Yuan religated and transformed GFP with Pgad

After talking to the advisors and troubleshooting our ligation and transformation problems, they came up with several ideas to help improve our transformation and ligation efficiency
First, they suspect our P restriction enzyme may not be digesting fully
Secondly, they think we should run our digestions/ligations for 2 hours/overnight respectively instead of 1 hour/30 minutes

Refer to the meeting notes for more information regarding the advisors advice

The lab digested for 2 hours at 37 degrees celsius:
Pgad E/S
Pbad E/S
GFP X/P
CLC E/S
Double Terminator X/P
Weak CP E/S
Medium CP E/S
Strongest CP E/S
Strong RBS E/X
Strongest RBS E/X
KanBB E/P

The lab ligated overnight at 16 degrees celsius:
Pgad/GFP
CLC/Double terminator
Strongest CP/Strongest RBS
Strongest CP/Strong RBS
Weak CP/Kan BB
Medium CP/ Kan BB
Kan BB control
Chlor BB control
Strongest RBS control
Strong RBS control

The lab overnighted(x3):
Phytases: EC and Cbac
Constitutive promoters
Strongest RBS
Pgad
Lysis
Pgad/Lysis
CLC
Pbad
GFP

Miniprep the overnighted parts from lastnight:
Phytases: EC and Cbac
Constitutive promoters
Strongest RBS
Pgad
Lysis
Pgad/Lysis
CLC
Pbad
GFP

Digested the parts with the following enzymes:
Phytases: EC and Cbac (E and S)
Strongest RBS (E and X)[messed up]
Pgad ( E and S)
Lysis (X and P)[could not find plate]
Pgad/Lysis (E and S)
CLC (E and S)
Pbad(E/S digest for sequencing)
GFP (X and P)[messed up]

nsCP(E/P)
wCP(E/P)
mCP(E/P)

Kbb:
6.53 uL DNA, 35.47 uL water
pGad1:
7.01 uL DNA , 35.49 uL water
pGad2:
8.91 uL DNA, 33.59 uL water
pGad3:
8.36 uL DNA, 34.14 uL water
citro1:
9.8 uL DNA, 32.7 uL water

The lysis plate could not be found for the overnight last night/miniprep this morning
The GFP, double terminator, and strongest RBS minipreps were all done incorrectly.

The GFP, double terminator, and strongest RBS parts were reovernighted to be reminiprepped and digested tomorrow

Mike and Lajja prepared the miniprepped DNA from monday’s overnights(pgad/lysis part and ECphy) for sequencing
The lab discovered that our plan of attack for the RBS/CP problem was wrong; the part we were using had RFP and an RBS and a CP in it

We will now digest the backbone (that has a CP/RBS/RFP in it) with S and P to get rid of the RFP and open up a slot for us to insert our part.

Then we will digest the part we want inserted with X and P. Finally, we will ligate the CP/RBS backbone part with the part we want inserted to make a CP/RBS/Part construct

We chose to follow two parallel routes:
1.) Digest/Gel extract/Ligate/Transform
2.) Digest/Ligate/Transform

Digested Ligated and transformed:
EC Phy(E/P) + KanaBB(E/P) ---> on kan
Citro Phy (X/P) + wCP(S/P) ----> on amp
Citro Phy (X/P) + mCP(S/P) ----> on amp
Citro Phy (X/P) + nsCP(S/P) ----> on amp

For route 1 the CP/RBS backbone part was gel extracted and eluted, making sure no RFP part could religate into the backbone.
For route 2 the CP/RBS backbone part was not gel extracted so some RFP can religate; however, because RFP should show up red we will pick non red colonies.

Ligation calculations:
707-27 = 680 bp mRFP
2948 - 680= 2268 backbone

CBPhy 1302bp

6(1302/2268)*10 = 34.44 ng CB Phy

ECPhy 1299 bp
Kbb 2204 bp

6(1299/2204)*10 = 35.36 ng ECPhy

10 ng/uL CP, 1 uL CP
10 ng/uL CBPhy, 3.44 uL CB Phy
10 ng/uL ECPhy, 3.54 uL EC Phy
10 ng/uL Kbb, 1 uL Kbb

CP+CBPhy
8.5 - 1- 3.44 = 4.06 uL water
Kbb+ECPhy
8.5-1-3.54 = 3.96 uL water

Yuan and Mike gel extracted the w. m, and nsCP backbones
Grant transformed the ligated parts from earlier

Tae and Lajja miniprepped the overnights from last night

Yuan transformed the CP + kan ligations (from 8/21), CP + phy ligations, and Pbad + rbs ligations.

Tae and Lajja PCR’d out the chloramphenicol backbone
Tae and Lajja however did not PCR correctly;

lysis(5.36 uL DNA, 37.14 uL water)
pgad(digested/gfp(digested already) ---> chloro(4 uL mix and 4uL
clc(digested)/xx(digested) ---------> kan(digested)
clc/xx+ wCP(g/e digested)
clc/xx+mCP
clc/xx+nsCP

Ligations
Pgad+GFP+ChlorBB

Pgad: 1251
GFP: 720
ChlorBB: 2070

6(1251/2070)*10 = 36.26 ng Pgad, 3.62 uL Pgad
6(720/2070)*10 = 20.87 ng GFP, 2.09 uL GFP

12.5 ng in 1 uL ChlorBB
.8uL ChlorBB

8.5 - 3.62 - 2.09 - .8 = 1.99 uL water

CLC+XX+Kan BB
CLC: 1564
XX: 129
Kan BB: 2204

6(1564/2204)*10 = 42.58 ng CLC, 4.26 uL CLC
6(129/2204)*10 = 3.51 ng XX, .351 uL XX

8.5 - 4.26 - .351 - 1 = 2.89 uL water

10 ng in 1 uL of KanBB
1 uL KanBB

CLC/XX: 1693
wCP: 2268

CLC/XX + wCP
1 uL CP
6(1693/2268)*10 = 44.79 ng, 4.48 uL CLC/XX

CLC/XX + mCP
6(1693/2268)*10 = 44.79 ng, 4.48 uL CLC/XX
3(1693/2268)*10 = 22.39ng, 2.24 uL CLC/XX
1(1693/2268)*10 = 7.46ng, .75 uL CLC/XX

8.5 - 4.48 - 1 = 3.02 uL water
8.5 - 2.24 - 1 = 5.26 uL water
8.5 - .75 - 1 = 6.75 uL water

CLC/XX + nsCP
6(1693/2268)*10 = 44.79 ng, 4.48 uL CLC/XX

Yuan transformed the ligated parts from yesterday. We chose not to ligate the clc + XX + wCP or clc + XX + nsCP parts because the gel yesterday revealed that our clc + XX part was not properly ligated.
Yuan transformed:
//on amp

• 6:1 clc + xx + mCP
• 3:1 clc + xx + mCP
• 1:1 clc + xx+ mCP
• clc + xx+ mCP control

//on chlor

• Pgad + GFP + chlor
• Pgad + GFP + chlor control

//on kan

• clc + xx + kan
• clc + xx + kan control
• gBlocks (5 uL DNA used, instead of 1 uL)
• gBlocks control

Yuan and Tae gel extracted EC phy, strong CP, new strong CP, medium CP, weak CP, and strongest RBS. The EC phy did not show on the gel under long wave UV light, and the strongest RBS had a very bad 260/280 ratio, prompting us to throw it out.

Lajja and Grant miniprepped RFP (for the chloramphenicol backbone), nsCP + CB phy, wCP + CB phy, mCP + CB phy, and EC phytase + kan. A number of them were thrown out when, in the process of miniprep, the colonies were shown to be red.

Yuan and Mike created plates from the overnights.

Lajja and Grant digested the parts they miniprepped.

• nsCP + CB phy x6 (EP)
• wCP + CB phy x6 (EP)
• mCP + CB phy x3 (EP)
• EC phy + kan x5 (XP)