Sbb14 Neeka Meshgin

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Tuesday Feb. 11

Searched web for thermophilic bacteria.

Options:

Borrelia burgdorferi

Aquifex aeolicus

Thermus thermophilus

Thermatoga maritima

Contacted Professor Wemmer to see if he has access to aquifex since we can't find it on atcg.

Thursday Feb. 13

Chose thermus thermophilus & thermatoga maritima. Split up 20 amino acids and 2 bacteria between 4 teams.


Friday Feb. 14

Split up 10 tRNA synthetases between group.


adrianna:M N neeka: P W daniel: R S kevin: T V akash:Q Y


Did Pubmed search for DNA sequences for prolyl and tryptophanyl tRNA synthetases of thermus thermophilus. Found them. No internal restriction sites for Nco1 and EcoR1.


Neeka Meshgin 13:38, 18 February 2014 (EST)

Designed oligos.


Forward prolyl tRNA synthetase ccaaaCCATGGatggcgaaggagaagggcct

Reverse prolyl tRNA synthetase gctagGAATTCtcagtacgccttggcgaaga


Forward tryptophanyl tRNA synthetase ccaaaCCATGGatgaagcgggtgctttccgg

Reverse tryptophanyl tRNA synthetase gctagGAATTCtcaccggaggaccggccgct


Neeka Meshgin 13:44, 20 February 2014 (EST)

Finished designing oligos and writing construction files. Edited my oligos for tryptophanyl tRNA synthetase to remove internal restriction site. Double-checked each other's oligos.


Construction file for tryptophanyl tRNA synthetase:

PCR TTHA1227F/ TTHA1227internalR on TTHA1227 ( 974 bp, A) PCR TTHA1227internalF/TTHA1227R on TTHA1227 (83 bp, B) PCR TTHA1227F/ TTHA1227R on A+B (1036 bp, pcrpdt) Digest prcpdt (Nco1/ EcoR1, L, 1005 pcrdig) Digest pBAD:myc-HisA (Nco1/EcoR1, 3736+317, plasdig) Ligate pcrdig and plasdig, product is TTHA1227-pBAD:myc-HisA

>TTHA1227F forward oligo

ccaaaCCATGGatgaagcgggtgctttccgg

>TTHA1227R reverse oligo

gctagGAATTCtcaccggaggaccggccgct

>TTHA1227internalF forward removing Nco1 site

caggcgacGatggaggaggtg

>TTHA1227internalR reverse removing Nco1 site

cacctcctccatCgtcgcctg

>TTHA1227 tryptophanyl tRNA synthetase of thermus thermophilus

>pBAD:myc-HisA



Construction file for prolyl tRNA synthetase

PCR TTHA0115-F/ TTHA0115-R (1456 bp, pcrpdt) Digest pcrpdt (Nco1/EcoRI, 1425+25+6, L, pcrdig) Digest pBAD:myc-HisA (Nco1/EcoRI, 3736+317, L, vectdig) Ligate pcrdig and vectdig (TTHA0115-pBAD:myc-HisA)

>TTHA0115F forward oligo

ccaaaCCATGGatggcgaaggagaagggcct

>TTHA0115R reverse oligo

gctagGAATTCtcagtacgccttggcgaaga

>TTHA0115 prolyl tRNA synthetase of thermus thermophilus

>pBAD:myc-HisA


Thursday Feb. 27

Watched demo of mini prep and gel purification.

Did mini prep protocol.


Tuesday March 4

Set up PCR. First made a "master mix" of ddH2O, buffer, and dNTP's for our group. Then diluted my oligos to 10 μM. Then combined "master mix," oligos, template DNA, and enzyme in a PCR reaction tube. Then placed the reaction tube in the thermocycler.


Thursday March 6

Ran a gel for our PCR products from Thursday. Out of my tRNA synthases, tryptophan worked, proline did not (lane 1).

Image:BioE140LG2Gel1.jpg


Friday March 7

Re-did PCR for proline. Did Zymo clean-up of my successful tryptophan.

Tuesday March 11

Did zymo cleanup of proline PCR product.

Digested tryptophan, and did zymo cleanup of digested tryptophan.

Thursday March 13

Ran a gel for proline PCR product. Gel displayed multiple products, ranging from 1400-2000 bp (should only have 1400). Image:3-13gel.jpg

Proline is shown above in lanes 4 and 6.

Re-did PCR for proline, (same protocol, except using 1 μL of DMSO and subtracting 1 μL of water) to prevent possibility that oligo's are annealing upstream of target sequence.

Tuesday March 18

Ran a gel for proline PCR product. 2 distinct bands were shown, and I cut out the 1400 bp band. Did zymo cleanup of proline PCR product from the gel.

Thursday March 20

Digested proline PCR product, and then did zymo cleanup of proline digest.

Ligated typtophan digest with vector digest following ligation protocol.

Followed transformation protocol. Also did a negative control (just vector, no ligated tRNA synth gene.) Streaked plasmids onto agar plates. Incubated at 37°C overnight.

Image:3-20platesG2.jpg

Friday March 21

Checked plates--none have colonies.

Tuesday April 1

Ligated proline digest with vector digest following ligation protocol. Followed transformation protocol for both proline and tryptophan. Streaked transformed cells on agar plates and incubated overnight.

Image:4-1platesG2.jpg

Thursday April 3

Colonies successfully grew on plates. Picked out round, well-isolated colonies to do miniprep later. Picked out 4 colonies for proline and 4 for tryptophan, and suspended each colony in 4 mL of LB media and ampicillin. Incubated overnight at 37°C.

Tuesday April 8

Colonies successfully grew in each test tube. Re-suspended cultures, and did miniprep purification of DNA following protocol.

Thursday April 10

Ran an analytical gel to check if cells were successfully transformed. Digested miniprep DNA samples (Trp #2, Trp $3, Pro #2, Pro #4) and vector DNA with NcoI/EcoRI using digestion protocol. Expecting to see 1400 bp band for proline trna synthetase gene and 1000 bp band for tryptophan. Gel shows unusual results--several small bands in each lane, resembling the ladder. Will re-do analytical gel.

From left: Trp #2, Trp $3, Pro #2, Pro #4. The rest belong to other group members. Image:4-10gel.jpg


Tuesday April 15

Since last gel didn't work, did another miniprep purification of DNA using protocol for all samples (Pro#1-4 and Trp#1-4)

Digested purified DNA samples with NcoI/EcoRI.

Thursday April 17

Ran a gel for digested samples.

Left to right: Pro#1, 2, 3, 4, ladder, Trp #1, 2, 3, 4, digested vector

Image:4-17gel.jpg


Tuesday April 22

Digested miniprep samples Trp#1-4 using NcoI/EcoRI.

Turned in my predicted sequence for Pro#1 to get sequenced.

Thursday April 24

Ran gel of digested products, Trp #1-4.

From left: vector digest, Trp#1, Trp #2. Lanes 4-9 are other group member's samples. Lane 10 is the ladder. Image: gel14-24.jpg


From left: Trp#3, Trp#4, Lanes 3-4 are other group member's samples. Lane 5 is vector dig. Lane 6 is ladder. Image:4_24_14_Gel2_pdtvectdig.jpg‎

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