Team:Wisconsin-Madison/notebook/Yue

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5-24 to 5-29 Summary
 
-
During this week it was mainly my goal to make good competent cells.  I went through the process twice. The first time, I got 12 aliquots of 40uL because I miss read the final OD measurement, which made the cells concentrated. The second time, I got 40 aliquots of 40 uL and the final OD measurement within the 0.08-0.12 range.
+
=Note book of July 12th,2010=
-
To test the transformation efficiency, I used pBAD18 ligation product to transform for both series of competent cells (5-26 and 5-27). However, after poor growth results on the plates with the 5-27 culture, I questioned if the ligation product was correct to use.  To test for the transformation efficiency I was supposed to use an intact plasmid, such as miniprep product. So I re-did the efficiency test with 5-27 cells (5-26 cells were used) and used the pBAD34 miniprep product. The 10^-2 dilution plate to have 463 colonies, with the efficiency to be 2.74 x10^7 CFUs/ug
+
1. Screening for the Rfal into LacI-RBS cloning
-
Also made point mutation primers for pBAD18 for the PstI second cut site in bla operon. I also made primers for the bile-induction promoter found in ''Lactobacillus acidophilus'' NCFM to use in its neotype strain, which is currently being sent to Prof. Jim Steele. 
+
Gel Picture
-
  6-01 to 6-04 Summary
+
[[File:IGEM 2010-07-12 13hr 39min.jpg]]
-
During this week, the team goal is to clone the encapsulation parts separately and together in one plasmid.  Also, to test these parts in an acid test separately and together.  
+
[[File:IGEM 2010-07-12 17hr 33min.jpg]]
-
For research: Find out about how to make our project safer, how to incorporate the encryption project into the lactose project, probiotics background information, how to improve the encapsulation process from Imperial College London.
+
2. Primer Design For the Cloning LacI-RBS-ygiV-RBS-Rfal-RBS-RFP-Terminator
-
  6-7 to 6-11 Summary
+
One is for Rfal: the forward primer has a XbaI and RBS in it; the reverse primer is on the backbone, and has a XmaI site on it.
-
During this week:
+
pRBS-Rfal_Fwd: TGCTCTAGA AAAGAGGAGAAAATGCTAACATCCTTTAAACT
-
*The Lactobacillus neotype strain has not arrived yet in Steele's lab. Wes has some MRS broth we can use to culture the strains.
+
pRBS-Rfal_Rev:   CCCCCCGGGTTAATTAATTGTATTGTTACGATTAT
-
*Contacted Steele for lactobacillus plasmids and expression strain to express the plasmid iGEM constructs
+
-
*Received primers on 6-7 and I helped elute the primers. We followed the formula: [(Amount of nm) x 1000pm/1nm] / 60pm total dilution. This gave us how much RNase free water to use for elution. After elution then we made 3x 30uL aliquots of the primers, except for the two sequencing primers.
+
-
* 6-08: Miniprep 2 pellets of pBAD18BB, 2 pellets of pBAD33BB, 2 pellets of YgiV, and 2 pellets of RcsB. Used the new Miniprep kit (6-08) and documented concentrations from the Nanodrop. Got the MRS premix broth from Wes.
 
-
* 6-09: The RcsB transformations Sarah and Hannah plated had about 10 colonies with the control plate with one colony. The YgiV transformation had no colonies on it. So Yue Wu and I re-did the ligation and transformation. We also ran a gel of the ligation Sarah did.
+
The other one is For RFP (I13507): The forward primer has a XmaI site on it and starts from the backbone; the reverse primer starts on the backbone too. [The original plasmid, I13507 has a structur of RBS-RFP-terminator]
-
Bile promoter research:  
+
pRFP(I13507)_Fwd: CCCCCCGGGgagaaagaggagaaatactagatgg
-
*The  ''Lactobacillus acidophilus'' neotype arrived today and will pick up 6-10. Need to make plates and broth to make freezer stocks of the culture.
+
-
*Lactobacillus bile promoter primers?
+
-
*Found an article about a bile-inducible efflux transporter in ''Bifidobacterium longum'' that is  induced 23 to 40 fold induction. [[File:Bile-Inducible_Efflux_Trans_in_B._longum_Gueimonde_2009.pdf]]
+
-
  6-14 to 6-18
+
pRFP(I13507)_Rev: AGTCAGTGAGCGAGGAAG
-
====6-13====
+
 
-
* Plated more acidophilus plates with and without parafilm. and tubes with and with out parafilm. One was in shaker and the other was in incubator.
+
=Note book of July 13th,2010=
 +
 
 +
1. Primer Designs:
 +
 
 +
Tyler helped me check the primers, and he get back to me with his comments:
 +
 
 +
pRBS-RfaI_Fwd - Tm for primer portion is too low(oligocalc says 44), add 4 bases from the gene sequence, try to have 3' end be G or C
 +
 
 +
pRBS-RfaI_Rev - GC content of the priming portion is way too low, try to get it to at least above 30%, you can move down the gene since you are at the ending site, also try to have the 3' end of the primer be a G or C
 +
 
 +
pRFP(I13507)_Fwd - good, I usually add a 5 base overhang, but 3 should be ok for the enzyme you are using
 +
 
 +
pRFP(I13507)_Fwd - good
 +
 
 +
So I redesigned my primers with
 +
 
 +
Detailed information are in:
 +
[[File:Primer Design for Rfal and RFP - 2.pdf]]
 +
 
 +
'''Primers For Rfal'''
 +
 
 +
Tm : 46  GC:41%
 +
Fwd: gagatgctaacatcctt (17bp)
 +
 
 +
pRBS-RfaI_Fwd : TGCTCTAGAAAAGAGGAGAAAtacgagatgctaacatcctt
 +
 
 +
Name Sequence                          Tm°C CG% nt A T C
 +
seq_1 tgctctagaaaagaggagaaatacgagatgctaacatcctt 75.1 39.0 41 16.0 9.0 7.0 9.0 417600.0                   12674.4             2.4 30.4
 +
 
 +
Name Sequence        Tm°C CG% nt A T C G
 +
seq_1 gagatgctaacatcctt 49.9 41.2 17 5.0 5.0 4.0 3.0 164400.0                   5169.4                     6.1 31.4
 +
 
 +
Tm:49  GC:40%
 +
Rev: taataatactagtagcggcc （17bp）
 +
 
 +
pRBS-RfaI_Rev: GCCGCTACTAGTATTATTA  [SpeI]
 +
 
 +
Name Sequence          Tm°C CG% nt A T C G
 +
seq_1 gccgctactagtattatta 49.6 36.8 19 5.0 7.0 4.0 3.0 185500.0                   5777.8                     5.4 31.1
 +
 
 +
'''Primers For RFP'''
 +
 
 +
pRFP(I13507)_Fwd: CTAGTCTAGAgagaaagaggagaaatactagatgg  [XbaI]
 +
 
 +
Name Sequence                          Tm°C CG% nt A T C G
 +
seq_1 ctagtctagagagaaagaggagaaatactagatgg 65.7 40.0 35 15.0 6.0 3.0 11.0 377400.0                   10950.2             2.6 29.0
 +
 
 +
Name Sequence                Tm°C CG% nt A T C G
 +
seq_1 gagaaagaggagaaatactagatgg 59.8 40.0 25 12.0 3.0 1.0 9.0 278000.0                   7861.2                     3.6 28.3
 +
 
 +
pRFP(I13507): AGTCAGTGAGCGAGGAAG  (cttcctcgctcactgact)
 +
 
 +
Name Sequence          Tm°C CG% nt A T C G
 +
seq_1 agtcagtgagcgaggaag 58.8 55.6 18 6.0 2.0 2.0 8.0 191900.0                   5637.7                     5.2 29.4
 +
 
 +
Name Sequence          Tm°C CG% nt A T C G
 +
seq_1 agtcagtgagcgaggaag 58.8 55.6 18 6.0 2.0 2.0 8.0 191900.0                   5637.7                     5.2 29.4
 +
 
 +
=Note book of July 14th,2010=
 +
 
 +
Cloning of LacI-RBS-ygiV with a terminator
 +
 
 +
a. Double digestion
 +
 
 +
b. Gel extraction and purificaiton
 +
 
 +
[[File:2.jpg]]
 +
 
 +
c. overnight ligation
 +
 
 +
=Note book of July 15th,2010=
 +
 
 +
Cloning of LacI-RBS-Rfal with a terminator
 +
 
 +
a. Double digestion
 +
 
 +
b. Gel extraction and purificaiton
 +
 
 +
[[File:1.jpg]]
 +
 
 +
c. 2 hours bench top ligation
 +
 
 +
d. Electroporation of LacI-RBS-ygiV-terminator and LacI-RBS-Rfal-terminator into DH10B cells.
 +
 
 +
e. Plate on Amp plates
 +
 
 +
f. run a gel to check the digestion of the backbone
 +
 
 +
[[File:3.jpg]]
 +
 
 +
=Note book of July 16th,2010=
 +
 
 +
A.Check the plate
 +
 
 +
1. LacI-RBS-ygiV-terminator
 +
 
 +
the pallet plate has about 200 colonies
 +
 
 +
the control has 8 colonies
 +
 
 +
 
 +
2.LacI-RBS-Rfal-terminator
 +
 
 +
the pallet plate has about 150 colonies
 +
 
 +
the control doesn't have colonies
 +
 
 +
 
 +
B.Screening
 +
 
 +
 
 +
 
 +
 
 +
=Note book of July 6th,2010=
 +
 
 +
A. Start the clone of Rfal into K200021(LacI+RBS)
 +
 
 +
1.cut the backbone, K200021, with PstI-HF and SpeI for 2 hours.
 +
cut the insert, Rfal, with PstI-HF and XbaI for 2 hours.
 +
 
 +
2.Gel Extract
 +
 
 +
K200021: 3264bp    Rfal:1263bp
 +
 
 +
[[File:IGEM 2010-07-06 13hr 05min.jpg]]
 +
 
 +
After the gel extraction, the concentrations are:
 +
 
 +
K200021: 33.1ng/ul
 +
Rfal: 53.9ng/ul
 +
 
 +
3.Ligation
 +
 
 +
for doing a K200021:Rfal is 1:4 ligation, 100ng of K200021 and 154.8ng of Rfal are needed. Based on the DNA concentration, add 3ul K200021 and 3ul Rfal for ligation.
 +
 
 +
Stored at 16C overnight.
 +
 
 +
 
 +
=Note book of July 7th,2010=
 +
 
 +
A. Cloning of LacI-RBS-Rfal
 +
 
 +
1.Electroporation
 +
 
 +
LacI-RBS-Rfal has a time constant 4.8;
 +
 
 +
The control has a time constant 4.7;
 +
 
 +
2.Plate on two Amp+Kan plate. Stored in a 37C incubator.
 +
 
 +
B. Cloning of LacI-RBS-ygiV into RBS
 +
 
 +
1.cut the backbone, RBS, with EcoRI-HF and XbaI, and cut the insert LacI-RBS-ygiV with EcoRI-HF and SpeI.
 +
 
 +
2.Gel Extraction
 +
 
 +
RBS:3200bp            LacI-RBS-ygiV:543bp
 +
 
 +
[[File:IGEM 2010-07-06 17hr 00min.jpg]]
 +
 
 +
after the gel extraction, the concentrations are:
 +
 
 +
RBS: 20.7ng/ul
 +
 
 +
lacI-RBS-ygiV: 19.8ng/ul
 +
 
 +
3.Ligation
 +
 
 +
For a 1 to 3 backbone:insert ratio, we need 100ng of RBS and 107.3ng of LacI-RBS-ygiV for Ligation. THerefore, add 3.7ul RBS and 4.3ul LacI-RBS-ygiV to ligate. Store at 4C overnight.
 +
 
 +
C.Run a gel to check the cutting peices
 +
 
 +
The orders are:
 +
 
 +
L-ygiV-ES; L-ygiV uncut; Rfal-PX; Rfal uncut; RBS-EX; RBS uncut; LacI-PS; LacI uncut; Ladder
 +
 
 +
unluckily, the first lane for L-ygiV-ES didn't show up any band.
 +
 
 +
[[File:IGEM 2010-07-07 11hr 24min.jpg]]
 +
 
 +
The rest of the bands are all correct.
 +
 
 +
Then run a gel for L-ygiV-ES and L-ygiV uncut by loading more DNA on the gel
 +
 
 +
[[File:IGEM 2010-07-07 13hr 09min.jpg]]
 +
 
 +
Now, this shows that the L-ygiV-ES is correct.
 +
 
 +
 
 +
=Note book of July 8th,2010=
 +
 
 +
A. Transformation of the Clone LacI-RBS-ygiV-RBS
 +
 
 +
1. Electroporation
 +
 
 +
LacI-RBS-ygiV-RBS: 4.7
 +
 
 +
Control: 4.9
 +
 
 +
2. Plate on two Amp plate.
 +
 
 +
B. Run a gel to check the Ligation of LacI-RBS-Rfal and LacI-RBS-ygiV-RBS
 +
 
 +
the orders are Ladder LacI-RBS-Rfal, control, LacI-RBS-ygiV-RBS, control
 +
 
 +
[[File:IGEM 2010-07-07 12hr 48min.jpg]]
 +
 
 +
C. Colony PCR for the LacI-RBS-Rfal Clone
 +
 
 +
1.The control plate only has one colony, compared with the regular plate with about 100 colonies.
 +
 
 +
2.picked 14 colonies for screening.
 +
 
 +
Gel for Colony PCR:
 +
 
 +
[[File:IGEM 2010-07-08 13hr 54min.jpg]]
 +
 
 +
Run for 5mins longer and add more EB
 +
 
 +
[[File:IGEM 2010-07-08 14hr 20min.jpg]]
 +
 
 +
No colony shows the correct band (around 1500bp)
 +
 
 +
=Note book of July 9th,2010=
 +
 
 +
A Cloning of LacI-RBS-ygiV into RBS, and Rfal into LacI-RBS.
 +
 
 +
Add 1.5mg of DNA into each tube:
 +
 
 +
RBS(B0034):5.3ul template; Add 1ul EcoRI-HF to digest 1 hour and inactivate at 65C for 20mins. Then Add 1ul of XbaI to digest an additional hour.
 +
 
 +
LacI-RBS(K200021): 9.5ul template; Add 1ul PstI-HF to digest 1 hour and inactivate at 80C for 20mins. Then Add 1ul of SpeI to digest an additional hour.
 +
 
 +
Rfal: 3.1ul template digest with 1ul PstI-HF and 1ul XbaI
 +
 
 +
LacI-ygiV: 4.4ul tempalte digest with 1ul EcoRI-HF and 1ul SpeI
 +
 
 +
Gel after 1 hour inactivation of EcoRI-HF in RBS and PstI-HF in LacI-RBS:
 +
 
 +
Orders are:
 +
 
 +
RBS cut with EcoRI-HF, RBS uncut, Ladder, LacI-RBS cut with PstI-HF, LacI-RBS uncut.
 +
 
 +
[[File:IGEM 2010-07-09 13hr 27min.jpg]]
 +
 
 +
 
 +
                                                                                             
 +
 
 +
=Note book of June 28th,2010=
 +
 
 +
a. Digestion of K200021 (LacI+RBS) with PstI-HF and SpeI, and digest ygiV and Rfal with PstI-HF and XbaI.
 +
 
 +
b. Gel Extraction and purification:
 +
 
 +
1. Gel
 +
 
 +
K200021-PS
 +
 
 +
ygiV and Rfal
 +
 
 +
[[File:IGEM 2010-06-27 13hr 19min.jpg]]
 +
 
 +
Concentration of the cutting peices:
 +
K200021-PS
 +
 
 +
ygiV-PX
 +
 
 +
Rfal-PX
 +
 
 +
Gel for the purification product
 +
 
 +
[[File:IGEM 2010-06-28 14hr 10min.jpg]]
 +
 
 +
c. Set up an overnight ligation
 +
 
 +
=Note book of June 29th,2010=
 +
 
 +
a. Transformation
 +
 
 +
pLacI-RBS-ygiV-PX: 4.1
 +
 
 +
pLacI-RBS-ygiV-PX(C): 3.9
 +
 
 +
pLacI-RBS-Rfal-PX: 4.0
 +
 
 +
pLacI-RBS-Rfal-PX(C):3.9
 +
 
 +
b. Plate on Amp100 plates.
 +
 
 +
 
 +
=Note book of June 30th,2010=
 +
 
 +
Re-plan the project.
 +
Figure out all the cloning need to be done by the end of the summer.
 +
Detailed information please see the spread sheet on Sarah's page.
 +
 
 +
=Note book of July 1st,2010=
 +
 
 +
A. Colony PCR of the LacI-RBS-ygiV and LacI-RBS-Rfal clone.
 +
 
 +
Upper: LacI-RBS-Rfal Clone
 +
 
 +
Lower: LacI-RBS-ygiV clone
 +
 
 +
[[File:IGEM 2010-07-01 14hr 49min-2.jpg]]
 +
 
 +
The LacI-RBS-ygiV clone shows up at the right length.  
 +
 
 +
 
 +
B. Innoculate 5 liquid culture of the ygiV clone.
 +
 
 +
 
 +
=Note book of July 2nd,2010=
 +
 
 +
A. Made 3 freezer stocks of LacI-RBS-ygiV clone
 +
 
 +
B. Miniprep LacI-RBS-ygiV
 +
 
 +
LacI-RBS-ygiV-1: 388.7ng/ul
 +
LacI-RBS-ygiV-2: 357.0ng/ul
 +
LacI-RBS-ygiV-3: 350.2ng/ul
 +
LacI-RBS-ygiV-4: 255.0ng/ul
 +
 
 +
 
 +
=Note book of June 21st,2010=
 +
 
 +
a.Minipreped 3 sets each for pBAD33BB, pBAD35BB, ygiV, and Rfal.
 +
 
 +
b.Set up a overnight digestions that cut pBAD33BB and pBAD35BB with PstI-HF and SpeI, and cut ygiV and Rfal with PstI-HF and XbaI. (37C overnight)
 +
 
 +
c.Screening for the 2nd time ygiV and Rfal cloning.
 +
 
 +
ygiV:
 +
 
 +
[[File:IGEM 2010-06-21 bads2 Peter ScanA.jpg]]
 +
 
 +
Rfal:
 +
 
 +
[[File:IGEM 2010-06-21 bads2 Peter ScanB.jpg]]
 +
 
 +
They all shows up at the same length as the positive control, which means we didn't get the desired cloning.
 +
 
 +
=Note book of June 22nd,2010=
 +
 
 +
a.Gel extraction of pBAD33BB, ygiV and Rfal.
 +
 
 +
For pBAD33BB
 +
 
 +
[[File:IGEM 2010-06-23 12hr 45min.jpg]]
 +
 
 +
For ygiV(486bp) and Rfal(1269bp)
 +
 
 +
[[File:IGEM 2010-06-23 12hr 44min.jpg]]
 +
 
 +
b.Gel purification of pBAD33BB, ygiV and Rfal.
 +
 
 +
after the gel purificaion, the concentrations are:
 +
 
 +
pBAD33BB-PX: 39.7ng/ul
 +
 
 +
ygiV: 25.7ng/ul
 +
 
 +
Rfal: 33.5ng/ul
 +
 
 +
c.Set up an overnight ligation with Insert/Vector ratio equals 4.
 +
 
 +
tubeA
 +
 
 +
pBAD33BB: 3.8ul
 +
 
 +
ygiV: 2.2ul
 +
 
 +
 
 +
tubeB
 +
 
 +
pBAD33BB: 3.8ul
 +
 
 +
Rfal: 4.2ul
 +
 
 +
 
 +
tube C (control)
 +
 
 +
pBAD33BB: 3.8ul
 +
 
 +
(16C overnight)
 +
 
 +
=Note book of June 23rd,2010=
 +
 
 +
a. Transformation
 +
 
 +
pBAD33BB-ygiV: 4.4  1.8
 +
 
 +
pBAD33BB-Rfal: 4.4  1.8
 +
 
 +
Control: 4.3  1.8
 +
 
 +
b. Plating on CM plate
 +
 
 +
c. run the gel for ligation product
 +
 
 +
[[File:Pflegerlab 2010-06-24 11hr 07min.jpg]]
 +
 
 +
 
 +
=Note book of June 24th,2010=
 +
 
 +
a. Check the transformation plate
 +
 
 +
pBAD33BB-ygiV:
 +
 
 +
[[File:Photo.JPG]]
 +
 
 +
 
 +
pBAD33BB-Rfal:
 +
 
 +
[[File:Photo2.JPG]]
 +
 
 +
 
 +
Control:
 +
 
 +
[[File:Photo1.JPG]]
 +
 
 +
b. pick 12 colonies from pBAD33BB-Rfal plate and 10 colonies from pBAD33BB-ygiV plate for the colony PCR screening.
 +
 
 +
ygiV-plate B
 +
 
 +
[[File:Plate B of ygiV-pBAD33BB screening pflegerlab 2010-06-25 14hr 45min.jpg]]
 +
 
 +
 
 +
Rfal-plate B
 +
 
 +
[[File:Plate A of Rfal-pBAD33BB screening pflegerlab 2010-06-25 14hr 42min.jpg]]
 +
 
 +
c. pick colonies from both plates into liquid cultures. Grow overnight. (5 from plateA and 5 from plateB)
-
====6-14====
 
   
   
-
::{|style="border: 1px solid green"
+
=Note book of June 25th,2010=
-
|''L. acidophilus''
+
 
-
|}
+
a. miniprep 10 liquid culture for Screening.
 +
 
 +
b. Digest with BamHI-HF and PstI-HF
 +
 
 +
c. run the gel for the screening
 +
 
 +
[[File:Screening.jpg]]
 +
 
 +
 
 +
 
 +
=Summary of Week June 14th to June 18th, 2010=
 +
 
 +
  06-14-10
 +
'''A. Miniprep'''
 +
 
 +
Miniprep All the new parts received
 +
 
 +
1. K082006  52.7ng/ul
 +
 
 +
2. K142001  196.7ng/ul
 +
 
 +
3. K173004  330.2ng/ul
 +
 
 +
4. pBAD35BB  102.4ng/ul
 +
 
 +
5. pBAD33BB  61.5ng/ul
 +
 
 +
6. pBAD35BB  105.8ng/ul
 +
 
 +
7. K142003  212.8ng/ul
 +
 
 +
8. K137113  174.8ng/ul
 +
 
 +
9. K142002  222.5ng/ul
 +
 
 +
10.pBAD35BB  117.2ng/ul
 +
 
 +
11.K200003  207.2ng/ul
 +
 
 +
12.K142000  177.4ng/ul
 +
 
 +
 
 +
'''B. Ligation for vgiV and pBAD33BB'''
 +
 
 +
Ligation of vgiV and pBAD33BB with a molar ratio 3:1
 +
 
 +
one for 2 hours, one for overnight
 +
 
 +
 
 +
'''C. Thansformation of the vgiV cloning'''
 +
 
 +
Transformed the 2-hour ligation and plated
 +
 
 +
  06-15-10
 +
'''A. Screening of the cloning of vgiV into pBAD33BB'''
 +
 
 +
The 2-hour ligation  only had two colonies.
 +
Screening with Colony PCR but didn't see any band.
 +
 
 +
'''B. Transformation of vgiV cloning'''
 +
 
 +
Transformed the overnight ligation and plated in CM argar plate.
 +
 
 +
'''C. Restriction Mapping of the Newly Receiving Parts'''
 +
 
 +
The Correct Parts are
 +
 
 +
Parts Band 1@ Band2@ Band3@
 +
 
 +
K112808 2500 1406
 +
 
 +
K137113 2200 650
 +
 
 +
K173004 3200 2100 1106
 +
 
 +
K142000 2100 1100
 +
 
 +
K142001 2100 1100
 +
 
 +
K142002 2100 1100
 +
 
 +
K142003 2100 1100
 +
 
 +
K082006 3200 750
 +
 
 +
K200003 3200 1300
 +
 
 +
 
 +
  06-16-10
 +
'''A. Screening of the cloning of vgiV into pBAD33BB'''
 +
 
 +
Saw a lot colony on both the 100ul plate and the pallet plate. The Control plate (backbone only) only had one colony.
 +
 
 +
Pick up 10 colonies from each plate( 100ul plate and pallet plate). Colony PCR for the screening
 +
 
 +
Run the Gel for the Coloy PCR, but didn't see the correct band.
 +
 
 +
(The band is about 200bp, which means the back bone ligate itself.)
 +
 
 +
'''B. Cloning of Rfal into pBAD35BB'''
 +
 
 +
Digest pBAD35BB with PstI-HF and XbaI, and digest Rfal with PstI-HF and SpeI
 +
 
 +
Gel extraction and purification.
 +
 
 +
Ligate with a insert/vector ratio 3:1
 +
 
 +
Ligate at 16C overnight
 +
 
 +
  06-17-10
 +
 
 +
'''A. Another Screening of the cloning of vgiV into pBAD33BB'''
 +
 
 +
Pick another 10 colonies from both the 100ul plate and the pallet plate.
 +
 
 +
Proceed the Colony PCR
 +
 
 +
Run the Gel, but still didn't see the correct band(about 700bp).
 +
 
 +
Do see a band down at 200bp, same length as the positive control. Therefore, confirmed that the colonies are just transformed with the backbone.
 +
 
 +
 
 +
'''B. Cloning of Rfal into pBAD35BB'''
 +
 
 +
Transform the Rfal cloning and the control into DH10B
 +
 
 +
Plated on Kan plate.
 +
 
 +
 
 +
 
 +
 
 +
Cloning of pET28b+RFP
 +
[[File:Cloning of pET28b-RFP.pdf]]
 +
 
 +
=Notebook May 25th, 2010=
 +
 
 +
'''BioBrick pBAD35'''
 +
a) Target Vector: pBAD35 (about 4200bp, 4.2kb)
 +
 
 +
  pBAD BioBrick Primers:
 +
  Tm value: Fwd: 72.0C
 +
            Rev: 68.1C
 +
 
 +
'''b) General Procedure'''
 +
1. PCR amplify
 +
2.  Pour 2 gels during this time
 +
3.  Check PCR on 1st Gel
 +
4.  Add 1ul DpnI/rxn for 1hr at 37C
 +
5.  PCR clean up or gel extraction (depends on whether you see a bright band at right place)
 +
6.  XbarI Digest
 +
7.  PCR clean up
 +
8.  Ligate
 +
 
 +
'''c) Cycle Design'''
 +
98C  1min
 +
98C  15s
 +
73C  30s  33X
 +
72c  126S
 +
72c  5mins
 +
4c  forever
 +
 
 +
'''d)Phusion Mix'''
 +
Water: 35ul
 +
5Xbuffer:10ul
 +
template:2ul
 +
Primer FW:0.5ul
 +
Primer RV:0.5ul
 +
dNTP:1ul
 +
Phusion Enzyme:1ul
 +
 
 +
Note: Keep the primers and Enzyme on ICE
 +
 
 +
'''RESULT'''
 +
1st Gel
 +
Lane    Band@
 +
35A    3-4kb
 +
35B    3-4kb
 +
18      4kb
 +
33      5kb
 +
34      4kb and 2.5kb(wrong)
 +
 
 +
[[File:IGEM Biobrick first 2010-05-25 15hr 09min.jpg]]
 +
 
 +
'''
 +
Protocol of PCR Purification:'''
 +
[[File:PCR purification-Qiagen.pdf]]
 +
 
 +
'''DIGESTION'''
 +
 
 +
'''pBAD35-A'''
 +
DNA: 40ul
 +
Buffer4: 5ul
 +
BSA: 0.5ul
 +
Water: 3.5ul
 +
XbaI: 1ul
 +
 
 +
'''pBAD35-B'''
 +
DNA: 40ul
 +
Buffer4: 5ul
 +
BSA: 0.5ul
 +
Water: 2.5ul
 +
XbaI: 2ul
 +
 
 +
Leave the digestion on a 37c water bath overnight
 +
 
 +
'''
 +
Inoculation for Encryption Project'''
 +
 
 +
10ml LB in each tube and add 10ul Antibiotic
 +
A: BBa_K118004
 +
B: BBa_K098995
 +
C: BBa_I718008
 +
D: BBa_B1006
 +
 
 +
=Notebook May 26th, 2010=
 +
a)PCR purification(refer to protocol on the protocol from Notebook May 25th 2010)
 +
DNA      Conc(ng/ul)
 +
pBAD35-A  57.6
 +
pBAD35-B  69.7
 +
 
 +
b)Ligation
 +
(Peter did all the ligation)
 +
Ligate on pBAD18,33,34,35A-B 100ng/10ul rxn
 +
2hrs bench ligation
 +
 
 +
c)Electroporation transformation
 +
 
 +
Use DH10B
 +
Add 1ul of plasmid
 +
Let it sits on ice for 5 mins
-
:::*''L. acidophilus'' plates with parafilm grew much quicker than the plate without parafilm. Most likely due to a more anaerobic environment, which Lb. a. likes better.
+
after the shock, immediately add 950ul LB and mix well
-
:::* No growth in the tubes yet.
+
Transfer to centrifuge tube
-
::{|style="border: 1px solid blue"
+
DNA  Time
-
|Ligation
+
35    4.2
-
|}
+
33    4.4
-
:::* Ligation of YgiV with pBAD33BB. One ligation was a two hour benchtop and the other was overnight at 15C to see which one would be better.
+
18    4.2
-
::: [[File:IGEM_two_hr_ligation_gel_3_6-14.jpg]]
+
-
# Ladder
+
-
# Control (pBAD33BB vector only)
+
-
# YgiV:pBAD33BB ligation
+
-
::{|style="border: 1px solid purple"
+
(control)
-
|Electroporation/Transformation
+
35    4.5
-
|}
+
33    4.2
-
:::* YgiV:pBAD33BB had a time constant of 4.40ms
+
18    4.2
-
:::* Control (pBAD33BB vector only) had a time constant of 5.40ms
+
-
:::* Incubated in a shaker for 1 hour and then plated pelleted cells on chlorophenicol plates.
+
-
::{|style="border: 1px solid orange"
+
Let them grow in the 37c shaker for 1hr.
-
|Competent cells
+
-
|}
+
-
:::* Prep: UV centrifuge bottles and put in freezer
+
Plate 50ul of cells
-
:::* Made 4 x 250ml and 2x 100ml salt free lb
+
-
====6-15====
 
-
::{|style="border: 1px solid pink"
 
-
|Screening
 
-
|}
 
-
:::*Screened two clones that grew from the transformation from 6-14. Followed the colony PCR. Did a 1:10 dilution of a 0.5 uL aliquot of pBAD seq primers, because the individual amount was too small to accurately pipette. After colony PCR, Yue Wu ran a gel of the colony PCR product (see below).
 
-
:::[[File:IGEM 2010-06-14 Colony PCR YgiV pBAD33BB.jpg]]
 
 +
'''Miniprep for Nate Pant's Project
 +
Miniprep'''
 +
Plasmid information
 +
A: BBa_K118004
 +
B: BBa_K098995
 +
C: BBa_I718008
 +
D: BBa_B1006
-
====6-16====
+
Centrifuge at 30000rpm for 10mins
-
Peter and I made competent cells 50 aliquots of 40 uL. Follow protocol on page 1 of notebook.
+
Follow the Miniprep Protocol:
 +
[[File:Qiagenmini.pdf]]
 +
'''
 +
Result'''
 +
Measured by Nanodrop
-
Tranformed competet cells with pBAD33BB miniprep (6-14) to test the efficency.
+
DNA    Conc(ng/ul)   260/280
 +
A      324.0          1.97
 +
B      288.3          1.98
 +
C      418.2          1.96
 +
D      276.6          1.97
-
====6-17====
 
-
Transformation efficency: 7 CFUs were on the 10^-5 plate, 87 CFUs were on the 10^-4 plate, ~640 CFUs were on the 10^-3 plate, 10^-2 had ~ 2,000, 10^-1 had a lawn. I used the 10^-4 for the effciency.  The efficiency was 1.41E10 CFUs/ug
+
=Notebook May 27th, 2010=
-
Nate did Alkyl lysis prep on the two potienal colonies used for colony PCR.
+
Check the colonies in the plates.
-
Then I digested with PstI-HF only and PstI-HF, XbaI double digest. After digest, I ran a gel (see below).
+
Picked 10 colonies from pBAD35B
-
:Lanes
+
Picked 5 colonies from pBAD33
-
::  2-log Ladder
+
Picked 5 colonies from pBAD18
-
::# Colony 1 Undigested Alkyl Lysis product
+
-
::# Colony 1 Digested (PstI-HF)
+
-
::# Colony 1 Digested (PstI-HF and XbaI)
+
-
::# Colony 2 Undigested Alkyl Lysis product
+
-
::# Colony 2 Digested (PstI-HF)
+
-
::# Colony 2 Digested (PstI-HF and XbaI)
+
-
::# YgiV gel extration purified cut product
+
-
::# pBAD33BB miniprep
+
-
[[File:IGEM_2010-06-17_post_screen_alkyl_lysis_digestion2.jpg]]
+
Inoculate them in 5ml LB with antibiotics
-
  6-21 to 6-22
+
'''
 +
Parts Checking
 +
'''
-
====6-21====
+
Concentration:
-
On Friday, 6-19, I did a colony PCR of RfaI:pBAD35BB from Richard's transformation colonies. I came in on Saturday morning to run a gel and didn't find anymore Loading Dye, so I put the colony PCR product into the freezer. Monday (6-21) I got a 1ml of loading dye from the bottle in Brian's lab and ran a gel of the colony PCR product.
+
BBa_K200000: 199.9ng/ul
 +
BBa_I716213: 186.5ng/ul
 +
BBa_S04114: 543.6ng/ul
 +
BBa_I718007: 146.1ng/ul
 +
BBa_I716212: 155.8ng/ul
 +
BBa_S03975:
 +
BBa_K200002: 309.3ng/ul
 +
BBa_K200003: 105.4ng/ul
 +
BBa_S09373: 243.1ng/ul
 +
BBa_J31000: 278.3ng/ul
 +
BBa_B1006: 276.6ng/ul
 +
BBa_K118004: 324.0ng/ul
 +
BBa_K098995: 288.3ng/ul
-
The gel didn't show any bands in the 700 region. The positive control (pBAD35BB [60.1 ng/ul]) had a faint band in the 3kB region and a fuzzy streak at the end (could be primer dimer) didn't not have a band in the 700 region. The negative control was negative of any bands. Since there was no bands in any of the colonies screened, I will not be doing Alkly lysis and digestion.
+
Single Digestion Test With EcoRI
 +
for
 +
1. BBa_K200000:
 +
2. BBa_I716213:
 +
3. BBa_S04114:
 +
4. BBa_I718007:
 +
5. BBa_I716212:
 +
6. BBa_S03975:(Don't have plasmid)
 +
7. BBa_K200002:
 +
8. BBa_K200003:
 +
9. BBa_S09373:
 +
10.BBa_J31000:
 +
11.BBa_B1006:
 +
12.BBa_K118004:
 +
13.BBa_K098995:
-
Peter and Sarah looked at the pBAD35BB sequence from the Biotech center and found that the PstI cut site had a point mutation that prohibited the PstI enzyme to cut. This could be one of the problems that lead to unsuccessful clones with the pBAD35BB vector.
+
Protocol
 +
1.8ul Buffer3(10X)
 +
0.18ul BSA(100X)
 +
12ul MiliQ H2O
 +
3ul DNA
 +
1ul EcoRI
-
====6-22====
+
Incubate in water bath at 37C for 2 hours (total volume 18ul)
-
Today, I did a miniprep of YgiV and RfaI encapsulation parts with Hannah. Since pBAD35BB PstI cut site is incorrect. We decided to clone both parts into pBAD33BB.  I ran a gel of the miniprep product to double check the nanodrop concentration.  From the nanodrop, the concentration YgiV [238.6 ng/uL] and RfaI [139.2 ng/uL]
+
add 3.6ul Dye (6X)
-
Richard and I did a overnight benchtop digestion of both parts and pBAD33BB vector. The parts were ones that he earlier minipreped, and not the ones Hannah and I did today.
+
Load 6ul into the GEL
 +
Gel:
 +
[[File:2010-05-27 Map - Single digest.jpg]]
-
====6-23====
+
Gel Description:
-
Today, I did research for the pH testing of L. acidophilus strain and MG1655 without any of the colanic acid genes to see how well they grow without the colanic acid genes.  In order to use the plate reader, we need to transform a RFP gene into L. acidophilus and MC1655. That is simple for MG1655, but for L. acidophilus conjugation might be nessarary, according to Prof. Steele.  The Registry of Parts have two plasmids that can mobilize between E. coli and Lactobacillus, but in the notes, they warn that certain strains are picky. So I am currently researching articles for acidophilus plasmids.
+
1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13
-
Another option is to use a chemical like Resazurin, which when the cells metabolize the chemical it becomes florescent.
+
Results:
 +
3, 9, 11, and 12 don't have the correct bands
-
Screened 14 colonies from pBAD35BB? transformation plate.  
+
So run the single digestion for all four again with 2 hours
 +
Gel:
 +
[[File:IGEM Single Digestion Second 2010-05-28 14hr 39min.jpg]]
 +
Gel Description:
 +
3, 9, Ladder, 11, 12
-
====6-24====
+
Result:
-
Prepped the 14 overnight cultures of the screens by Alkyl lysis miniprep.
+
Still not correct
-
Made solution for competent cells.
+
 +
Double Digestion
 +
Find one enzyme cutting site with the gene
 +
1. BBa_K200000:
 +
Buffer:4
 +
Enzyme:AflII EcoRI
 +
2. BBa_I716213:
 +
Buffer:2
 +
Enzyme:AflIII SpeI
 +
3. BBa_S04114:
 +
Buffer:3
 +
Enzyme:EcoRV EcoRI
 +
4. BBa_I718007:
 +
Buffer:3
 +
Enzyme:EcoRV EcoRI
 +
5. BBa_I716212:
 +
Buffer:3
 +
Enzyme:EcoRV EcoRI
 +
6. BBa_S03975:(Don't have plasmid)
 +
7. BBa_K200002: (don't have enzyme stock that can cut within the gene)
 +
8. BBa_K200003:
 +
Buffer:4
 +
Enzyme:NheI EcoRI
 +
9. BBa_S09373:
 +
Buffer:3
 +
Enzyme:EcoRV EcoRI
 +
10.BBa_J31000:
 +
Buffer:3
 +
Enzyme:BglI EcoRI
 +
11.BBa_B1006: (Too small, cannot cut within the gene)
 +
12.BBa_K118004:
 +
Buffer:3
 +
Enzyme:EcoRV EcoRI
 +
13.BBa_K098995:
 +
Buffer:2
 +
Enzyme:HindIII EcoRI
-
====6-25====
+
Protocol
-
*Making competent cells today!
+
1.8ul NEB Buffer(10X)
-
*More iGEM parts came in and making Streptomycin plates for one of the parts.
+
0.18ul BSA(100X)
 +
11ul MiliQ H2O
 +
3ul DNA
 +
1ul Enzyme1
 +
1ul Enzyme2
-
*Here is the file on a different mechanism of induction by bile. [[File:AcrAB_efflux_pump_by_RamA_Nlkaldo_2008.pdf]]
+
Incubate in water bath at 37C for 2 hours (total volume 18ul)
 +
add 3.6ul Dye (6X)
-
It is found in ''Salmonella enterica'' serovar Typhimurium. The protien, RamA binds to the acrAB promoter in the presence of bile. The transcription of RamA is induced by indole. Indole is found in the intestines due to bacterial metabolism of the amino acid tryptophan.  The paper suggests that RamA is a major regulator of the operon acrAB and that when bile is present the bile some how changes the RamA from an inactive state to an active state thus increasing the expression. On page 24252, they have a diagram of how they think the indole and bile coordinate the induction.
+
Load 6ul into the GEL
-
The paper also mentions that ''E. coli'' does not have RamA to control acrAB expression and instead uses multiple proteins to control acrAB expression. If you put the Salmonella acrAB gene into E. coli will the native proteins also induce expression or will the Salmonella acrAB gene be different enough that the native proteins won't recognize and induce expression?
+
Gel
 +
[[File:IGEM Parts Double Digestion 2010-05-28 14hr 54min.jpg]]
 +
[[File:IGEM Parts Double Digestion White 2010-05-28 14hr 57min.jpg]]
-
To help with this question, I plan on doing a BLAST of the Salmonella acrAB operon and the ramA gene to see how similar it is with ''E. coli''. Also I plan on doing the same BLAST but for Lactobacillus. If Lactobacillus doesn't have a acrAB gene, this might prove beneficial because we won't have to worry about other native proteins also transcribing the engineered gene.
+
Result
 +
Only 1, 2, 4, 5, 8, 13 show the correct bands
-
  6-28 to 7-2
 
-
====6-28====
+
=Notebook May 28th, 2010=
-
In the group meeting with Wes, we discussed about the pBAD35BB mutated PstI site. need to cut with SpeI and PstI. PstI is on both parent and Biobricked clone (this would be to check if the PstI site worked from the PCR amplification on new pBAD35 miniprep.
+
-
Also we talked about the logistics of the pH acid test on ''L. acidophilus'' and MG1655 ''E. coli''. If we are using a GFP/RFP protien, we need to transform it into the two hosts. Easy for ''E. coli'', but for ''L. acidophilus'' better to use conjugation.  There is a plasmid in the Registry of Parts that the MIT 2008 team used to transform into a Lactobacillus spieces from ''E. coli''. So we plan on trying to use this plasmid.  Also Wes suggested chemical transformation.
+
Miniprep for the screening
-
In lab today, I did a transformation of pBAD33BB (miniprepped 6-21-10) to test the competent cell transformation efficiency. The time constant was 4.6 ms
+
Restriction Enzyme Mapping for all the 20 screening plasmid
-
Also the Lactose-Drug team decided to use the plasmids the parts came in to clone (ie pSB1AK3). Richard still had no clonig success with pBAD33BB and Nate has had greater success using the plamids the parts come in. So we started overnight cultures to beginning cloning in these vectors.
+
1.8ul Buffer3(10X)
 +
0.18ul BSA(100X)
 +
12ul MiliQ H2O
 +
3ul DNA
 +
1ul SpeI
-
====6-29====
+
Incubate in water bath at 37C for 2 hours (total volume 18ul)
-
Competent cell transformation plates:
+
add 3.6ul Dye (6X)
-
*10^-1 >~2,000 CFUs
+
-
*10^-2 ~2,000 CFUs
+
-
*10^-3 ~356 CFUs
+
-
*10^-4 22 CFUs
+
-
*10^-5 2 CFUs
+
-
To calculate the transformation efficiency, I used 10^-3, 356 CFUs
+
Load 6ul into the GEL
 +
Run the GEL at 100V for 30mins
 +
[[File:IGEM Biobrick 2010-05-28 16hr 55min.jpg]]
 +
GEL upper
 +
18-1 18-2 18-3 18-4 18-5 Ladder 33-1 33-2 33-3 33-4 33-5
-
Miniprep
+
Gel Lower
 +
35-1 35-2 35-3 35-4 35-5 Ladder 35-6 35-7 35-8 35-9 35-10
-
Hannah and I miniprepped 2 tubes of K200021 (LacI promoter), which we are going to use as our vector, and one tube of K137113 (RcsA, colanic acid encapsulation gene). 
 
-
:Concentrations from nanodrop:
 
-
::K200021 A [138.6 ng/uL]
 
-
::K137113  [207.2 ng/uL]
 
-
::K200021 B [91.4 ng/uL]
 
-
Digest of LacI A and RscA parts above:  Want 1.5ng for 50uL Rxn, for 2 hours at 37C in water bath.
+
=Notebook June 1st, 2010=
-
For LacI A vector, I digested with SpeI and PstI. For RscA, I digested with XbaI and PstI. This will make a scar with SpeI and XbaI and the two PstI will create a PstI site. This will keep the four biobrick site intact to clone in the next part.  The vector length should be 3.2kb and the insert length should be 625 bp.
 
-
Gel Extraction of the two parts:
+
Search the Detailed info of the Parts we received
 +
1. BBa_K200000: Colanic acid global regulator (RcsB)
 +
2. BBa_I716213: Cre(TTG Start)
 +
3. BBa_S04114: Lysis
 +
4. BBa_I718007: RBS-Cre
 +
5. BBa_I716212: Cre(GTG Start)
 +
6. BBa_S03975: Lacl+pL Luxr+ LuxpR(Don't have plasmid)
 +
7. BBa_K200002: ygiV
 +
8. BBa_K200003: Ligase(Rfal)
 +
9. BBa_S09373: LacZ
 +
10.BBa_J31000: Invertase Hin from Salmonella Typhimurium
 +
11.BBa_B1006: Stop
 +
12.BBa_K118004: rbs+dxs
 +
13.BBa_K098995: Heat sensitive cl QPI with high promoter
-
Ran 2 cut products (digest product) and 2 uncut products (miniprep product). The gel was run at 75V for 45 min. After a couple of minutes I notice it was going towards the negative electrodes (it was about 2mm past the wells). I had accidentally switched the electrodes in the machine, even though the chamber direction was correct, but it seemed it didn't effect any of the bands.
+
Extra:
-
See gel picture below.
+
BBa_J31001: DNA Invertase
-
[[File:IGEM_2010-06-29_K200021_RscA_gel_extraction.jpg]]
+
 +
=Sequences for all the parts=
 +
>BBa_K200000 Part-only sequence (654 bp)
 +
atgaacaatatgaacgtaattattgccgatgaccatccgatagtcttgttcggtattcgcaaatcacttgagcaaattgagtgggtgaatgttgtcggcg
 +
aatttgaagactctacagcactgatcaacaacctgccgaaactggatgcgcatgtgttgattaccgatctctccatgcctggcgataagtacggcgatgg
 +
cattaccttaatcaagtacatcaagcgccatttcccaagcctgtcgatcattgttctgactatgaacaacaacccggcgattcttagtgcggtattggat
 +
ctggatatcgaagggatcgtgctgaaacaaggtgcaccgaccgatctgccgaaagctctcgccgcgctccagaaagggaagaaatttaccccggaaagcg
 +
tttctcgcctgttggaaaaaatcagtgctggtggttacggtgacaagcgtctctcgccaaaagagagtgaagttctgcgcctgtttgcggaaggcttcct
 +
ggtgaccgagatcgctaaaaagctgaaccgcagtattaaaaccatcagtagccagaagaaatctgcgatgatgaagctgggtgtcgagaacgatatcgcc
 +
ctgctgaattatctctcttcagtgaccttaagtccggcagataaagactaataa
-
Ligation
 
-
Concentration from gel purification were K200021 [12.9ng/uL] and RcsA [5.5ng/uL].  I used the Clontech website to calculate the ng ratio. Vector was 100ng and insert 58.8ng. I divided these by the respective concentrations, giving 7.7uL and 10.7uL respectively. However, I want to do a 10uL reaction so I divided 7.7uL and 10.7uL by 2 to keep the ratio. This gave me 3uL for the vector and 5 uL for the insert. I ran the ligation in the PCR machine at 16C overnight.
+
>BBa_I716213 Part-only sequence (1032 bp)
 +
ttgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacctgatggacatgttcagggatc
 +
gccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataaccggaaatggtttcccgcaga
 +
acctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgt
 +
cggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggattcgaaaagaaaacgttgatgccggtgaacgtgcaaaacagg
 +
ctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgc
 +
ttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacg
 +
aaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatc
 +
cgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagc
 +
aactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcc
 +
cgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaacctggatagtgaaacagggg
 +
caatggtgcgcctgctggaagatggcgattaa
-
====6-30====
 
-
Gel of ligation:
 
-
1uL of product was used for all wells. For the ladder, 6uL was used of the premix ladder. There is a light smear in the LacI + RcsA ligation, so it possible it worked.
 
-
[[File:IGEM_2010-06-30_LacI_RscA_ligation_product.jpg]]
 
-
Transformation:
+
>BBa_S04114 Part-only sequence (1410 bp)
 +
gattgttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaaaaacatgacctgttggc
 +
cgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaaaaaca
 +
gtaatcgacgcaacgatgtgcgccattatcgcctggttcattcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgt
 +
ttatcggctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataatcaacgta
 +
aggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggagagctatt
 +
tactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacgctaccagcttctttcccgttggtgggat
 +
gcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgattg
 +
atcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggctgacagcct
 +
gattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcgattatctccgctctggttatctgcatcatcgtct
 +
gcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaat
 +
tactgacatgcagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgat
 +
gatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtggataatgcagcctcccccc
 +
gactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggatactagagccaggcatcaaataaaacg
 +
aaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttc
 +
tgcgtttata
-
For LacI + RcsA, the time constant was 3.4ms. For the vector only control, the time constant was 3.8ms.
 
-
Plating:  I plated 100uL and the pellet of both the ligation and control
 
-
====7-1====
+
>BBa_I718007 Part-only sequence (1058 bp)
 +
attaaagaggagaaatactagatgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacc
 +
tgatggacatgttcagggatcgccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataa
 +
ccggaaatggtttcccgcagaacctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggc
 +
cagctaaacatgcttcatcgtcggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggatccgaaaagaaaacgttgatg
 +
ccggtgaacgtgcaaaacaggctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaa
 +
tctggcatttctggggattgcttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatg
 +
ttaatccatattggcagaacgaaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccg
 +
tctctggtgtagctgatgatccgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgc
 +
cctggaagggatttttgaagcaactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtc
 +
ggagccgcgcgagatatggcccgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgta
 +
acctggatagtgaaacaggggcaatggtgcgcctgctggaagatggcgattaagaatt
-
Transformation plates:
 
-
:100uL aliquot
 
-
::Control 3CFUs
 
-
::LacI + RcsA 25CFUs
 
-
:Pellet
 
-
::Control 15 CFUs
 
-
::LacI + RcsA 221 CFUs
 
-
Since the transformation results had a significant different between the control and variable, I did a colony PCR on 10 random colonies from the 100uL plate. After PCR, I ran a gel which had 8 successful clones. 1,3-5, 7-10. The RcsA insert was 624bp and the LacI promoter is 75bp, which is a total of 699bp. The successful clones hand one band around between the 600 and 700 region on the ladder. So it is most likely a clone with the RcsA insert and sequencing needs to be done to confirm it.  See gel below.
+
>BBa_I716212 Part-only sequence (1032 bp)
-
[[File:IGEM_2010-07-01_LacI+RcsA_Colony_PCR_gel.jpg]]
+
ttgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacctgatggacatgttcagggatc
 +
gccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataaccggaaatggtttcccgcaga
 +
acctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgt
 +
cggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggattcgaaaagaaaacgttgatgccggtgaacgtgcaaaacagg
 +
ctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgc
 +
ttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacg
 +
aaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatc
 +
cgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagc
 +
aactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcc
 +
cgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaacctggatagtgaaacagggg
 +
caatggtgcgcctgctggaagatggcgattaa
-
I inoculated 5 and 7 for overnight cultures.
 
-
====7-2====
+
>BBa_S03975 Part-only sequence (1082 bp)
-
Peter and I made 52 aliquots of 40uL competent cells. Sarah also minipreped the two overnight cultures along with her overnight cultures.  The next step to do is to insert LacI and RcsA construct in front of two terminators (B0015) to complete the construct.
+
aattgtgagcggataacaattgacattgtgagcggataacaagatactgagcacatactagagaaagaggagaaatactagatgaaaaacataaatgccg
-
To do this I want to clone the previous construct in front of the B0015 part in the pSB1AK3 plasmid. So I digested the LacI + RcsA construct with EcoRI and SpeI. The pSB1AK3:B0015 was digested with EcoRI and XbaI.  DNA digested was 1.5 ug each as a 50uL reaction for 2 hours in a 37C water bath.
+
acgacacatacagaataattaataaaattaaagcttgtagaagcaataatgatattaatcaatgcttatctgatatgactaaaatggtacattgtgaata
 +
ttatttactcgcgatcatttatcctcattctatggttaaatctgatatttcaatcctagataattaccctaaaaaatggaggcaatattatgatgacgct
 +
aatttaataaaatatgatcctatagtagattattctaactccaatcattcaccaattaattggaatatatttgaaaacaatgctgtaaataaaaaatctc
 +
caaatgtaattaaagaagcgaaaacatcaggtcttatcactgggtttagtttccctattcatacggctaacaatggcttcggaatgcttagttttgcaca
 +
ttcagaaaaagacaactatatagatagtttatttttacatgcgtgtatgaacataccattaattgttccttctctagttgataattatcgaaaaataaat
 +
atagcaaataataaatcaaacaacgatttaaccaaaagagaaaaagaatgtttagcgtgggcatgcgaaggaaaaagctcttgggatatttcaaaaatat
 +
taggttgcagtgagcgtactgtcactttccatttaaccaatgcgcaaatgaaactcaatacaacaaaccgctgccaaagtatttctaaagcaattttaac
 +
aggagcaattgattgcccatactttaaaaattaataacactgatagtgctagtgtagatcactactagagccaggcatcaaataaaacgaaaggctcagt
 +
cgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatat
 +
actagagacctgtaggatcgtacaggtttacgcaagaaaatggtttgttatagtcgaataaatactagagaaagaggagaaa
-
After digestion, I ran a gel to extract the desired DNA fragments. For pSB1AK3:B0015, I wanted the 3318bp length and for LacI + RcsA construct I wanted the fragment at 700bp. See below.
 
-
[[File:IGEM_2010-07-02_LacI+RcsA_pSB1AK3.jpg]]
 
 +
>BBa_K200002 Part-only sequence (486 bp)
 +
atgacaaacctgacactggatgtaaacattatcgatttcccatcaatacctgtggcgatgttgccgcaccgctgtagccctgaattgctcaactacagcg
 +
tggcgaaatttatcatgtggcgtaaagagacggggctttctcctgttaaccaaagccagacttttggcgtcgcctgggacgaccctgccaccaccgcacc
 +
ggaagcgtttcgctttgatatctgcggcagcgttagcgaaccgattcccgataatcgttatggtgtgagcaatggtgaacttaccggtggacgttatgcc
 +
gtggcccgccacgttggcgagctggacgatatttcacacacggtatggggcatcattcgccactggctgcctgcaagcggcgagaaaatgcgtaaagcac
 +
cgattctgtttcactacaccaatcttgccgaaggggtgacagagcagcgactggaaacggatgtttatgtgccgttggcgtgataa
-
After gel extraction, I did a gel purification and a 10uL overnight ligation reaction, using 3uL of vector (pSB1AK3:B0015) and 5uL of insert (LacI+RcsA).
 
-
I also received and plated part BBa_I742123 (pTG262) plasmid part that is a shuttle vector between ''E.coli'' and ''Lactobacillus'' species.
 
-
====7-3====
+
>BBa_K200003 Part-only sequence (1263 bp)
-
I froze my ligation for a transformation on Monday.  The pTG262 didn't grow.
+
atgctaacatcctttaaacttcattcattgaaaccttacactctgaaatcatcaatgattttagagataataacttatatattatgttttttttcaatga
 +
taattgcattcgtcgataatactttcagcataaaaatatataatatcactgctatagtttgcttattgtcactaattttacgtggcagacaagaaaatta
 +
taatataaaaaaccttattcttcccctttctatatttttaataggcttgcttgatttaatttggtattctgcgtttaaagtagataattcgccatttcgt
 +
gctacttaccatagttatttaaatactgccaaaatatttatatttggttcttttattgttttcttgacactaactagccagctaaaatcaaaaaaagaga
 +
gtgtattatacactttgtattctctgtcatttctaattgctggatatgcaatgtatattaatagcattcatgaaaatgaccgcatttcttttggtgtagg
 +
aacggcaacaggagcagcatattcaacaatgctaatagggatagttagtggcgttgcgattctttatactaagaaaaatcatccttttttatttttatta
 +
aatagttgcgcggtactttatgttctggcgctaacacaaaccagagcaaccctactcctgttccctataatttgtgttgctgcattaatagcttattata
 +
ataaatcacccaagaaattcacttcctctattgttctactaattgctatattagctagcattgttattatatttaataaaccaatacagaatcgctataa
 +
tgaagcattaaatgacttaaacagttataccaatgctaatagtgttacttccctaggtgcaagactggcaatgtacgaaattggtttaaatatattcata
 +
aagtcacctttttcatttagatcagcagagtcacgcgctgaaagtatgaatttgttagttgcagaacacaataggctaagaggggcattggagttttcta
 +
acgtacatctacataatgagataattgaagcagggtcactgaaaggtctgatgggaattttttccacacttttcctctatttttcactattttatatagc
 +
atataaaaaacgagctttgggtttgttgatattaacgcttggcattgtggggattggactcagtgatgtgatcatatgggcacgcagcattccaattatc
 +
attatatccgctatagtcctcttactcgtcattaataatcgtaacaatacaattaattaataa
-
====7-5====
 
-
Transformation
+
>BBa_S03973 Part-only sequence (3230 bp)
 +
aaagaggagaaatactagatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc
 +
ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt
 +
tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcac
 +
ggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacat
 +
ttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcgg
 +
ttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgac
 +
ggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatg
 +
ttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttcttt
 +
atggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctg
 +
aacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaag
 +
cctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatca
 +
tcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccg
 +
aaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctga
 +
ccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaa
 +
tgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacacc
 +
acggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctac
 +
ctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatcc
 +
ccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggc
 +
gatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttt
 +
tccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatgg
 +
taagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaa
 +
ctctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctca
 +
gtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccg
 +
ccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacatt
 +
ggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacgg
 +
cagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatgg
 +
tagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagag
 +
cgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtata
 +
ccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcag
 +
ccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggatt
 +
ggtggcgacgactcctggagcccgtcagtatcggcggaatttcagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataatactaga
 +
gccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggct
 +
caccttcgggtgggcctttctgcgtttata
-
Transformed the ligation done 7-2/7-3.
 
-
:Time constants
 
-
::Control 4.20ms
 
-
::LacI+RcsA+B0015 4.10ms
 
-
Transformation of ygiV:pSB1AK3 for tranformation efficiency of compet. cells.
 
-
:Time constant 5.10
 
-
Also ran a ligation gel. See below.  There is a little smear, which means it might have worked.
+
>BBa_J31000 Part-only sequence (573 bp)
-
:Lane 1  Ligation product
+
atggctactattgggtatattcgggtgtcaacaattgaccaaaatatcgatttacagcgtaatgcgcttaccagtgcaaattgtgaccgcatttttgaag
-
:Lane 2  Control (vector B0015:pSB1AK3 only)
+
accgtatcagtggcaagattgcaaaccgccccggcctgaaacgggcgttaaagtatgtaaataaaggcgatactcttgtcgtctggaaattagacagact
-
:Lane 3  B0015:pSB1AK3 miniprep product
+
gggccgtagcgtgaaaaatctggtggcgttaatatcagaattacatgaacgtggagctcacttccattctttaaccgatagtattgataccagtagcgcg
-
:Lane 4  B0015:pSB1AK3 digested (gel extraction product)
+
atggggcgattcttttttcatgtaatgtcagcactggccgagatggagcgagaattaatcgtcgagcgaacccttgccggactggctgccgccagagcgc
-
:Lane 5  LacI+RcsA digested (gel extraction product)
+
aaggacgactgggagggcgccctcgggcgatcaacaaacatgaacaggaacagattagtcggctattagagaaaggccatcctcggcagcaattagctat
 +
tatttttggtattggcgtatccaccttatacagatactttccggcaagcagtataaaaaaacgaatgaattaa
-
[[File:IGEM_2010-07-05_Ligation_(21_+_13)_+_B0015.jpg]]
 
-
====7-6====
 
-
Transformation plates for Trans. efficiency
+
>BBa_K118004 Part-only sequence (1882 bp)
 +
ctcaaggaggtactagatgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagttta
 +
ccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtgg
 +
cgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaat
 +
cggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagt
 +
gccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttg
 +
aagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaacca
 +
tctggcacagctgctttccggtaagctttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgtgccgccaattaaagagctgctcaaacgc
 +
accgaagaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggc
 +
ttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagaccc
 +
gatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactgg
 +
ttgtgcgaaacggcagcgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatc
 +
gctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccacttt
 +
cctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggt
 +
caaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctct
 +
ataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattgg
 +
caaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacg
 +
ctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccatta
 +
tgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaagg
 +
aactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataataa
-
:10^-1 solid lawn
 
-
:10^-2 speckled lawn
 
-
:10^-3 >~2,000
 
-
:10^-4 ~2,000
 
-
:10^-5 ~380 CFUs
 
-
Transformation plates for ligation
 
-
Control 8 CFUs
 
-
Ligation ~800 CFUs
 
-
Colony PCR
+
>BBa_K098995 Part-only sequence (935 bp)
 +
tttatggctagctcagtcctaggtacaatgctagctactagagaaagaggagaaatactagatgagcacaaaaaagaaaccattaacacaagagcagctt
 +
gaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatcccaggaatctgtcgcagacaagatggggatggggcagtcag
 +
gcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcccttc
 +
aatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagtgagtatgagtaccctgttttttctcatgttcaggcaggg
 +
atgttctcacctgagcttagaacctttaccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcattctggcttgaggttg
 +
aaggtaattccatgaccgcaccaacaggctccaagccaagctttcctgacggaatgttaattctcgttgaccctgagcaggctgttgagccaggtgattt
 +
ctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggatagcggtcaggtgtttttacaaccactaaacccacagtacccaatg
 +
atcccatgcaatgagagttgttccgttgtggggaaagttatcgctagtcagtggcctgaagagacgtttggctgatactagagtcacactggctcacctt
 +
cgggtgggcctttctgcgtttatatactagagagagaatataaaaagccagattattaatccggcttttttattattttactagagtaacaccgtgcgtg
 +
ttgactattttacctctggcggtgataatggttgc
-
I picked 10 random colonies from the ligation plate and used them for colony PCR. I used the pSB1AK3:B0015 miniprepped product as the positive control. I also had a negative control of just water in place of the DNA.
 
-
Then I ran a gel of the colony PCR. The successful clones showed a band around 1 kb, which should have LacI (75bp) + RcsA (625bp) + B0015 (129bp)= 825bp + the length of the primers. See picture below.  
+
=Summary for Week June 7th to June 11th=
 +
  6-7-10:
 +
a) Miniprep 2 pellet each for pBAD33, pBAD35, pBAD18, ygiV (attach to the cell wall), RcsB(expression factor that increase the clonic acid).
-
[[File:IGEM_2010-07-06_(21+13)_+B0015_colony_PCR.jpg]]
+
b) enzyme digestion for pBAD33, vgiV, pBAD18, and RcsB.  
-
I chose 4 and 8 for overnight cultures.
+
c) Gel extract for vgiV and RcsB and purification
-
====7-7====
+
d) PCR purification for pBAD18 and pBAD33
-
Miniprepped the two overnight cultures. Then digested one of them (#8) and the LacI+ RcsA construct in a 10uL reaction for 2 hours at 37C as a second check to see if the the right parts ligated together. 
+
-
Then I ran 5uL of product on a 3% gel to see the two diffent lengths. The difference should be ~129bp. See below.
+
e) overnight ligation (set up by Sarah)
-
:Lane 1 LacI+RcsA digest
+
  6-8-10:
-
:Lane 2 LacI+RcsA+B0015(TT)  
+
a) transformation pBAD18-RcsB and pBAD33-vgiV into DH10B E. Coli.
-
:Lane 3 LacI+RcsA uncut
+
(Sarah and Hannah plates the cloning of pBAD18-RcsB and pBAD33-vgiV)
-
:Lane 4 LacI+RcsA+B0015(TT)uncut
+
-
[[File:IGEM_2010-07-07_Digest_for_sequencing.jpg]]
+
b) Miniprep
 +
For Encryption Project: BBa_I11020, BBa_K199021, BBa_K2902006, BBa_200021, BBa_B1006-A, BBa_B1006-B, BBa_E0240
 +
For Drug Project: 2 pellets of each, pBAD18, pBAD33, pBAD35, vgiV, RcsB
-
====7-8====
+
  6-9-10:
-
Hannah and I did sequencing PCR reaction for sending to the Biotech center.
+
-
  7-19 to 7-23 Summary
+
a) For the RcsB and VgiV plates, only a few colonies growed (they are really tiny, kindda hard to see, so we put it back to the incubator growing for couple of more hours)
-
Making primers for isolating the ramA gene, RamA binding site,arcR?,pacrAB? from Salmonella enterica LT2.  
+
b) Did the cloning of RcsB into pBAD18 and vgiV into pBAD33 again with different insert/vector ratio.
-
Trying to decide whether to use the acrR:acrAB repression system or the cI phage repressor system. I know I don't want to use the pLacI promoter because the system will be in the system will be in the presence of lactose and will prevent the repressor from binding. If I do use the acrR:acrAB system I will probably need to knock out MarA because its a transcriptional activator for the acrAB promoter.
+
c) Plate the pellet and control.
-
Making acid dunking and acid growth curve assays. Also making pH 2-7 solutions of LB/MRS broth to use next week.
+
  6-10-10:
-
====7-26====
+
a) The pBAD18-RcsB plate has colonies growed.
-
I decided to use the acrR:acrAB repression system because it will be easier to use since it already has the ramA binding site and encodes for a repressor. making primers ro amplify the ramA and acrRAB region.
+
-
====7-28====
+
b) Set up the screening with colony PCR
-
Made competent cells and ordered primers
+
-
====8-6====
+
c)     Made MRS Broth, MRS plates, no antibiotic plates, Amp+Kan plates, and CM plates with Mary.
-
*Made pH-media gradient with Peter(has data)
+
-
*Made PCR reactions for temperture gradients for amplify ramA and acrRAB. Made two master mixes for 8,10ul reactions. Range for ramA was 72C to 80C and the range for acrRAB was 71C to 81C.
+
-
*Ran gel of PCR reactions. See below.
+

Latest revision as of 22:03, 24 October 2010

Contents

Note book of July 12th,2010

1. Screening for the Rfal into LacI-RBS cloning

Gel Picture

File:IGEM 2010-07-12 13hr 39min.jpg

File:IGEM 2010-07-12 17hr 33min.jpg

2. Primer Design For the Cloning LacI-RBS-ygiV-RBS-Rfal-RBS-RFP-Terminator

One is for Rfal: the forward primer has a XbaI and RBS in it; the reverse primer is on the backbone, and has a XmaI site on it.

pRBS-Rfal_Fwd: TGCTCTAGA AAAGAGGAGAAAATGCTAACATCCTTTAAACT

pRBS-Rfal_Rev: CCCCCCGGGTTAATTAATTGTATTGTTACGATTAT


The other one is For RFP (I13507): The forward primer has a XmaI site on it and starts from the backbone; the reverse primer starts on the backbone too. [The original plasmid, I13507 has a structur of RBS-RFP-terminator]

pRFP(I13507)_Fwd: CCCCCCGGGgagaaagaggagaaatactagatgg

pRFP(I13507)_Rev: AGTCAGTGAGCGAGGAAG

Note book of July 13th,2010

1. Primer Designs:

Tyler helped me check the primers, and he get back to me with his comments:

pRBS-RfaI_Fwd - Tm for primer portion is too low(oligocalc says 44), add 4 bases from the gene sequence, try to have 3' end be G or C

pRBS-RfaI_Rev - GC content of the priming portion is way too low, try to get it to at least above 30%, you can move down the gene since you are at the ending site, also try to have the 3' end of the primer be a G or C

pRFP(I13507)_Fwd - good, I usually add a 5 base overhang, but 3 should be ok for the enzyme you are using

pRFP(I13507)_Fwd - good

So I redesigned my primers with

Detailed information are in: File:Primer Design for Rfal and RFP - 2.pdf

Primers For Rfal

Tm : 46 GC:41% Fwd: gagatgctaacatcctt (17bp)

pRBS-RfaI_Fwd : TGCTCTAGAAAAGAGGAGAAAtacgagatgctaacatcctt

Name Sequence Tm°C CG% nt A T C seq_1 tgctctagaaaagaggagaaatacgagatgctaacatcctt 75.1 39.0 41 16.0 9.0 7.0 9.0 417600.0 12674.4 2.4 30.4

Name Sequence Tm°C CG% nt A T C G seq_1 gagatgctaacatcctt 49.9 41.2 17 5.0 5.0 4.0 3.0 164400.0 5169.4 6.1 31.4

Tm:49 GC:40% Rev: taataatactagtagcggcc （17bp）

pRBS-RfaI_Rev: GCCGCTACTAGTATTATTA [SpeI]

Name Sequence Tm°C CG% nt A T C G seq_1 gccgctactagtattatta 49.6 36.8 19 5.0 7.0 4.0 3.0 185500.0 5777.8 5.4 31.1

Primers For RFP

pRFP(I13507)_Fwd: CTAGTCTAGAgagaaagaggagaaatactagatgg [XbaI]

Name Sequence Tm°C CG% nt A T C G seq_1 ctagtctagagagaaagaggagaaatactagatgg 65.7 40.0 35 15.0 6.0 3.0 11.0 377400.0 10950.2 2.6 29.0

Name Sequence Tm°C CG% nt A T C G seq_1 gagaaagaggagaaatactagatgg 59.8 40.0 25 12.0 3.0 1.0 9.0 278000.0 7861.2 3.6 28.3

pRFP(I13507): AGTCAGTGAGCGAGGAAG (cttcctcgctcactgact)

Name Sequence Tm°C CG% nt A T C G seq_1 agtcagtgagcgaggaag 58.8 55.6 18 6.0 2.0 2.0 8.0 191900.0 5637.7 5.2 29.4

Name Sequence Tm°C CG% nt A T C G seq_1 agtcagtgagcgaggaag 58.8 55.6 18 6.0 2.0 2.0 8.0 191900.0 5637.7 5.2 29.4

Note book of July 14th,2010

Cloning of LacI-RBS-ygiV with a terminator

a. Double digestion

b. Gel extraction and purificaiton

2.jpg

c. overnight ligation

Note book of July 15th,2010

Cloning of LacI-RBS-Rfal with a terminator

a. Double digestion

b. Gel extraction and purificaiton

1.jpg

c. 2 hours bench top ligation

d. Electroporation of LacI-RBS-ygiV-terminator and LacI-RBS-Rfal-terminator into DH10B cells.

e. Plate on Amp plates

f. run a gel to check the digestion of the backbone

3.jpg

Note book of July 16th,2010

A.Check the plate

1. LacI-RBS-ygiV-terminator

the pallet plate has about 200 colonies

the control has 8 colonies


2.LacI-RBS-Rfal-terminator

the pallet plate has about 150 colonies

the control doesn't have colonies


B.Screening



Note book of July 6th,2010

A. Start the clone of Rfal into K200021(LacI+RBS)

1.cut the backbone, K200021, with PstI-HF and SpeI for 2 hours. cut the insert, Rfal, with PstI-HF and XbaI for 2 hours.

2.Gel Extract

K200021: 3264bp Rfal:1263bp

File:IGEM 2010-07-06 13hr 05min.jpg

After the gel extraction, the concentrations are:

K200021: 33.1ng/ul Rfal: 53.9ng/ul

3.Ligation

for doing a K200021:Rfal is 1:4 ligation, 100ng of K200021 and 154.8ng of Rfal are needed. Based on the DNA concentration, add 3ul K200021 and 3ul Rfal for ligation.

Stored at 16C overnight.


Note book of July 7th,2010

A. Cloning of LacI-RBS-Rfal

1.Electroporation

LacI-RBS-Rfal has a time constant 4.8;

The control has a time constant 4.7;

2.Plate on two Amp+Kan plate. Stored in a 37C incubator.

B. Cloning of LacI-RBS-ygiV into RBS

1.cut the backbone, RBS, with EcoRI-HF and XbaI, and cut the insert LacI-RBS-ygiV with EcoRI-HF and SpeI.

2.Gel Extraction

RBS:3200bp LacI-RBS-ygiV:543bp

File:IGEM 2010-07-06 17hr 00min.jpg

after the gel extraction, the concentrations are:

RBS: 20.7ng/ul

lacI-RBS-ygiV: 19.8ng/ul

3.Ligation

For a 1 to 3 backbone:insert ratio, we need 100ng of RBS and 107.3ng of LacI-RBS-ygiV for Ligation. THerefore, add 3.7ul RBS and 4.3ul LacI-RBS-ygiV to ligate. Store at 4C overnight.

C.Run a gel to check the cutting peices

The orders are:

L-ygiV-ES; L-ygiV uncut; Rfal-PX; Rfal uncut; RBS-EX; RBS uncut; LacI-PS; LacI uncut; Ladder

unluckily, the first lane for L-ygiV-ES didn't show up any band.

File:IGEM 2010-07-07 11hr 24min.jpg

The rest of the bands are all correct.

Then run a gel for L-ygiV-ES and L-ygiV uncut by loading more DNA on the gel

File:IGEM 2010-07-07 13hr 09min.jpg

Now, this shows that the L-ygiV-ES is correct.


Note book of July 8th,2010

A. Transformation of the Clone LacI-RBS-ygiV-RBS

1. Electroporation

LacI-RBS-ygiV-RBS: 4.7

Control: 4.9

2. Plate on two Amp plate.

B. Run a gel to check the Ligation of LacI-RBS-Rfal and LacI-RBS-ygiV-RBS

the orders are Ladder LacI-RBS-Rfal, control, LacI-RBS-ygiV-RBS, control

File:IGEM 2010-07-07 12hr 48min.jpg

C. Colony PCR for the LacI-RBS-Rfal Clone

1.The control plate only has one colony, compared with the regular plate with about 100 colonies.

2.picked 14 colonies for screening.

Gel for Colony PCR:

File:IGEM 2010-07-08 13hr 54min.jpg

Run for 5mins longer and add more EB

File:IGEM 2010-07-08 14hr 20min.jpg

No colony shows the correct band (around 1500bp)

Note book of July 9th,2010

A Cloning of LacI-RBS-ygiV into RBS, and Rfal into LacI-RBS.

Add 1.5mg of DNA into each tube:

RBS(B0034):5.3ul template; Add 1ul EcoRI-HF to digest 1 hour and inactivate at 65C for 20mins. Then Add 1ul of XbaI to digest an additional hour.

LacI-RBS(K200021): 9.5ul template; Add 1ul PstI-HF to digest 1 hour and inactivate at 80C for 20mins. Then Add 1ul of SpeI to digest an additional hour.

Rfal: 3.1ul template digest with 1ul PstI-HF and 1ul XbaI

LacI-ygiV: 4.4ul tempalte digest with 1ul EcoRI-HF and 1ul SpeI

Gel after 1 hour inactivation of EcoRI-HF in RBS and PstI-HF in LacI-RBS:

Orders are:

RBS cut with EcoRI-HF, RBS uncut, Ladder, LacI-RBS cut with PstI-HF, LacI-RBS uncut.

File:IGEM 2010-07-09 13hr 27min.jpg



Note book of June 28th,2010

a. Digestion of K200021 (LacI+RBS) with PstI-HF and SpeI, and digest ygiV and Rfal with PstI-HF and XbaI.

b. Gel Extraction and purification:

1. Gel

K200021-PS

ygiV and Rfal

File:IGEM 2010-06-27 13hr 19min.jpg

Concentration of the cutting peices: K200021-PS

ygiV-PX

Rfal-PX

Gel for the purification product

File:IGEM 2010-06-28 14hr 10min.jpg

c. Set up an overnight ligation

Note book of June 29th,2010

a. Transformation

pLacI-RBS-ygiV-PX: 4.1

pLacI-RBS-ygiV-PX(C): 3.9

pLacI-RBS-Rfal-PX: 4.0

pLacI-RBS-Rfal-PX(C):3.9

b. Plate on Amp100 plates.


Note book of June 30th,2010

Re-plan the project. Figure out all the cloning need to be done by the end of the summer. Detailed information please see the spread sheet on Sarah's page.

Note book of July 1st,2010

A. Colony PCR of the LacI-RBS-ygiV and LacI-RBS-Rfal clone.

Upper: LacI-RBS-Rfal Clone

Lower: LacI-RBS-ygiV clone

File:IGEM 2010-07-01 14hr 49min-2.jpg

The LacI-RBS-ygiV clone shows up at the right length.


B. Innoculate 5 liquid culture of the ygiV clone.


Note book of July 2nd,2010

A. Made 3 freezer stocks of LacI-RBS-ygiV clone

B. Miniprep LacI-RBS-ygiV

LacI-RBS-ygiV-1: 388.7ng/ul LacI-RBS-ygiV-2: 357.0ng/ul LacI-RBS-ygiV-3: 350.2ng/ul LacI-RBS-ygiV-4: 255.0ng/ul


Note book of June 21st,2010

a.Minipreped 3 sets each for pBAD33BB, pBAD35BB, ygiV, and Rfal.

b.Set up a overnight digestions that cut pBAD33BB and pBAD35BB with PstI-HF and SpeI, and cut ygiV and Rfal with PstI-HF and XbaI. (37C overnight)

c.Screening for the 2nd time ygiV and Rfal cloning.

ygiV:

File:IGEM 2010-06-21 bads2 Peter ScanA.jpg

Rfal:

File:IGEM 2010-06-21 bads2 Peter ScanB.jpg

They all shows up at the same length as the positive control, which means we didn't get the desired cloning.

Note book of June 22nd,2010

a.Gel extraction of pBAD33BB, ygiV and Rfal.

For pBAD33BB

File:IGEM 2010-06-23 12hr 45min.jpg

For ygiV(486bp) and Rfal(1269bp)

File:IGEM 2010-06-23 12hr 44min.jpg

b.Gel purification of pBAD33BB, ygiV and Rfal.

after the gel purificaion, the concentrations are:

pBAD33BB-PX: 39.7ng/ul

ygiV: 25.7ng/ul

Rfal: 33.5ng/ul

c.Set up an overnight ligation with Insert/Vector ratio equals 4.

tubeA

pBAD33BB: 3.8ul

ygiV: 2.2ul


tubeB

pBAD33BB: 3.8ul

Rfal: 4.2ul


tube C (control)

pBAD33BB: 3.8ul

(16C overnight)

Note book of June 23rd,2010

a. Transformation

pBAD33BB-ygiV: 4.4 1.8

pBAD33BB-Rfal: 4.4 1.8

Control: 4.3 1.8

b. Plating on CM plate

c. run the gel for ligation product

File:Pflegerlab 2010-06-24 11hr 07min.jpg


Note book of June 24th,2010

a. Check the transformation plate

pBAD33BB-ygiV:

Photo.JPG


pBAD33BB-Rfal:

File:Photo2.JPG


Control:

File:Photo1.JPG

b. pick 12 colonies from pBAD33BB-Rfal plate and 10 colonies from pBAD33BB-ygiV plate for the colony PCR screening.

ygiV-plate B

File:Plate B of ygiV-pBAD33BB screening pflegerlab 2010-06-25 14hr 45min.jpg


Rfal-plate B

File:Plate A of Rfal-pBAD33BB screening pflegerlab 2010-06-25 14hr 42min.jpg

c. pick colonies from both plates into liquid cultures. Grow overnight. (5 from plateA and 5 from plateB)


Note book of June 25th,2010

a. miniprep 10 liquid culture for Screening.

b. Digest with BamHI-HF and PstI-HF

c. run the gel for the screening

File:Screening.jpg


Summary of Week June 14th to June 18th, 2010

 06-14-10

A. Miniprep

Miniprep All the new parts received

1. K082006 52.7ng/ul

2. K142001 196.7ng/ul

3. K173004 330.2ng/ul

4. pBAD35BB 102.4ng/ul

5. pBAD33BB 61.5ng/ul

6. pBAD35BB 105.8ng/ul

7. K142003 212.8ng/ul

8. K137113 174.8ng/ul

9. K142002 222.5ng/ul

10.pBAD35BB 117.2ng/ul

11.K200003 207.2ng/ul

12.K142000 177.4ng/ul


B. Ligation for vgiV and pBAD33BB

Ligation of vgiV and pBAD33BB with a molar ratio 3:1

one for 2 hours, one for overnight


C. Thansformation of the vgiV cloning

Transformed the 2-hour ligation and plated 

 06-15-10

A. Screening of the cloning of vgiV into pBAD33BB

The 2-hour ligation only had two colonies. Screening with Colony PCR but didn't see any band.

B. Transformation of vgiV cloning

Transformed the overnight ligation and plated in CM argar plate.

C. Restriction Mapping of the Newly Receiving Parts

The Correct Parts are

Parts Band 1@ Band2@ Band3@

K112808 2500 1406

K137113 2200 650

K173004 3200 2100 1106

K142000 2100 1100

K142001 2100 1100

K142002 2100 1100

K142003 2100 1100

K082006 3200 750

K200003 3200 1300 


 06-16-10

A. Screening of the cloning of vgiV into pBAD33BB

Saw a lot colony on both the 100ul plate and the pallet plate. The Control plate (backbone only) only had one colony.

Pick up 10 colonies from each plate( 100ul plate and pallet plate). Colony PCR for the screening

Run the Gel for the Coloy PCR, but didn't see the correct band.

(The band is about 200bp, which means the back bone ligate itself.)

B. Cloning of Rfal into pBAD35BB

Digest pBAD35BB with PstI-HF and XbaI, and digest Rfal with PstI-HF and SpeI

Gel extraction and purification.

Ligate with a insert/vector ratio 3:1

Ligate at 16C overnight 

 06-17-10

A. Another Screening of the cloning of vgiV into pBAD33BB

Pick another 10 colonies from both the 100ul plate and the pallet plate.

Proceed the Colony PCR

Run the Gel, but still didn't see the correct band(about 700bp).

Do see a band down at 200bp, same length as the positive control. Therefore, confirmed that the colonies are just transformed with the backbone.


B. Cloning of Rfal into pBAD35BB

Transform the Rfal cloning and the control into DH10B

Plated on Kan plate.



Cloning of pET28b+RFP File:Cloning of pET28b-RFP.pdf

Notebook May 25th, 2010

BioBrick pBAD35 a) Target Vector: pBAD35 (about 4200bp, 4.2kb) 

  pBAD BioBrick Primers:
  Tm value: Fwd: 72.0C
            Rev: 68.1C

b) General Procedure 1. PCR amplify 2. Pour 2 gels during this time 3. Check PCR on 1st Gel 4. Add 1ul DpnI/rxn for 1hr at 37C 5. PCR clean up or gel extraction (depends on whether you see a bright band at right place) 6. XbarI Digest 7. PCR clean up 8. Ligate

c) Cycle Design 98C 1min 98C 15s 73C 30s 33X 72c 126S 72c 5mins 4c forever

d)Phusion Mix Water: 35ul 5Xbuffer:10ul template:2ul Primer FW:0.5ul Primer RV:0.5ul dNTP:1ul Phusion Enzyme:1ul

Note: Keep the primers and Enzyme on ICE

RESULT 1st Gel Lane Band@ 35A 3-4kb 35B 3-4kb 18 4kb 33 5kb 34 4kb and 2.5kb(wrong)

File:IGEM Biobrick first 2010-05-25 15hr 09min.jpg

Protocol of PCR Purification: File:PCR purification-Qiagen.pdf

DIGESTION

pBAD35-A DNA: 40ul Buffer4: 5ul BSA: 0.5ul Water: 3.5ul XbaI: 1ul

pBAD35-B DNA: 40ul Buffer4: 5ul BSA: 0.5ul Water: 2.5ul XbaI: 2ul

Leave the digestion on a 37c water bath overnight

Inoculation for Encryption Project

10ml LB in each tube and add 10ul Antibiotic A: BBa_K118004 B: BBa_K098995 C: BBa_I718008 D: BBa_B1006

Notebook May 26th, 2010

a)PCR purification(refer to protocol on the protocol from Notebook May 25th 2010) DNA Conc(ng/ul) pBAD35-A 57.6 pBAD35-B 69.7

b)Ligation (Peter did all the ligation) Ligate on pBAD18,33,34,35A-B 100ng/10ul rxn 2hrs bench ligation

c)Electroporation transformation

Use DH10B Add 1ul of plasmid Let it sits on ice for 5 mins

after the shock, immediately add 950ul LB and mix well Transfer to centrifuge tube

DNA Time 35 4.2 33 4.4 18 4.2

(control) 35 4.5 33 4.2 18 4.2

Let them grow in the 37c shaker for 1hr.

Plate 50ul of cells


Miniprep for Nate Pant's Project Miniprep Plasmid information A: BBa_K118004 B: BBa_K098995 C: BBa_I718008 D: BBa_B1006

Centrifuge at 30000rpm for 10mins

Follow the Miniprep Protocol: File:Qiagenmini.pdf Result Measured by Nanodrop

DNA Conc(ng/ul) 260/280 A 324.0 1.97 B 288.3 1.98 C 418.2 1.96 D 276.6 1.97


Notebook May 27th, 2010

Check the colonies in the plates.

Picked 10 colonies from pBAD35B Picked 5 colonies from pBAD33 Picked 5 colonies from pBAD18

Inoculate them in 5ml LB with antibiotics

Parts Checking

Concentration: BBa_K200000: 199.9ng/ul BBa_I716213: 186.5ng/ul BBa_S04114: 543.6ng/ul BBa_I718007: 146.1ng/ul BBa_I716212: 155.8ng/ul BBa_S03975: BBa_K200002: 309.3ng/ul BBa_K200003: 105.4ng/ul BBa_S09373: 243.1ng/ul BBa_J31000: 278.3ng/ul BBa_B1006: 276.6ng/ul BBa_K118004: 324.0ng/ul BBa_K098995: 288.3ng/ul

Single Digestion Test With EcoRI for 1. BBa_K200000: 2. BBa_I716213: 3. BBa_S04114: 4. BBa_I718007: 5. BBa_I716212: 6. BBa_S03975:(Don't have plasmid) 7. BBa_K200002: 8. BBa_K200003: 9. BBa_S09373: 10.BBa_J31000: 11.BBa_B1006: 12.BBa_K118004: 13.BBa_K098995:

Protocol 1.8ul Buffer3(10X) 0.18ul BSA(100X) 12ul MiliQ H2O 3ul DNA 1ul EcoRI

Incubate in water bath at 37C for 2 hours (total volume 18ul) add 3.6ul Dye (6X)

Load 6ul into the GEL Gel: File:2010-05-27 Map - Single digest.jpg

Gel Description: 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13

Results: 3, 9, 11, and 12 don't have the correct bands

So run the single digestion for all four again with 2 hours Gel: File:IGEM Single Digestion Second 2010-05-28 14hr 39min.jpg

Gel Description: 3, 9, Ladder, 11, 12

Result: Still not correct

Double Digestion Find one enzyme cutting site with the gene 1. BBa_K200000: Buffer:4 Enzyme:AflII EcoRI 2. BBa_I716213: Buffer:2 Enzyme:AflIII SpeI 3. BBa_S04114: Buffer:3 Enzyme:EcoRV EcoRI 4. BBa_I718007: Buffer:3 Enzyme:EcoRV EcoRI 5. BBa_I716212: Buffer:3 Enzyme:EcoRV EcoRI 6. BBa_S03975:(Don't have plasmid) 7. BBa_K200002: (don't have enzyme stock that can cut within the gene) 8. BBa_K200003: Buffer:4 Enzyme:NheI EcoRI 9. BBa_S09373: Buffer:3 Enzyme:EcoRV EcoRI 10.BBa_J31000: Buffer:3 Enzyme:BglI EcoRI 11.BBa_B1006: (Too small, cannot cut within the gene) 12.BBa_K118004: Buffer:3 Enzyme:EcoRV EcoRI 13.BBa_K098995: Buffer:2 Enzyme:HindIII EcoRI

Protocol 1.8ul NEB Buffer(10X) 0.18ul BSA(100X) 11ul MiliQ H2O 3ul DNA 1ul Enzyme1 1ul Enzyme2

Incubate in water bath at 37C for 2 hours (total volume 18ul) add 3.6ul Dye (6X)

Load 6ul into the GEL

Gel File:IGEM Parts Double Digestion 2010-05-28 14hr 54min.jpg File:IGEM Parts Double Digestion White 2010-05-28 14hr 57min.jpg

Result Only 1, 2, 4, 5, 8, 13 show the correct bands


Notebook May 28th, 2010

Miniprep for the screening

Restriction Enzyme Mapping for all the 20 screening plasmid

1.8ul Buffer3(10X) 0.18ul BSA(100X) 12ul MiliQ H2O 3ul DNA 1ul SpeI

Incubate in water bath at 37C for 2 hours (total volume 18ul) add 3.6ul Dye (6X)

Load 6ul into the GEL

Run the GEL at 100V for 30mins File:IGEM Biobrick 2010-05-28 16hr 55min.jpg GEL upper 18-1 18-2 18-3 18-4 18-5 Ladder 33-1 33-2 33-3 33-4 33-5

Gel Lower 35-1 35-2 35-3 35-4 35-5 Ladder 35-6 35-7 35-8 35-9 35-10


Notebook June 1st, 2010

Search the Detailed info of the Parts we received 1. BBa_K200000: Colanic acid global regulator (RcsB) 2. BBa_I716213: Cre(TTG Start) 3. BBa_S04114: Lysis 4. BBa_I718007: RBS-Cre 5. BBa_I716212: Cre(GTG Start) 6. BBa_S03975: Lacl+pL Luxr+ LuxpR(Don't have plasmid) 7. BBa_K200002: ygiV 8. BBa_K200003: Ligase(Rfal) 9. BBa_S09373: LacZ 10.BBa_J31000: Invertase Hin from Salmonella Typhimurium 11.BBa_B1006: Stop 12.BBa_K118004: rbs+dxs 13.BBa_K098995: Heat sensitive cl QPI with high promoter

Extra: BBa_J31001: DNA Invertase

Sequences for all the parts

>BBa_K200000 Part-only sequence (654 bp) atgaacaatatgaacgtaattattgccgatgaccatccgatagtcttgttcggtattcgcaaatcacttgagcaaattgagtgggtgaatgttgtcggcg aatttgaagactctacagcactgatcaacaacctgccgaaactggatgcgcatgtgttgattaccgatctctccatgcctggcgataagtacggcgatgg cattaccttaatcaagtacatcaagcgccatttcccaagcctgtcgatcattgttctgactatgaacaacaacccggcgattcttagtgcggtattggat ctggatatcgaagggatcgtgctgaaacaaggtgcaccgaccgatctgccgaaagctctcgccgcgctccagaaagggaagaaatttaccccggaaagcg tttctcgcctgttggaaaaaatcagtgctggtggttacggtgacaagcgtctctcgccaaaagagagtgaagttctgcgcctgtttgcggaaggcttcct ggtgaccgagatcgctaaaaagctgaaccgcagtattaaaaccatcagtagccagaagaaatctgcgatgatgaagctgggtgtcgagaacgatatcgcc ctgctgaattatctctcttcagtgaccttaagtccggcagataaagactaataa


>BBa_I716213 Part-only sequence (1032 bp) ttgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacctgatggacatgttcagggatc gccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataaccggaaatggtttcccgcaga acctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgt cggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggattcgaaaagaaaacgttgatgccggtgaacgtgcaaaacagg ctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgc ttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacg aaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatc cgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagc aactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcc cgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaacctggatagtgaaacagggg caatggtgcgcctgctggaagatggcgattaa


>BBa_S04114 Part-only sequence (1410 bp) gattgttctatcagtaatcgaccttattcctaattaaatagagcaaatccccttattgggggtaagacatgaagatgccagaaaaacatgacctgttggc cgccattctcgcggcaaaggaacaaggcatcggggcaatccttgcgtttgcaatggcgtaccttcgcggcagatataatggcggtgcgtttacaaaaaca gtaatcgacgcaacgatgtgcgccattatcgcctggttcattcgtgaccttctcgacttcgccggactaagtagcaatctcgcttatataacgagcgtgt ttatcggctacatcggtactgactcgattggttcgcttatcaaacgcttcgctgctaaaaaagccggagtagaagatggtagaaatcaataatcaacgta aggcgttcctcgatatgctggcgtggtcggagggaactgataacggacgtcagaaaaccagaaatcatggttatgacgtcattgtaggcggagagctatt tactgattactccgatcaccctcgcaaacttgtcacgctaaacccaaaactcaaatcaacaggcgccggacgctaccagcttctttcccgttggtgggat gcctaccgcaagcagcttggcctgaaagacttctctccgaaaagtcaggacgctgtggcattgcagcagattaaggagcgtggcgctttacctatgattg atcgtggtgatatccgtcaggcaatcgaccgttgcagcaatatctgggcttcactgccgggcgctggttatggtcagttcgagcataaggctgacagcct gattgcaaaattcaaagaagcgggcggaacggtcagagagattgatgtatgagcagagtcaccgcgattatctccgctctggttatctgcatcatcgtct gcctgtcatgggctgttaatcattaccgtgataacgccattacctacaaagcccagcgcgacaaaaatgccagagaactgaagctggcgaacgcggcaat tactgacatgcagatgcgtcagcgtgatgttgctgcgctcgatgcaaaatacacgaaggagttagctgatgctaaagctgaaaatgatgctctgcgtgat gatgttgccgctggtcgtcgtcggttgcacatcaaagcagtctgtcagtcagtgcgtgaagccaccaccgcctccggcgtggataatgcagcctcccccc gactggcagacaccgctgaacgggattatttcaccctcagagagaggctgatcactatgcaaaaacaactggatactagagccaggcatcaaataaaacg aaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttc tgcgtttata


>BBa_I718007 Part-only sequence (1058 bp) attaaagaggagaaatactagatgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacc tgatggacatgttcagggatcgccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataa ccggaaatggtttcccgcagaacctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggc cagctaaacatgcttcatcgtcggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggatccgaaaagaaaacgttgatg ccggtgaacgtgcaaaacaggctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaa tctggcatttctggggattgcttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatg ttaatccatattggcagaacgaaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccg tctctggtgtagctgatgatccgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgc cctggaagggatttttgaagcaactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtc ggagccgcgcgagatatggcccgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgta acctggatagtgaaacaggggcaatggtgcgcctgctggaagatggcgattaagaatt


>BBa_I716212 Part-only sequence (1032 bp) ttgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacctgatggacatgttcagggatc gccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataaccggaaatggtttcccgcaga acctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgt cggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggattcgaaaagaaaacgttgatgccggtgaacgtgcaaaacagg ctctagcgttcgaacgcactgatttcgaccaggttcgttcactcatggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgc ttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacg aaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatc cgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagc aactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcc cgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaacctggatagtgaaacagggg caatggtgcgcctgctggaagatggcgattaa


>BBa_S03975 Part-only sequence (1082 bp) aattgtgagcggataacaattgacattgtgagcggataacaagatactgagcacatactagagaaagaggagaaatactagatgaaaaacataaatgccg acgacacatacagaataattaataaaattaaagcttgtagaagcaataatgatattaatcaatgcttatctgatatgactaaaatggtacattgtgaata ttatttactcgcgatcatttatcctcattctatggttaaatctgatatttcaatcctagataattaccctaaaaaatggaggcaatattatgatgacgct aatttaataaaatatgatcctatagtagattattctaactccaatcattcaccaattaattggaatatatttgaaaacaatgctgtaaataaaaaatctc caaatgtaattaaagaagcgaaaacatcaggtcttatcactgggtttagtttccctattcatacggctaacaatggcttcggaatgcttagttttgcaca ttcagaaaaagacaactatatagatagtttatttttacatgcgtgtatgaacataccattaattgttccttctctagttgataattatcgaaaaataaat atagcaaataataaatcaaacaacgatttaaccaaaagagaaaaagaatgtttagcgtgggcatgcgaaggaaaaagctcttgggatatttcaaaaatat taggttgcagtgagcgtactgtcactttccatttaaccaatgcgcaaatgaaactcaatacaacaaaccgctgccaaagtatttctaaagcaattttaac aggagcaattgattgcccatactttaaaaattaataacactgatagtgctagtgtagatcactactagagccaggcatcaaataaaacgaaaggctcagt cgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggctcaccttcgggtgggcctttctgcgtttatat actagagacctgtaggatcgtacaggtttacgcaagaaaatggtttgttatagtcgaataaatactagagaaagaggagaaa


>BBa_K200002 Part-only sequence (486 bp) atgacaaacctgacactggatgtaaacattatcgatttcccatcaatacctgtggcgatgttgccgcaccgctgtagccctgaattgctcaactacagcg tggcgaaatttatcatgtggcgtaaagagacggggctttctcctgttaaccaaagccagacttttggcgtcgcctgggacgaccctgccaccaccgcacc ggaagcgtttcgctttgatatctgcggcagcgttagcgaaccgattcccgataatcgttatggtgtgagcaatggtgaacttaccggtggacgttatgcc gtggcccgccacgttggcgagctggacgatatttcacacacggtatggggcatcattcgccactggctgcctgcaagcggcgagaaaatgcgtaaagcac cgattctgtttcactacaccaatcttgccgaaggggtgacagagcagcgactggaaacggatgtttatgtgccgttggcgtgataa


>BBa_K200003 Part-only sequence (1263 bp) atgctaacatcctttaaacttcattcattgaaaccttacactctgaaatcatcaatgattttagagataataacttatatattatgttttttttcaatga taattgcattcgtcgataatactttcagcataaaaatatataatatcactgctatagtttgcttattgtcactaattttacgtggcagacaagaaaatta taatataaaaaaccttattcttcccctttctatatttttaataggcttgcttgatttaatttggtattctgcgtttaaagtagataattcgccatttcgt gctacttaccatagttatttaaatactgccaaaatatttatatttggttcttttattgttttcttgacactaactagccagctaaaatcaaaaaaagaga gtgtattatacactttgtattctctgtcatttctaattgctggatatgcaatgtatattaatagcattcatgaaaatgaccgcatttcttttggtgtagg aacggcaacaggagcagcatattcaacaatgctaatagggatagttagtggcgttgcgattctttatactaagaaaaatcatccttttttatttttatta aatagttgcgcggtactttatgttctggcgctaacacaaaccagagcaaccctactcctgttccctataatttgtgttgctgcattaatagcttattata ataaatcacccaagaaattcacttcctctattgttctactaattgctatattagctagcattgttattatatttaataaaccaatacagaatcgctataa tgaagcattaaatgacttaaacagttataccaatgctaatagtgttacttccctaggtgcaagactggcaatgtacgaaattggtttaaatatattcata aagtcacctttttcatttagatcagcagagtcacgcgctgaaagtatgaatttgttagttgcagaacacaataggctaagaggggcattggagttttcta acgtacatctacataatgagataattgaagcagggtcactgaaaggtctgatgggaattttttccacacttttcctctatttttcactattttatatagc atataaaaaacgagctttgggtttgttgatattaacgcttggcattgtggggattggactcagtgatgtgatcatatgggcacgcagcattccaattatc attatatccgctatagtcctcttactcgtcattaataatcgtaacaatacaattaattaataa


>BBa_S03973 Part-only sequence (3230 bp) aaagaggagaaatactagatgaccatgattacggattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcc ttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgctt tgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccctcaaactggcagatgcac ggttacgatgcgcccatctacaccaacgtgacctatcccattacggtcaatccgccgtttgttcccacggagaatccgacgggttgttactcgctcacat ttaatgttgatgaaagctggctacaggaaggccagacgcgaattatttttgatggcgttaactcggcgtttcatctgtggtgcaacgggcgctgggtcgg ttacggccaggacagtcgtttgccgtctgaatttgacctgagcgcatttttacgcgccggagaaaaccgcctcgcggtgatggtgctgcgctggagtgac ggcagttatctggaagatcaggatatgtggcggatgagcggcattttccgtgacgtctcgttgctgcataaaccgactacacaaatcagcgatttccatg ttgccactcgctttaatgatgatttcagccgcgctgtactggaggctgaagttcagatgtgcggcgagttgcgtgactacctacgggtaacagtttcttt atggcagggtgaaacgcaggtcgccagcggcaccgcgcctttcggcggtgaaattatcgatgagcgtggtggttatgccgatcgcgtcacactacgtctg aacgtcgaaaacccgaaactgtggagcgccgaaatcccgaatctctatcgtgcggtggttgaactgcacaccgccgacggcacgctgattgaagcagaag cctgcgatgtcggtttccgcgaggtgcggattgaaaatggtctgctgctgctgaacggcaagccgttgctgattcgaggcgttaaccgtcacgagcatca tcctctgcatggtcaggtcatggatgagcagacgatggtgcaggatatcctgctgatgaagcagaacaactttaacgccgtgcgctgttcgcattatccg aaccatccgctgtggtacacgctgtgcgaccgctacggcctgtatgtggtggatgaagccaatattgaaacccacggcatggtgccaatgaatcgtctga ccgatgatccgcgctggctaccggcgatgagcgaacgcgtaacgcgaatggtgcagcgcgatcgtaatcacccgagtgtgatcatctggtcgctggggaa tgaatcaggccacggcgctaatcacgacgcgctgtatcgctggatcaaatctgtcgatccttcccgcccggtgcagtatgaaggcggcggagccgacacc acggccaccgatattatttgcccgatgtacgcgcgcgtggatgaagaccagcccttcccggctgtgccgaaatggtccatcaaaaaatggctttcgctac ctggagagacgcgcccgctgatcctttgcgaatacgcccacgcgatgggtaacagtcttggcggtttcgctaaatactggcaggcgtttcgtcagtatcc ccgtttacagggcggcttcgtctgggactgggtggatcagtcgctgattaaatatgatgaaaacggcaacccgtggtcggcttacggcggtgattttggc gatacgccgaacgatcgccagttctgtatgaacggtctggtctttgccgaccgcacgccgcatccagcgctgacggaagcaaaacaccagcagcagtttt tccagttccgtttatccgggcaaaccatcgaagtgaccagcgaatacctgttccgtcatagcgataacgagctcctgcactggatggtggcgctggatgg taagccgctggcaagcggtgaagtgcctctggatgtcgctccacaaggtaaacagttgattgaactgcctgaactaccgcagccggagagcgccgggcaa ctctggctcacagtacgcgtagtgcaaccgaacgcgaccgcatggtcagaagccgggcacatcagcgcctggcagcagtggcgtctggcggaaaacctca gtgtgacgctccccgccgcgtcccacgccatcccgcatctgaccaccagcgaaatggatttttgcatcgagctgggtaataagcgttggcaatttaaccg ccagtcaggctttctttcacagatgtggattggcgataaaaaacaactgctgacgccgctgcgcgatcagttcacccgtgcaccgctggataacgacatt ggcgtaagtgaagcgacccgcattgaccctaacgcctgggtcgaacgctggaaggcggcgggccattaccaggccgaagcagcgttgttgcagtgcacgg cagatacacttgctgatgcggtgctgattacgaccgctcacgcgtggcagcatcaggggaaaaccttatttatcagccggaaaacctaccggattgatgg tagtggtcaaatggcgattaccgttgatgttgaagtggcgagcgatacaccgcatccggcgcggattggcctgaactgccagctggcgcaggtagcagag cgggtaaactggctcggattagggccgcaagaaaactatcccgaccgccttactgccgcctgttttgaccgctgggatctgccattgtcagacatgtata ccccgtacgtcttcccgagcgaaaacggtctgcgctgcgggacgcgcgaattgaattatggcccacaccagtggcgcggcgacttccagttcaacatcag ccgctacagtcaacagcaactgatggaaaccagccatcgccatctgctgcacgcggaagaaggcacatggctgaatatcgacggtttccatatggggatt ggtggcgacgactcctggagcccgtcagtatcggcggaatttcagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataatactaga gccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctctactagagtcacactggct caccttcgggtgggcctttctgcgtttata


>BBa_J31000 Part-only sequence (573 bp) atggctactattgggtatattcgggtgtcaacaattgaccaaaatatcgatttacagcgtaatgcgcttaccagtgcaaattgtgaccgcatttttgaag accgtatcagtggcaagattgcaaaccgccccggcctgaaacgggcgttaaagtatgtaaataaaggcgatactcttgtcgtctggaaattagacagact gggccgtagcgtgaaaaatctggtggcgttaatatcagaattacatgaacgtggagctcacttccattctttaaccgatagtattgataccagtagcgcg atggggcgattcttttttcatgtaatgtcagcactggccgagatggagcgagaattaatcgtcgagcgaacccttgccggactggctgccgccagagcgc aaggacgactgggagggcgccctcgggcgatcaacaaacatgaacaggaacagattagtcggctattagagaaaggccatcctcggcagcaattagctat tatttttggtattggcgtatccaccttatacagatactttccggcaagcagtataaaaaaacgaatgaattaa


>BBa_K118004 Part-only sequence (1882 bp) ctcaaggaggtactagatgagttttgatattgccaaatacccgaccctggcactggtcgactccacccaggagttacgactgttgccgaaagagagttta ccgaaactctgcgacgaactgcgccgctatttactcgacagcgtgagccgttccagcgggcacttcgcctccgggctgggcacggtcgaactgaccgtgg cgctgcactatgtctacaacaccccgtttgaccaattgatttgggatgtggggcatcaggcttatccgcataaaattttgaccggacgccgcgacaaaat cggcaccatccgtcagaaaggcggtctgcacccgttcccgtggcgcggcgaaagcgaatatgacgtattaagcgtcgggcattcatcaacctccatcagt gccggaattggtattgcggttgctgccgaaaaagaaggcaaaaatcgccgcaccgtctgtgtcattggcgatggcgcgattaccgcaggcatggcgtttg aagcgatgaatcacgcgggcgatatccgtcctgatatgctggtgattctcaacgacaatgaaatgtcgatttccgaaaatgtcggcgcgctcaacaacca tctggcacagctgctttccggtaagctttactcttcactgcgcgaaggcgggaaaaaagttttctctggcgtgccgccaattaaagagctgctcaaacgc accgaagaacatattaaaggcatggtagtgcctggcacgttgtttgaagagctgggctttaactacatcggcccggtggacggtcacgatgtgctggggc ttatcaccacgctaaagaacatgcgcgacctgaaaggcccgcagttcctgcatatcatgaccaaaaaaggtcgtggttatgaaccggcagaaaaagaccc gatcactttccacgccgtgcctaaatttgatccctccagcggttgtttgccgaaaagtagcggcggtttgccgagctattcaaaaatctttggcgactgg ttgtgcgaaacggcagcgaaagacaacaagctgatggcgattactccggcgatgcgtgaaggttccggcatggtcgagttttcacgtaaattcccggatc gctacttcgacgtggcaattgccgagcaacacgcggtgacctttgctgcgggtctggcgattggtgggtacaaacccattgtcgcgatttactccacttt cctgcaacgcgcctatgatcaggtgctgcatgacgtggcgattcaaaagcttccggtcctgttcgccatcgaccgcgcgggcattgttggtgctgacggt caaacccatcagggtgcttttgatctctcttacctgcgctgcataccggaaatggtcattatgaccccgagcgatgaaaacgaatgtcgccagatgctct ataccggctatcactataacgatggcccgtcagcggtgcgctacccgcgtggcaacgcggtcggcgtggaactgacgccgctggaaaaactaccaattgg caaaggcattgtgaagcgtcgtggcgagaaactggcgatccttaactttggtacgctgatgccagaagcggcgaaagtcgccgaatcgctgaacgccacg ctggtcgatatgcgttttgtgaaaccgcttgatgaagcgttaattctggaaatggccgccagccatgaagcgctggtcaccgtagaagaaaacgccatta tgggcggcgcaggcagcggcgtgaacgaagtgctgatggcccatcgtaaaccagtacccgtgctgaacattggcctgccggacttctttattccgcaagg aactcaggaagaaatgcgcgccgaactcggcctcgatgccgctggtatggaagccaaaatcaaggcctggctggcataataa



>BBa_K098995 Part-only sequence (935 bp) tttatggctagctcagtcctaggtacaatgctagctactagagaaagaggagaaatactagatgagcacaaaaaagaaaccattaacacaagagcagctt gaggacgcacgtcgccttaaagcaatttatgaaaaaaagaaaaatgaacttggcttatcccaggaatctgtcgcagacaagatggggatggggcagtcag gcgttggtgctttatttaatggcatcaatgcattaaatgcttataacgccgcattgcttgcaaaaattctcaaagttagcgttgaagaatttagcccttc aatcgccagagaaatctacgagatgtatgaagcggttagtatgcagccgtcacttagaagtgagtatgagtaccctgttttttctcatgttcaggcaggg atgttctcacctgagcttagaacctttaccaaaggtgatgcggagagatgggtaagcacaaccaaaaaagccagtgattctgcattctggcttgaggttg aaggtaattccatgaccgcaccaacaggctccaagccaagctttcctgacggaatgttaattctcgttgaccctgagcaggctgttgagccaggtgattt ctgcatagccagacttgggggtgatgagtttaccttcaagaaactgatcagggatagcggtcaggtgtttttacaaccactaaacccacagtacccaatg atcccatgcaatgagagttgttccgttgtggggaaagttatcgctagtcagtggcctgaagagacgtttggctgatactagagtcacactggctcacctt cgggtgggcctttctgcgtttatatactagagagagaatataaaaagccagattattaatccggcttttttattattttactagagtaacaccgtgcgtg ttgactattttacctctggcggtgataatggttgc


Summary for Week June 7th to June 11th

 6-7-10:

a) Miniprep 2 pellet each for pBAD33, pBAD35, pBAD18, ygiV (attach to the cell wall), RcsB(expression factor that increase the clonic acid).

b) enzyme digestion for pBAD33, vgiV, pBAD18, and RcsB.

c) Gel extract for vgiV and RcsB and purification

d) PCR purification for pBAD18 and pBAD33

e) overnight ligation (set up by Sarah) 

 6-8-10:

a) transformation pBAD18-RcsB and pBAD33-vgiV into DH10B E. Coli. (Sarah and Hannah plates the cloning of pBAD18-RcsB and pBAD33-vgiV)

b) Miniprep For Encryption Project: BBa_I11020, BBa_K199021, BBa_K2902006, BBa_200021, BBa_B1006-A, BBa_B1006-B, BBa_E0240 For Drug Project: 2 pellets of each, pBAD18, pBAD33, pBAD35, vgiV, RcsB 

 6-9-10:

a) For the RcsB and VgiV plates, only a few colonies growed (they are really tiny, kindda hard to see, so we put it back to the incubator growing for couple of more hours)

b) Did the cloning of RcsB into pBAD18 and vgiV into pBAD33 again with different insert/vector ratio.

c) Plate the pellet and control. 

 6-10-10:

a) The pBAD18-RcsB plate has colonies growed.

b) Set up the screening with colony PCR

c) Made MRS Broth, MRS plates, no antibiotic plates, Amp+Kan plates, and CM plates with Mary.

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