Team:Penn/Notebook/Biofilms

From 2012.igem.org

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Latest revision as of 19:00, 18 July 2012

6/6/12

Set up equipment
Autoclaved 2x 1L Sterile ddH₂O
Autoclaved 500 mL LB Broth for scale up of luxS (Plate 3 Well 2H, pSB1A2), plsr (Plate 2 Well 14H in pSB1A2 resistance to Amp)
Added iGEM logo to wiki

6/7/12

Resuspended available biobricks luxS & plsr (resuspended in 10uL ddH₂O)
Resuspended DNA transformed into DH5α (40uL DH5α+2uL DNA) & plated on LB plates w/ 15 uL of 100 mg/mL Amp spread on surface
Requested lsrR & lsrK from iGEM HQ

6/8/12

iGEM Wiki now split into two separate Notebooks
Plenty of colonies, too many to count(TMTC) grew from plsr & luxS transformation from 6/7/12
- Selected one colony each
- Grew in 10 mL LB+Amp (100 ug/mL)
Contacted researchers for V. harveyi bioassay & lysostaphin, V. harveyi must be purchased through ATCC ($300), lysostaphin obtained thru MTA.

6/9/12

~2mL of LB evaporated during incubation
Cells spun down @ 5000 rpm for 10 min
Miniprepped 4x Lysis into 1 column
[plsr]=112 ng/uL
[luxS]=114 ng/uL

6/11/12-6/16/12

Dry lab work

6/18/12

Obtained & resuspended primers for eGFP & plsr

6/19/12

performed PCR w/ NEB standard taq kit w/ primers for eGFP & plsr
Tested 2 annealing temperatures, 55C and 50C
Ran gel w/ PCR product (2% agarose), saw clear eGFP bands, but possible .1kb obscured by loading dye.

6/20/12

Performed qiagen PCR purification & nanodropped (it like it's hot)

6/21/12

Ran gel of yesterday's PCR purification

Image of 2% Agarose Gel of post-purification PCR products of eGFP (.85kb) & plsr (.1kb)

- EtBr Gel 2% Agarose
  - Lane 1: NEB 2-log ladder (1 ug loaded)
  - Lane 2: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 55C annealing temp.
  - Lane 3: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 55C annealing temp.
  - Lane 4: PCR of eGFP using plsr_XhoI Fwd & eGFP_EcoRI Rev primers w/ 50C annealing temp.
  - Lane 5: PCR of plsr using plsr_BglII Fwd & plsr_XhoI Rev primers w/ 50C annealing temp.
  - Lane6/7: Null
  - Lane 8: 1kb ladder left at rt o/n
Obtained & transformed pET-26b

6/22/12

Miniprepped pet26b and digested with BglII and EcorR

6/25/12

Ran gel of eGFP/plsr ligation
Ordered restriction enzymes from the Cell Center

6/26/12

Attempted Gel Purification of digest products from 2/25/12
- Yields for Gel purification were too low to be useful (>1 ng/uL)
Digested purified PCR rxns (annealing temperature=55C)

6/27/12

Ran gel of digested PCR rxns (55C), found no evidence of successful digestion.

6/28/12

Purified digest and attempted

6/29/12

Performed digest of pET-26b, eGFP, and plsr
Spread Biobrick shipment on Amp plates (lsrR, lsrK)

7/2/12

Selected colonies from lsrK and lsrR streaked plates
- Grew in 10mL TB Broth

7/3/12

Ran gel of ligated product, no size shift, unlikely that ligation was successful.
Ligation rxn transformed to make sure

7/5/12

Ligation transformation of pET-26-plsr-GFP failed, no colonies present
Talked to Dan:
- When using primers to introduce restriction sites into

7/6/12

Redesigned primers for plsr & egfp
Set up TC Incubator & hood
Contacted people in the FDA thru personal contacts for human practices

7/9/12

Made Inactivating Solution for restriction digest
- 50mM EDTA (pH=8.0)
- 50% Glycerol
Planned test restriction digest pET-26b troubleshooting tomorrow
Trained new users for Tissue Culture

7/10/12

Troubleshooting of pET-26 digestion
- Digest of pET-26b w/ BglII and XhoI
- Time course:
- 10 min
- 30 min
- 3 hr
- 6 hr
- o/n
- Inactivate w/ Inactivating Solution (10uL/50uL of restriction reaction)
Nikita, Ashwin, Avin all have acceptable sterile practice
Resuspended IDT luxS synthesized DNA
Transformed synthesized luxS into DH5a

7/11/12

Ran 2% gel of digest, did not see signs of any restriction
Re-planned pET-26b digest troubleshooting
Selected luxS colonies

7/12/12

Thawed out 293T-CMV-Gaussia cells from Sarkar LN2
Miniprepped luxS colony, digested w/ BamHI-HF & EcoRI-HF
Performed gel extraction of luxS fragment

7/13/12

Passaged 293T-CMV-Gaussia cells 1:10 & 1:5 for weekend
Ligation of luxS w/ pET-26b
Ligation rxn transformed into DH5a

7/14/12

Ligation showed successful transformants

7/15/12

Colonies selected by Mike, grown in 10mL LB

7/16/12

SAAST Presentation
Miniprep of 10mL pET-26b-luxS cultures
Ran gel of minipreps from 3 pET-26b-luxS colonies to confirm successful ligation
pET-26b-luxS #1/#2 are slightly larger, indicating successful ligation, not as sure for pET-26b-luxS #3
Last lane is unligated pET-26b vector

Image of 1% Agarose Gel of Miniprep products of pET-26b-luxS from three colonies

7/17/12

Ran gel to determine if insertion of pET-26b-luxS was correctly ligated
- Gel showed there was a possible size shift corresponding to insertion in all three selected colonies of pET-26b-luxS colonies

Image of 2% Agarose Gel of PCR products of eGFP (.85kb) & plsr (.1kb)

- From left to right (optimum temperatures bolded): Ladder, plsr @45C,52C,58C,65C, egfp @52C,58C,65C
Performed 100uL PCR of plsr @65 & 100uL PCR of egfp @58 for further ligation experiments.

@@ Line 66: / Line 66: @@
 ==7/2/12==
-*Performed a ligation
+*Selected colonies from lsrK and lsrR streaked plates
+**Grew in 10mL TB Broth
 ==7/3/12==
-*Performed a ligation
+*Ran gel of ligated product, no size shift, unlikely that ligation was successful.
+*Ligation rxn transformed to make sure
 ==7/5/12==
-Unknown
+*Ligation transformation of pET-26-plsr-GFP failed, no colonies present
+*Talked to Dan:
+**When using primers to introduce restriction sites into
 ==7/6/12==
 *Redesigned primers for plsr & egfp
+*Set up TC Incubator & hood
+*Contacted people in the FDA thru personal contacts for human practices
+==7/9/12==
+*Made Inactivating Solution for restriction digest
+**50mM EDTA (pH=8.0)
+**50% Glycerol
+*Planned test restriction digest pET-26b troubleshooting tomorrow
+*Trained new users for Tissue Culture
+==7/10/12==
+*Troubleshooting of pET-26 digestion
+**Digest of pET-26b w/ BglII and XhoI
+**Time course:
+**10 min
+**30 min
+**3 hr
+**6 hr
+**o/n
+**Inactivate w/ Inactivating Solution (10uL/50uL of restriction reaction)
+*Nikita, Ashwin, Avin all have acceptable sterile practice
+*Resuspended IDT luxS synthesized DNA
+*Transformed synthesized luxS into DH5a
+==7/11/12==
+*Ran 2% gel of digest, did not see signs of any restriction
+*Re-planned pET-26b digest troubleshooting
+*Selected luxS colonies
+==7/12/12==
+*Thawed out 293T-CMV-Gaussia cells from Sarkar LN2
+*Miniprepped luxS colony, digested w/ BamHI-HF & EcoRI-HF
+*Performed gel extraction of luxS fragment
+==7/13/12==
+*Passaged 293T-CMV-Gaussia cells 1:10 & 1:5 for weekend
+*Ligation of luxS w/ pET-26b
+*Ligation rxn transformed into DH5a
+==7/14/12==
+*Ligation showed successful transformants
+==7/15/12==
+*Colonies selected by Mike, grown in 10mL LB
+==7/16/12==
+*SAAST Presentation
+*Miniprep of 10mL pET-26b-luxS cultures
+*Ran gel of minipreps from 3 pET-26b-luxS colonies to confirm successful ligation
+*pET-26b-luxS #1/#2 are slightly larger, indicating successful ligation, not as sure for pET-26b-luxS #3
+*Last lane is unligated pET-26b vector
+[[File:pET-26b-luxSnocut071712.JPG|thumb|120px|northeast|alt=SCIENCE.|Image of 1% Agarose Gel of Miniprep products of pET-26b-luxS from three colonies]]
+==7/17/12==
+*Ran gel to determine if insertion of pET-26b-luxS was correctly ligated
+**Gel showed there was a possible size shift corresponding to insertion in all three selected colonies of pET-26b-luxS colonies
+[[File:PCRoptimization071812.JPG|thumb|120px|right|alt=SCIENCE.|Image of 2% Agarose Gel of PCR products of eGFP (.85kb) & plsr (.1kb)]]
+**From left to right (optimum temperatures bolded): Ladder, plsr @45C,<b>52C</b>,58C,<b>65C</b>, egfp @52C,<b>58C</b>,65C
+*Performed 100uL PCR of plsr @65 & 100uL PCR of egfp @58 for further ligation experiments.