Team:Calgary/Notebook/Calendar/Week16

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Genomic Library Expression

Project Participants

Peter Qi, Saeed Qureshi

Author: Peter

July 7-14th, 2011

Miniprepped the B0015 plasmid (iGEM) from overnight cultures in LB/AK. Plasmid yield is acceptable, with relatively high purity in most samples. The plasmids are cut with EcoRI and blunted ended with T4 polymerase. A streak plate (Gent15) of the pUCP30T backbone was acquired from the Schryvers lab glycerol stocks, and overnight cultures were made to collect the backbone plasmid. The yield was high with good purity. pUCP30T plasmid DNA was digested with SfiI with recommended reaction conditions, and frozen/stored at -20C, to stop but not inactivate the SfiI enzyme. Further cloning steps to be continued in the next week.

Author: Peter

July 14-21st, 2001

Cleaned up SfiI restriction digest reaction with PCR purification kit, and measured DNA concentration on Nanodrop, the concentration was low, but the purity was relatively high for most samples. The product is the pUC30T plasmid cut and blunt-ended (still 1 linear piece of DNA).

Blunt ended the products of B0015 (iGEM plasmid) EcoRI digest, and blunt-ended by T4 reaction, the products subsequently purified by gel extraction. Concentration measured as before, but is too low and impure. However, it is acceptable to use these samples to do the ligation step with product from above, introducing the T7 double terminator in B0015 into the pUCP30T backbone.

Plasmid DNA was prepared from overnight P. putida cultures, two preps were made. A genomic prep was made as well. The plasmid prep samples contained high nucleotide concentration, but is likely contaminated with RNA, confirmed by RNase treatment results. There is also a large amount of undissolved material, which is likely RNA. The genomic prep yielded little DNA. Once all samples are properly solubilized, they will all be treated with RNase, and concentration measured. The DNA prepared here is for the genomic and plasmid promoter libraries, which can start once all DNA are collected.

More plasmid DNA of the parts to be cloned will be prepped, since low DNA concentration has been an issue. The pUCP30T-ter plasmid should constructed and transformed next week.

Author: Peter

July 21-28th

Performed 10 minipreps each for the B0015 and pUCP30T plasmid, and DNA/nucleotide concentration of each prep is measured on Nanodrop. All B0015 preps are digested with EcoRI for 2 hours, while all pUCP30T preps are digested with SfiI for 2 hours. The pUCP30T digest is purified using a PCR purification kit, with modification to the original protocol (see list of protocols). All digests are blunt-ended by Invitrogen T4 DNA polymerase, and purified again using PCR purification. The purified blunt-end fragments are all digested in PstI for 2 hours, and run on a 1% agarose gel for electrophoresis. The proper fragments from pUCP30T (~4.1kb) is isolated using Qiagen gel extraction kit. The fragment from the B0015 plasmid (129kb) was lost due to over elution. The B0015 fragment will be recovered again next week using a 1.5% agarose gel, and a ladder appropriate for its size. Gel extraction protocols will also be refined to give higher and purer yields of cloned parts.

Author: Peter

July 28th – Aug 5th, 2011

OBJECTIVES:

1) To purify the B0015 part (T7 double terminator element, 129bp) from restriction digests. 2) Ensure that the B0015 terminator part is flanked by one sticky end made by PstI, and a blunt ended with a poly-A tail. 3) To purify the pUCT30T plasmid (-lacZ a) from restriction digests. PROJECT/SUBPROJECT: Making a genetic tool/plasmid to make a genomic & plasmid library of P. putida. OBJECTIVE: See above REFERENCE: July 28th – Aug 5th, 2011 WHAT DID YOU DO: For objectives 1) & 2): 1. Restriction digest of 10µg of B0015 plasmid DNA by EcoRI (incubation at 37C for 2 hrs, inactivate enzyme by incubation at 80C for 20min). 2. Blunt end the restriction digest products using NEB T4 DNA polymerase. 3. Use Qiagen PCR purification kit to remove T4 DNA polymerase and nucleotides (dNTPs) 4. Added a poly-A tail to the blunt ends. For objectives 3): 1. Restriction digest of 10µg of pUCP30T plasmid DNA by SfiI (incubation at 50C for 2 hrs, remove enzyme by PCR purification). 2. Blunt end the restriction digest products using NEB T4 DNA polymerase. 3. Use Qiagen PCR purification kit to remove T4 DNA polymerase and nucleotides (dNTPs) 4. Added a poly-T tail to the blunt ends. RESULTS: Purified results show 50-60% yields from PCR purifications at the end of step 3 (for both sections), or approximately 5 µg of DNA. INTERPRETATION The amount of DNA retrieved after each cloning step may be too little eventually to acquire enough DNA, especially for the B0015. Potentially, PCR amplification of the iGEM part

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