures and magnesium concentrations. All twenty four unique reactions produced a band at the expected size of xylE.
Week 15 August 8 - 14
The xylE PCR products had the original host plasmids removed in a digest before they were cloned using the newly arrived pGEM-T vector blue/white colony cloning system from Promega. PCR producing adenine overhangs for mms6 were completed successfully before they were gel extracted and cloned using the new system. Colonies of both cloning were picked, grown in liquid media, and mini-prepped. Restricting these isolated plasmids and running on a gel shows bands of expected sizes.
Week 16 August 15 - 21
An assembly to attach the arginine tag to xylE is done with red/white colony screening. White colonies are picked, grown in liquid media, and mini-prepped. When restricted and run on a gel no colony has a band at the expected size. Sequencing data from Genewiz confirms k331022, a ribosomal bind site and yellow fluorescent protein with an n-terminally tagged arginine, and k331024, a ribosomal binding site and cyan fluorescent protein with an n-terminally tagged arginine.
Week 17 August 22 - 28
Ryan is on vacation on Vancouver island. The arginine tag is assembled onto xylE.
Week 18 August 29 - September 4
Primers add or alter the Silver fusion standard to xylF and xylT arrive. Initial PCR produces expected bands when run on a gel, even for xylT which is in the genomic DNA. Using pGEM-T both genes are cloned, however, the plates are bad and no selection pressure exists.
Week 19 September 5 - 11
New plates are made and the two genes are re-cloned. Colonies picked, grown in liquid media, and mini-prepped.
Week 20 September 12 - 18
The genes for xylT and xylF were sent for sequencing.
Week 21 September 19 - 25
Final Half Week September 26 - 28