Team:Penn State/Notebook

From 2014.igem.org

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   <td><center><b>Monday, June 2, 2014</b></center></td>
   <td><center><b>Monday, June 2, 2014</b></center></td>
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   <td>Constructed dCas9 gene cassette and plasmid backbone with replication origin ColE1 via PCR Rescue. Gel purified dCas9 and ColE1 cassettes.</td>
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   <td>Constructed dCas9 gene cassette and plasmid backbone with replication origin ColE1 via PCR Rescue. Gel purified dCas9 and ColE1 cassettes. 1 out of 4 dCas9 PCR's were successful, and 2 out of 4 colE1's were successful, all of which were Ashlee's. We attributed this to Emily's lack of cloning experience.</td>
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   <td><center><b>Sunday, June 8, 2014</b></center></td>
   <td><center><b>Sunday, June 8, 2014</b></center></td>
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   <td>One 4-part, two 3-part, and two 2-part Gibson CBA's were conducted to assemble the kanamycin resistance cassette, two genome overlaps, and the colE1 replication origin. </td>
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   <td>One 4-part, two 3-part, and two 2-part Gibson CBA's were conducted to assemble the kanamycin resistance cassette, two genome overlaps, and the colE1 replication origin. This completed plasmid will be termed "plasmid 1". </td>
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   <td><center><b>Wednesday, June 11, 2014</b></center></td>
   <td><center><b>Wednesday, June 11, 2014</b></center></td>
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   <td>3 successful colonies were sent for sequencing. In order to insert the dCas9 system into plasmid 1, dCas9 was digested with XhoI and ClaI. 4 successful colonies were digested with ClaI for 3 hours, heat inactivated at 65 degrees C, and then digested with SalI-HF restriction enzyme. SalI and XhoI are compatible sites. These digestions were gel purified, resulting in low concentrations of plasmid DNA. Only two colonies were used to continue further.</td>
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   <td><center><b>Thursday, June 12, 2014</b></center></td>
   <td><center><b>Thursday, June 12, 2014</b></center></td>
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   <td>The plasmid backbone was digested with phosphotase enzyme. These backbones were ligated to dCas9 over 18 hours at 16 degrees C to ensure maximum ligation product.</td>
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   <td><center><b>Friday, June 13, 2014</b></center></td>
   <td><center><b>Friday, June 13, 2014</b></center></td>
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   <td>Emily purified the ligation product.</td>
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  <td><center><b>Saturday, June 14, 2014</b></center></td>
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   <td><center><b>Sunday June 15, 2014</b></center></td>
   <td><center><b>Sunday June 15, 2014</b></center></td>
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   <td>Ashlee transformed the ligation into <i>E. coli</i> DH10B electrocompetent cells via electroporation, and plated them on kanamycin antibiotic agar plates to grow overnight.</td>
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   <td><center><b>Monday, June 16, 2014</b></center></td>
   <td><center><b>Monday, June 16, 2014</b></center></td>
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   <td>The ligation failed. We amplified more of the dCas9 system using PCR Rescue, in which 2 out of 4 PCR's were successful - <b>both Emily's!</b>. We are evaluating this difficult PCR and will be altering the annealing temperature.  </td>
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Revision as of 18:56, 20 June 2014


WELCOME TO iGEM 2014!

Your team has been approved and you are ready to start the iGEM season!
On this page you can document your project, introduce your team members, document your progress
and share your iGEM experience with the rest of the world!


Click here to edit this page!

Home Team Official Team Profile Project Parts Modeling Notebook Safety Human Practices Attributions

Penn State iGEM 2014 Notebook Page

Here you will find weekly summaries of our wet laboratory progress, team updates, and accomplishments outside the laboratory. Below [link this] is our detailed, day-to-day progress laboratory notebook.

Weekly Summaries

will create links to each week, but will just list down the page Week 1, Week 2, etc etc

Laboratory Notebook

Biodetoxification
Codon Optimization
Tuesday, May 20, 2014
 First iGEM meeting with Dr. Richard and Dr. Salis. ...................
Wednesday, May 21, 2014
 Emily's first experience with cloning! Ashlee led Emily through several practice experiments from designs made earlier in the year: making a gel, loading samples, gel purifying DNA. ...................
Thursday, May 22, 2014
 Ashlee and Emily performed a transformation.
Friday, May 23, 2014
 We picked several colonies for overnight growth to do more cloning tomorrow.
Saturday, May 24, 2014
 Memorial Day Weekend? How about lab cloning weekend! Ashlee conducted plasmid preparation and digestion.
Monday, May 26, 2014
 Due to a string of failed clonings with the broadhost vector pSEVA251 and the large inserts, new designs are evaluated! Instead of creating a plasmid with the HMF pathway (7.5 kb) and dCas9 system (5.5 kb), we shall add the HMF pathway and dCas9 to the P. putida genome using homologous recombination.
Tuesday, May 27, 2014
 Inoculated LB broth with ampicillin and dCas9 plasmid from cryogenic storage; inoculated Lb broth with chloramphenicol and FTV vector from Ashlee's past experiment; streaked the HMF vector on a kanamycin plate.
Wednesday, May 28, 2014
 Emily and Ashlee made cryogenic storage of the dCas9 plasmid; plasmid prepared the FTV and dCas9 vectors; digested FTV vector; inoculated LB broth with a colony from the HMF plate for overnight growth and plasmid preparation tomorrow.
Thursday, May 29, 2014
 Prepared plasmid containing the HMF pathway; inoculated LB broth with Lambda Red Recombinase plasmid from cryogenic storage. Ashlee, Emily, and graduate student Iman Farasat ordered primers for three plasmids that will be constructed via Gibson Chew-Back and Annealing Assembly, two of which will be inserted into the genome by homologous recombination.
Friday, May 30, 2014
 Ashlee and Emily made cryogenic storage of the Lambda Red Recombinase plasmid.
Monday, June 2, 2014
 Constructed dCas9 gene cassette and plasmid backbone with replication origin ColE1 via PCR Rescue. Gel purified dCas9 and ColE1 cassettes. 1 out of 4 dCas9 PCR's were successful, and 2 out of 4 colE1's were successful, all of which were Ashlee's. We attributed this to Emily's lack of cloning experience.
Tuesday, June 3, 2014
 Conducted Colony PCR using P. putida KT2440 strain as DNA template to construct two ~1 kb genome overlaps. Plasmid prepared the Lambda Red Recombinase plasmid, DH10B-PKD46, FTV-ptac-LacI-CmR plasmid, and NoHP_15A_Plmra_CmR plasmid containing RFP with a strong, unique promoter. Stock of NoHP_15A_Pkmra_CmR and FTV_ptac_LacI_CmR for cryogenic storage was also made. Lambda Red Recombinase cassette was amplified using PCR Rescue and gel purified.
Wednesday, June 4, 2014
 We made ampicillin agar plates and ampicillin antibiotic stock for cloning. The PCR Rescue of Lambda Red Recombinase was also gel purified.
Thursday, June 5, 2014
 We conducted PCR Rescue to amplify the kanamycin resistance cassette (specifically the neomycin cassette, which also confers resistance to kanamycin) from pSEVA251 KanR plasmid. Two different sets of primers for kanamycin were tested, and the first set was successful - all 4 PCR's were correct. The second set of primers all failed. However, Emily had her first PCR success! Kanamycin cassette was gel purified.
Friday, June 6, 2014
 Conducted colony PCR using P. putida KT2440 strain as the DNA template to construct 1 kb overlaps for homologous recombination. All four of the first genome overlaps with gene PP_0747 were successful; only 2 overlaps with upp gene were successful. These were gel purified. We learned Gibson Chew-Back Annealing Assembly (CBA) protocol.
Sunday, June 8, 2014
 One 4-part, two 3-part, and two 2-part Gibson CBA's were conducted to assemble the kanamycin resistance cassette, two genome overlaps, and the colE1 replication origin. This completed plasmid will be termed "plasmid 1".
Monday, June 9, 2014
 The two 2-part and two 3-part CBA's were amplified using PCR Rescue and gel purified. The original 4-part CBA was transformed into E. coli electrocompetent cells using electroporation and plated on kanamycin antibiotic agar plates. The 4-part CBA was repeated to ensure accuracy. Because the CBA parts contained no plasmid DNA, the 4-part CBA could be digested by restriction enzyme Dpn1. Dpn1 binds and cuts methylated DNA sites, thus destroying any plasmid DNA remaining as a contaminant.
Tuesday, June 10, 2014
 The original 4-part CBA worked! Many colonies appeared on the plate after incubation at 37 degrees C for 18 hours, and 12 colonies were selected for plasmid preparation. These were digested with AatII and XbaI, two restriction sites that are only both contained in the final assembled 4-part plasmid. 6/12 colonies showed the correct bands on the gel. We also prepared more 1 kb ladder from concentrate.
Wednesday, June 11, 2014
 3 successful colonies were sent for sequencing. In order to insert the dCas9 system into plasmid 1, dCas9 was digested with XhoI and ClaI. 4 successful colonies were digested with ClaI for 3 hours, heat inactivated at 65 degrees C, and then digested with SalI-HF restriction enzyme. SalI and XhoI are compatible sites. These digestions were gel purified, resulting in low concentrations of plasmid DNA. Only two colonies were used to continue further.
Thursday, June 12, 2014
 The plasmid backbone was digested with phosphotase enzyme. These backbones were ligated to dCas9 over 18 hours at 16 degrees C to ensure maximum ligation product.
Friday, June 13, 2014
 Emily purified the ligation product.
Sunday June 15, 2014
 Ashlee transformed the ligation into E. coli DH10B electrocompetent cells via electroporation, and plated them on kanamycin antibiotic agar plates to grow overnight.
Monday, June 16, 2014
 The ligation failed. We amplified more of the dCas9 system using PCR Rescue, in which 2 out of 4 PCR's were successful - both Emily's!. We are evaluating this difficult PCR and will be altering the annealing temperature.
Tuesday, June 17, 2014
Wednesday, June 18, 2014
Thursday, June 19, 2014
Friday, June 20, 2014
Saturday, June 21, 2014
Sunday, June 22, 2014
Monday, June 23, 2014
Tuesday, June 24, 2014
Wednesday, June 25, 2014
Thursday, June 26, 2014
Friday, June 27, 2014
Saturday, June 28, 2014
Sunday, June 29, 2014
Monday, June 30, 2014
Tuesday, July 1, 2014
Wednesday, July 2, 2014
Thursday, July 3, 2014
Friday, July 4, 2014
Saturday, July 5, 2014
Sunday, July 6, 2014
Monday, July 7, 2014
Tuesday, July 8, 2014
Wednesday, July 9, 2014
Thursday, July 10, 2014
Friday, July 11, 2014
Saturday, July 12, 2014
Sunday, July 13, 2014
Monday, July 14, 2014
Tuesday, July 15, 2014
Wednesday, July 16, 2014
Thursday, July 17, 2014
Friday, July 18, 2014
Saturday, July 19, 2014
Sunday, July 20, 2014
Monday, July 21, 2014
Tuesday, July 22, 2014
Wednesday, July 23, 2014
Thursday, July 24, 2014
Friday, July 25, 2014
Saturday, July 26, 2014
Sunday, July 27, 2014
Monday, July 28, 2014
Tuesday, July 29, 2014
Wednesday, July 30, 2014
Thursday, July 31, 2014

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