Reporter: Week 2 May 23-27

==Monday==
The reporters continued research on the linkers to hold the fusion proteins.
[[File:Jim_and_Alex_research.jpg‎|right|500px|caption]]

==Tuesday==
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The reporter group decided on a few linkers to test in a fusion protein construct. These linkers are K243004 (4 amino acids), K105102 (10 amino acids) as well as the linker used in the Imperial College part, K316007. The reporters spent the rest of the time designing primers for PCR. We needed primers for the XylE part, the linker, the tev cleavage site and the GFP/flag tag complex of the Imperial College part, K316007, the CI repressor/RecA cleavage site, the LacZ part (J33210), the K243004 linker, and the K105012 linker. 
===Primers===
Note:

<blockquote> <span style="color:#04B404">Green font indicates junk DNA ends (binding sites).</span></blockquote><blockquote><span style="color:#FF0000">Red font indicates annealing sites.</span></blockquote><blockquote>CAPITAL LETTERS INDICATE PREFIX AND SUFFIX.</blockquote><blockquote>lowercase letters indicate gene of interest. </blockquote>

====Linker used in K316007====

*''designed by Ben''

'''Annealing Temperature:''' 54.13&deg;C

'''Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5’ <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCG<span style="color:#FF0000">GCggaggttcagg</span>aggcagcACCGGTTAATACTAGTAGCGGCCGCTGCAG<span style="color:#04B404">TATT</span> 3’

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5’ <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCG<span style="color:#FF0000">GCggaggttcagg</span> 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5’ <span style="color:#04B404">AATA</span>CTGCAGCGGCCGCTACTAGTATTAACCGGTgctgcct<span style="color:#FF0000">cctgaacctccGC</span> 3’

====K105102: 10 Amino Acid Linker====

*''designed by Brian''

'''Annealing Temperature:''' 56.3&deg;C

'''Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span style="color:#FF0000">ggtgaaaatttgtattttcaa</span>tctggtggtACCGGTTAATACTAGTAGCGGCCGCTGCAG<span style="color:#04B404">TATT</span> 3'

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span style="color:#FF0000">ggtgaaaatttgtattttcaa</span> 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">AATA</span>CTGCAGCGGCCGCTACTAGTATTAACCGGTaccaccaga<span style="color:#FF0000">ttgaaaatacaaattttcacc</span> 3'

====K243004: 4 Amino Acid Linker====
 
*''designed by Alex''

'''Annealing Temperature:''' 57.78&deg;C

'''Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGG<span style="color:#FF0000">CggtggttctggtACC</span>GGTTAATACTAGTAGCGGCCGCTGCAG<span
style="color:#04B404">TATT</span> 3'

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGG<span style="color:#FF0000">CggtggttctggtACC</span>

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">AATA</span>CTGCAGCGGCCGCTACTAGTATTAACC<span style="color:#FF0000">GGTaccagaaccaccG</span> 3'

====tev Cleave Site====

*''designed by Jim''

'''Annealing Temperature:''' 55.83&deg;C

'''Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">ATTA</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span style="color:#FF0000">gagaatttgtattttcaggg</span>tACCGGTTAATACTAGTAGCGGCCGCTGCAG<span style="color:#04B404">ATTA</span> 3’

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">ATTA</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span style="color:#FF0000">gagaatttgtattttcaggg</span> 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TAAT</span>TAATCTGCAGCGGCCGCTACTAGTATTAACCGGTa<span style="color:#FF0000">ccctgaaaatacaaattctc</span> 3'

====cI Cleave Site====

*''designed by Jim''

'''Annealing Temperature:''' 56.13&deg;C

'''Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">ATTA</span>GAATTCGCGGCCGCTTCTAGATGGCCGGCgtt<span style="color:#FF0000">caggcagggatgttc</span>tcaACCGGTTAATACTAGTAGCGGCCGCTGCAG<span style="color:#04B404">ATTA</span> 3’

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">ATTA</span>GAATTCGCGGCCGCTTCTAGATGGCCGGCgtt<span style="color:#FF0000">caggcagggatgttc</span>

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5’ <span style="color:#04B404">TAAT</span>CTGCAGCGGCCGCTACTAGTATTAACCGGTtga<span style="color:#FF0000">gaacatccctgcctg</span> 3’

====XylE Part of K316007====

*''designed by Ben''

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5’ <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span style="color:#FF0000">aacaaaggtgtaatgcgac</span> 3’

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5’ <span style="color:#04B404">TATT</span>CTGCAGCGGCCGCTACTAGTATTAACCGGT<span
style="color:#FF0000">ggtcagcacggtcatg</span> 3’

====J33210: LacZ====

*''designed by Brian''

'''Annealing Temperatures:''' Forward=57.74&deg;C     Reverse=55.05&deg;C

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span
style="color:#FF0000">accatgattacggattcac</span> 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">ATAA</span>CTGCAGCGGCCGCTACTAGTATTAACCGGT<span
style="color:#FF0000">tcactccagccagc</span> 3'

====GFP and Flag Tag from K316007====

*''designed by Alex''

'''Annealing Temperatures:''' Forward=56.88&deg;C     Reverse=59.95&deg;C

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">TATT</span>GAATTCGCGGCCGCTTCTAGATGGCCGGC<span
style="color:#FF0000">cgtaaaggagaagaacttttc</span> 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' <span style="color:#04B404">AATA</span>CTGCAGCGGCCGCTACTAGTATTAACCGGT<span
style="color:#FF0000">cttgtcgtcatcatctttataat</span> 3'

==Wednesday==

&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The reporters designed primers for site-directed gene mutagenesis for the XylE gene in order to eliminate the restriction sites from the XylE gene. These are the primers:

Note: Red font shows the mutagenesis site

====Mutation 1: bp315 NgoMIV Site I====
'''Annealing Temperature:''' 79.49&deg;C

'''Original Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' GGC CGG CGC GTG CGC TTC C 3'

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' GGC CG<span
style="color:#FF0000">A</span> CGC GTG CGC TTC C 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' G GAA GCG CAC GCG <span
style="color:#FF0000">A</span>TCG GCC 3'

====Mutation 2: bp 486 XylE NgoMIV Site II====
'''Annealing Temperature:''' 70.54&deg;C

'''Original Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' C GAC GAA TTG CCG GCG ACC TAT GAC C 3'

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' C GAC GAA TTG CC<span
style="color:#FF0000">C</span> GCG ACC TAT GAC C 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' G GTC ATA GGT CGC <span
style="color:#FF0000">G</span>GG CAA TTC GTC G 3' 

====Mutation 3: bp 837 XylE Agel====
'''Annealing Temperature:''' 79.16&deg;C

'''Original Sequence:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' CAC AAA CCG GTG ACC TGG ACC ACC G 3'

'''Forward:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' CAC AAA CC<span
style="color:#FF0000">C</span> GTG ACC TGG ACC ACC G 3'

'''Reverse:'''
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;5' C GGT GGT CCA GGT CAC <span
style="color:#FF0000">G</span>GG TTT GTG 3'

==Thursday==
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The reporter group also began assembly of '''a construct featuring the XylE gene (with RBS) and a lac inducible promoter. This construct will be used to test the reporting system''' before the sensor system is created. Ben and Alex transformed the P<sub>lac</sub> and the XylE gene. The P<sub>lac</sub> part is R0010 and the XylE part is J33204.

==Friday==
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;The reporter group ran colony PCR on the created colonies then ran the results on a gel. The gel showed that the construct consisted of less than 500 base pairs, but should have been over 1100 base pairs. We think that only the lac inducible promoter showed up in our colonies. As a result of this set back, we will have to redo assembly next week.