Team:Alberta/Notebook June

June 1, 2010

KanA/B' and KanB/A' fragements PCRed on 11-05-2010, digested with BsaI-HF at 37^oC for 1.5hours, heat inactivated at 65^oC for 30 minutes. Tried to ligate KanA/B' fragments to each other and tried to ligate KanB/A' fragments to each other. Also tried to ligate KanA/B' fragments with KanB/A'. Ligated with T4 DNA ligase for 3 hours at 21^oC.

Set up liquid cultures of KanRA/B'-Bsa and KanR B/A'-Bsa in pSB1C3 from plates streaked with on 30-05-2010.

June 2, 2010

Miniprepped liquid cultures from 01-06-2010. Ran a 1% agarose gel of the ligations performed 01-06-2010.

To optimize the Restriction and ligation of BsaI-HF, digested KanA/B' and KanB/A' fragements PCRed on 11-05-2010 with the following recipe:

Digestion Recipe:

14μL either A/B' or B/A' Kanamycin resistance cassette (approx. 100ng/μL)

5μL 1/10 dillution of 100X BSA

5μL 10X NEBuffer4

1.5&mu:L BsaI-HF

24.5μL MilliQ

Digested at 50^oC for 1hour, heat inactivated the enzyme at 65^oC for 20 minutes PCR purified the digests.

Mike

* Got inline forms to work, and ajax to work

Steve

* Added simple construct validation
* Added simple sequence annotation
* Added reverse strand display

June 3, 2010

Tried to ligate the KanA/B' fragments to itself. Tried to ligate the KanB/A' fragments to itself. Tried to ligate the KanA/B' fragments to the KanB/A' fragments.

Ligation Recipe:

8μL of digest from 02-06-2010 (either 8μL of one of the fragments or 4μL of each)

1μL T4 DNA ligase

6μL 5X Buffer

15μL MilliQ H₂O

Took aliquots of ligations at varying times and ran 1% agarose gels to test ligation.

Digested some of Minipreps of the KanA/B' fragment inserted into pSB1C3 from 02-06-2010.

Digestion Recipe:

14μL plasmid (approx 300-400ng/&mu:L)

5μL 10X NEBuffer 4

5μL 1/10 dilution of 100X BSA

1.5μL BsaI-HF

24.5μL MilliQ H₂O

Construct of Amp Resistance in pSB1C3 Backbone

Made a 1/100 dilution of AmpR and TetR PCR Products from 27-05-2010 and performed PCR reactions to produce antibiotic inserts to make parts.

PCR Recipe:

35.5μL MilliQ H₂O

1μL 10 uM dNTPs

5μL 10X PCR Buffer

2μL 50 uM MgCl₂

2.5μL Primer + (ApR 1/10+ or TR 1/10+)

2.5μL Primer - (ApR 1/10- or TR 1/10-)

1μL Template (1/100 diluted AmpR or TetR)

0.5μL Taq Polymerase

PCR Program:

5 min-94^oC

45 sec-94^oC

1 min-60^oC

1 min-72^oC

Repeat 2 through 4 35 times

5 min-72^oC

Experiment continue on 09-06-2010.

June 4, 2010

Tried to test the limits of ligation reaction. Ligation Recipe of plasmids cut on 03-06-2010:

8μL digestion mixture

6μL 5X T4 ligase buffer

1μL T4 ligase

15μL MilliQ

Tried to further reaction of KanA/B' fragments to KanB/A' fragments. To the existing Ligase reactions from 03-06-2010 added:

1μL T4 ligase

6μL 5X T4 ligase buffer

23μL MilliQ

Tried to set limits of Kan fragments that would ligate:

24μL digestion from 02-06-2010 (either 24μL of one of the fragments or 12μL of each)

6μL 5X T4 ligase buffer

1μL T4 ligase

June 7, 2010

Steve

* Added more complicated custom sequence annotation
* Integrating Jacqueline's login stuff with construct & biobyte owner/admin permissions and junk

June 8, 2010

To test the function of E.coli smell variant experiments for our kit, BBa_J45120 (Wintergreen) and BBa_J45200 (Banana) from the MIT 2006 BioBrick Registry were transformed in DH5α cells.

Steve

* Integrating construct designer with mike's electronic lab book back-end
* Created a StepGenerator that generates a crude protocol based on the contents of an experiments associated constructs

June 9, 2010

Construct of Amp Resistance in pSB1C3 Backbone

Restriction Digested AmpR and TetR inserts from 03-06-2010 to be ligated with psB1C3 vector later on.

Digestion Recipe for AmpR:

13.4μL MilliQ H₂O

0.60&mu:L AmpR (333.3 ng/μL, determined by nanodrop)

2μL 10X BSA

2μL 10X Buffer 4

2μL BsaI

Digestion Recipe fro TetR:

13.3μL MilliQ H₂O

0.7&mu:L TetR (571.3 ng/μL, determined by nanodrop)

2μL 10X BSA

2μL 10X Buffer 4

2μL BsaI

Both digestions were incubated at 50^oC for 1hour, heat inactivated the enzyme at 70^oC for 20 minutes.

Experiment continue on 10-06-2010.

Mike

* Added FlexImage plugin and integrated with lab book

June 10, 2010

Double Digested Kan/Chlor minipreps to determine orientation.

Digestion Recipe:

5μL 10X Buffer 3

1μL XbaI

1μL PstI

5μL Kan/Chlor fragments

33μL MilliQ H₂O

5μL 10X BSA

Incubated at 37^oC for 1hour, heat inactivated the enzyme at 80^oC for 20 minutes.

Construct of Amp Resistance in pSB1C3 Backbone

Restriction Digested A/B' psB1C3 vector.

Digestion Recipe:

10μL MilliQ H₂O

5&mu:L psB1C3

2μL 10X BSA

2μL 10X Buffer 4

1μL BsaHF

Incubated at 37^oC for 1hour, heat inactivated the enzyme at 80^oC for 20 minutes.

Ligated digested A/B' psB1C3 with AmpR from 09-06-2010.

Ligation Recipe:

9μL A/B' psB1C3 vector

5μL AmpR insert

6μL 5X Ligase Buffer

1μL Ligase

9μL MilliQ H₂O

Incubated at room temperature for 45 minutes. Transformed with DH5α cells using 15μL of the ligated A/B' psB1C3 with AmpR.

Experiment continue on 11-06-2010.

Mike

* Fixed all bugs with image uploading, cleaned up routing for lab book
* added inline upload forms to experiment view

June 11, 2010

Construct of Amp Resistance in pSB1C3 Backbone

We got colonies of psB1C3 with AmpR from the DH5α transformations from 10-06-2010. We made overnights.

Experiment continue on 12-06-2010.

We experimented with L'Oreal skin toner LB media to see if BBa_J45120 plasmids from 08-06-2010 would produce a wintergreen odor. We incubated cultures in L'Oreal LB in a 5%, 10% and 15% concentration.

Mike

* synced up with Steve
* added ordering functionality to steps
* added ability to insert steps between other steps

June 12, 2010

Construct of Amp Resistance in pSB1C3 Backbone

Miniprep of psB1C3 with AmpR was made from the overnights from 11-06-2010.

Experiment continue on 16-06-2010.

L'Oreal LB cultures from 11-06-2010 had little growth success. Two of the 5% L'Oreal LB cultures grew.

June 14, 2010

Mike

* fixed up step model code
* improved javascript on experiment page

June 15, 2010

Mike

* added associations between users and experiments
* added code for changing views based on ownership
* added user profile page

To do:
* add publish experiment checkbox to profile page
* move javascript for experiment view to its own file
* fix weird bug that reappeared in form on Experiment view
* add user "notes" to steps
* add image caching to FlexImage stuff

June 16, 2010

Construct of Amp Resistance in pSB1C3 Backbone

psB1C3 with AmpR miniprep from 12-06-2010 ran undigested on a 1% agarose gel.

We digested psB1C3 with AmpR to see if the AmpR insert would be released from the psB1C3 vector.

Digestion Recipe:

14.1μL MilliQ H₂O

0.9μL psB1C3 with AmpR (222.5 ng/μL, determined by nanodrop)

2μL 10X BSA

2μL 10X Buffer 4

1μL BsaI

Incubated at 50^oC for 1hour, heat inactivated the enzyme at 65^oC for 30 minutes.

Also, we made more overnights of psB1C3 with AmpR.

Experiment continue on 17-06-2010.

June 17, 2010

Construct of Amp Resistance in pSB1C3 Backbone

Digested psB1C3 with AmpR from 16-06-2010 was ran on a 1% agarose gel.

June 28, 2010

Transformed 2009 Cambridge color series parts: Bba_K274100, BBa_K274200, BBa_K274002, BBa_K274003, BBa_K274004, BBa_J23100. Also transformed 2004 UTAustin BBa_M30109. Transformed using 5uL of DNA.

Made starter culture of Dbl3 in LB at 11:03am. Inoculated large overnight liquid culture as per specifications in Inoue method at 6:00pm

Performed an enzyme efficiency experiment to determine which of BbsI, BfuAI, BsaI, or BsaI-HF works most efficiently.

June 29, 2010

Made a glycerol stock of PL5 from 1.5mL liquid overnight made 28-06-2010.

Took remaining transformation of Dbl3 cells with ccdB BfuA/B' and ccdB BfuB/A' from 28-06-2010 out of the fridge and plated 25, 50 and 100μL of each on chloramphenicol LB plates

From the chloramphenicol and chloramphenicol/Kanamycin plates streaked with ccdB BbsA/B', ccdB BbsB/A',ccdB BsaA/B' and ccdB BsaB/A' on [[#28-06-2010|28-06-2010]], only ccdB BsaB/A' #6,8,10,11,12,14,15,18, ccdB BbsA/B' #8,15 and 20 grew on only the chloramphenicol plate. Made 5mL overnight liquid cultures with chloramphenicol of the some of the streaks that worked (ccdB BsaB/A' #6,8,10,11,12, ccdB BbsA/B' # 15, 20 and ccdB BsaB/A'#5 which didn't work but is a positive control).

Miniprepped liquid culture grown 28-06-2010 of Kan BsaA/B' #7. Concentration is 293.4ng/μL.

All the transormations of Cambridge 1009 color series performed 28-06-2010 worked. Made 5mL liquid cultures of each part.

Made competent Dbl3 cells by the Inuoe method from liquid cultures grown overnight. (set up on 28-06-2010). The OD of the culture was 0.775.

June 30, 2010

We Ran enzyme efficiency experiment with the same specifications as on 28-06-2010 but omitting BsaI-HF from the experiment because BsaI, BbsI and BfuAI appear to be the best of the enzymes. Also we ran the experiment with 10 Units of each enzyme not 20 Units.

Miniprepped liquid cultures of ccdB Bbs A/B' # 15 and 20 and of ccdB BsaB/A'#6, 8, 10, 11, 12 and 5 (which did grow on both Kan/Chlor and Chlor plates, so it is a positive control for the incorrect plasmid) grown 29-06-2010. The concentrations of the minipreps were between 200 and 400ng/mu;L and the curves did not show contamination. Digested approximately 500ng of minipreps with EcoRI and PstI with the following recipe:

19.5μL MilliQ H₂O

2.5&mu:L Miniprep

3μL 10X BSA

3μL 10X NEBuffer 3

1μL EcoRI

1μL PstI

made 5mL culture of Kan BbsA/B' #1 from <--Find date-->

Ya! We got colonies of the ccdB BfuA/B' an ccdB BfuB/A' part plasmids that were plated 29-06-2010.

Miniprep of liquid cultures made 29-06-2010 of transformations of Cambridge 2009 color series. Concentrations are as follows: J23100, 397.7 ng/μL; K274100, 388.9ng/μL; K274200, 556.5ng/μL; K274004, 106.1ng/μL; K130109, 82.5ng/μL; K274003, 161.0ng/μL.

Constructing Kan, Chlor and Tet Parts in pSB1A3 Backbone

Strategy: to insert Kan into pSB1A3 after cutting both with NotI. Then, digest with BsaI to take Kan out and insert chlor and tet parts.

Restriction Digest with NotI Protocol:

Kan AB PCR product (82.3 ng/ul) 1ul

pSB1A3 plasmid (30.5 ng/ul) 3ul

NotI enzyme 1ul

10x REact3 buffer 1ul

MilliQ H2O 4ul

Total 10ul

Incubated at 37C for 1 1/2 hours.

Ligation Protocol:

Digested pSB1A3 and Kan AB fragment 8ul

T4 DNA ligase 1ul

5X T4 DNA ligase buffer 6ul

MilliQ H20 5ul

Total 20ul

Incubated at room temperature for 1 hour.

Gel Electrophoresis:

Lanes 2-4 are digests of pSB1A3 and kan AB fragment with NotI. Lanes 5-7 are ligations of the digested pSB1A3 and kan fragments. Note that no ligated DNA shows up on the gel due to a small volume of digestion (3ul) loaded. Lanes 2-4 are digests of pSB1A3 and kan AB fragment with NotI. Lanes 5-7 are ligations of the digested pSB1A3 and kan fragments. Note that no ligated DNA shows up on the gel due to a small volume of digestion (3ul) loaded.

Transformation: Followed Inoue Protocol using 50ul DH5-alpha competent cells and 2ul of ligated pSB1A3 and kan AB part. Plated 100ul, 100ul, and 400ul on Kan/Amp plates overnight at 37C. Results:

Plate 1 (100ul): TNTC white colonies and ~20 red colonies

Plate 2 (100ul): TNTC white colonies and ~3 red colonies

Plate 3 (400ul): TNTC white colonies and ~30 red colonies

Experiment continues on 06-07-2010