Team:Caltech/Week 13

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Latest revision as of 18:31, 13 September 2011

Caltech iGEM 2011

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September 4

Overnights of: K123001 (bisdB) x2, pNT001 (construct vector) x2, and pSB3k3(BioBrick submission vector) x3

September 5

Miniprepped overnights of K123001, pNT001, and pSB3k3
PCR assembly of K123001 with prefix-promoter and terminator-suffix ultrimers to make bisdB construct
Gel of PCR assembly product
Overnights of J23119-lacZ-MG-1655 for x-gal analysis

Results

Miniprep concentrations:

Miniprep	Nanodrop conc. (ng/uL)
pNT001-a	41
pNT001-b	83.5
K123001-a	101.8
K123001-b	68.0
pSB3k3-a	69.3
pSB3k3-b	83.4
I372017	0
pSB3k3-c	54.1

Gel reveals correct sized product of ~1.5kbp

September 6

Assembly of bisdB pcr'ed construct into pNT001 (with bisdA) and pSB3k3 (BioBrick submission vector, with unwanted RFP)
Plate all assembled & transformed constructs and controls
Grow overnights of J23119-lacZ-MG-1655 from two different plate sources and one glycerol stock

September 7

Plated the rest of transformed pSB3k3-bisdB
Add the J23119-lacZ-MG-1655 to autoclaved LB+glass beads to incubate biofilm at 37&degC
Performed qualitative X-gal experiment to pick best J23119-lacZ-MG-1655 overnight (turned most blue in least time)
Restreak pNT001-bisdB colonies onto another plate for clearer single colonies

Results

Plate results:

Plate	Number of colonies
pNT001-bisdB	15
pSB3k3-bisdB	0
pSB3k3-control #1 (just digestion)	20, all red
pSB3k3-control #2 (digestion + ligation)	6, one white
pSB3k3-control #3 (digest, dephosphorylate, ligate)	0

September 8

Overnights of all pNT001-bisdB and pSB3k3-bisdB colonies for sequencing tomorrow
Transformed DDT-pET protein overexpression vector into BL21D3 strain
Mix 40mg/mL X-gal (100x) in preparation for X-gal-biofilm experiment
PCR amplification for BisdB parts mix using phusion:
Volume: 50 ul

Temp 98°C for 30 seconds
Temp 98°C for 10 seconds
Temp 55°C for 20 seconds
Temp 72°C for 30 seconds
Go to 2, 9
Temp 98°C for 10 seconds
Temp 65°C for 20 seconds
Temp 72°C for 30 seconds
Go to 6, 19
Temp 79°C for 3 minutes
Temp 4°C forever
END

Results

Transformation plating of pSB3k3-bisdB yielded 4 red colonies and one white colony

September 9

Minipreps and quikchange kit
Quikchange kit pcr for a 25 ul reaction:

2.5 ul of 10x buffer
5 ul of plasmid
1 ul of primer A
1 ul of primer B
1 ul of dNTP mix
1 ul of lightning
0.75 ul of Quik Solution
12.75 ul of water

@@ Line 49: / Line 49: @@
 <td>54.1</td>
 </tr>
-</table>
+</table><br/>
-Gel reveals correct sized product of ~1.5kbp<br/>
+<gallery>File:BisdB_assembly_gel.jpg|Gel reveals correct sized product of ~1.5kbp</gallery>
 ==September 6==
@@ Line 95: / Line 96: @@
 Transformed DDT-pET protein overexpression vector into BL21D3 strain<br/>
 Mix 40mg/mL X-gal (100x) in preparation for X-gal-biofilm experiment<br/>
+PCR amplification for BisdB parts mix using phusion: <br/>
+Volume: 50 ul <br/>
+* Temp 98°C for 30 seconds
+* Temp 98°C for 10 seconds
+* Temp 55°C for 20 seconds
+* Temp 72°C for 30 seconds
+* Go to 2, 9
+* Temp 98°C for 10 seconds
+* Temp 65°C for 20 seconds
+* Temp 72°C for 30 seconds
+* Go to 6, 19
+* Temp 79°C for 3 minutes
+* Temp 4°C forever
+* END
 ===Results===
 Transformation plating of pSB3k3-bisdB yielded 4 red colonies and one white colony<br/>
+==September 9==
+Minipreps and quikchange kit<br/>
+Quikchange kit pcr for a 25 ul reaction:<br/>
+* 2.5 ul of 10x buffer
+* 5 ul of plasmid
+* 1 ul of primer A
+* 1 ul of primer B
+* 1 ul of dNTP mix
+* 1 ul of lightning
+* 0.75 ul of Quik Solution
+* 12.75 ul of water
 }}